Biomedical subjects

M Wagner

Publications and source records attributed to M Wagner.

At least 19 recordsLinked to original sources

PubMed1992

Specificity of the sialic acid-binding lectin from the snail Cepaea hortensis.

The specificity of the sialic acid-binding lectin from the snail Cepaea hortensis, purified by affinity chromatography on fetuin-Sepharose, was studied by hemagglutination inhibition assay applying 32 sialic acid derivatives and 14 glycoproteins. 2-alpha-Methyl-9-O-acetyl-NeuAc was the most potent inhibitor, followed closely by 2-alpha-methyl-NeuAc and 2-alpha-benzyl-NeuAc. An axially orientated carboxyl group is a prerequisite for maximal lectin-sugar binding. Neither size nor polarity of the alpha-anomeric substituent significantly influenced inhibition potency. An intact sialic acid N-acetyl group is essential for optimal lectin-sugar interaction. The trihydroxypropyl side chain also is of great importance. However, a bulky hydrophobic substituent at the side chain like a 9-O-tosyl residue did not decrease binding to the lectin. The lectin did not distinguish between NeuAc alpha 2----3Gal beta 1----4Glc and NeuAc alpha 2----6Gal beta 1----4Glc. Among other sugars tested, only N-acetylglucosamine showed inhibition, although 50-fold less. The most potent glycoprotein inhibitors were those carrying O-chains only or preferentially, as ovine submaxillary mucin, bovine submaxillary mucin, and glycophorin A. Tamm-Horsfall protein was an exception being a strong inhibitor, although carrying only N-chains. Asialoglycoproteins were inactive. Glycoproteins containing the NeuAc alpha 2----3Gal sequence inhibited the lectin as well as those with NeuAc alpha 2----6GalNAc. From the results a model of the lectin's binding site for sialic acid is suggested.

Animals↗

PubMed1992

Sensitive detection of RNAs in single cells by flow cytometry.

A rapid and sensitive fluorescent in situ hybridization method has been developed to probe RNA contents of individual cells by flow cytometry. Fixed cells in suspension were hybridized with 5' end-fluorophore-labeled oligodeoxynucleotides complementary to defined regions of the RNA of interest and analyzed by flow cytometry. With this method, we monitored combinations of histone H4 mRNA, 18S rRNA and 28S rRNA levels in synchronized HeLa S3 cells by multicolor analysis. A fluorescence signal equivalent to 1800 copies of histone H4 mRNA per cell was detected with signal-to-background ratio of 5.4. If non-specific binding of the fluorophore-labeled probe can be reduced, as few as 100 copies of mRNA of the size of H4 could be detected in individual cells by flow cytometry.

Base Sequence↗

PubMed1992

Localization of the steroid 1-dehydrogenase in Rhodococcus erythropolis IMET 7030 by immunoelectron microscopy.

The steroid 1-dehydrogenase of Rhodococcus erythropolis IMET 7030, an active steroid-transforming strain, was localized by immunogold labelling both in cells induced with 17-alpha-methyl-testosterone and in noninduced cells. The labelling intensity was much higher in induced cells than in noninduced cells, indicating increased enzyme production in the case of induction. Using the postembedding procedure, the main portion of the enzyme was found in the peripheral region of the cytoplasm. A considerable amount was bound in clusters to the inner side of the cytoplasmic membrane. The enzyme was also detected in channels connecting the cytoplasm with the cell surface. By means of the preembedding labelling, a few gold clusters could be detected on the cell surface. The significance of this observation was discussed.

Cytoplasm↗

PubMed1992

Overexpression of a Rhodococcus erythropolis protein in Escherichia coli with immunological identity to the Rhodococcus steroid 1-dehydrogenase. Immunoelectron microscopic localization and electrophoretic studies.

The recombinant Escherichia coli K-12 strain chi 6060 harbouring the plasmid pYA 1201 with a gene from Rhodococcus erythropolis IMET 7030 overexpressed a protein which reacts with a monospecific antiserum against the steroid 1-dehydrogenase (Sdh) from the same Rhodococcus strain. It was shown previously that this recombinant protein exhibits no enzymatic activity. By immunogold labelling the protein was localized on ultrathin sections of the recombinant E. coli strain. After cultivation at 37 degrees C it was found within large cytoplasmic compartments (inclusion bodies). The inclusion bodies occupied 40% to 75% of the sectioned cell area. The highest amount of protein was observed after induction of the culture with isopropyl-beta-D-thiogalactopyranoside. Approximately 20% of the induced cells became enlarged (up to 5-fold of the normal size) and deformed; multiplication of the Rhodococcus protein producing cells was inhibited. After ultrasonic cell disintegration the inclusion bodies were found only in the fraction of the sedimented cell debries and did still react with anti-Sdh. When recombinant E. coli cells were cultivated at 28 degrees C, inclusion bodies appeared very seldom and the immunoreactive protein was distributed throughout the whole cytoplasm.

Animals↗

PubMed1992

Spirometric and endoscopic evaluation of airway collapse in infants with bronchopulmonary dysplasia.

We evaluated eight infants with bronchopulmonary dysplasia (BPD) at ages from 2 to 13 months who had repeated episodes of clinical respiratory deterioration associated with agitation. These episodes limited further weaning from ventilation or necessitated recurrent intubation and reinstitution of ventilation. All infants underwent spirometric evaluation and six also had endoscopic examination during simulated agitation episodes (elicited by toe pinching). All babies were found to have a very prolonged near zero expiratory airflow pattern, accompanied by vigorous diaphragmatic and abdominal muscle activity and rapid development of hypoxia. Six patients had endoscopically documented tracheal collapse under the same simulated circumstances. The episodes ceased with calming or sedation of the infants.

Airway Obstruction↗

PubMed1992

Omission of exon 12 in cystic fibrosis transmembrane conductance regulator (CFTR) gene transcripts.

Cystic fibrosis transmembrane conductance regulator (CFTR) mRNA transcripts isolated from both expressing and "non-expressing" cell types of normal individuals exhibit differential splicing to a variable extent in a region encoding the putative nucleotide binding fold of the CFTR polypeptide. Sequence analysis of the aberrant fragments obtained after cDNA polymerase chain reaction amplification confirmed the in-frame joining of exons 11 and 13. The proportion of alternative splicing is reproducible and constant in a given individual. The omission of exon 12 in a significant proportion of transcripts supports the hypothesis that a minimal amount of correctly expressed CFTR is sufficient for the maintenance of a clinically normal phenotype.

Base Sequence↗

PubMed1992

Interaction of the yeast pleiotropic drug resistance genes PDR1 and PDR5.

The network of genes which mediates multiple drug resistance in yeast includes, among others, the PDR1 gene, which encodes a putative regulator of gene expression, and PDR5, a locus whose amplification leads to resistance. We demonstrate that disruption of PDR5 causes marked hypersensitivity not only to cycloheximide but also to sulphometuron methyl and the mitochondrial inhibitors chloramphenicol, lincomycin, erythromycin and antimycin. Genetic analysis of double mutants containing an insertion in PDR5 (pdr5:Tn5), which renders cells hypersensitive to cycloheximide, and a pdr1 mutation, which confers resistance to this inhibitor, indicates that the expression of resistance requires a functional PDR5 gene. The same interdependency is observed for chloramphenicol, but not for oligomycin, lincomycin, erythromycin or sulphometuron methyl. Northern analysis of PDR1 and PDR5 transcripts reveals that the 5.2 kbp PDR5 transcript is overexpressed in pdr1 (resistant) mutants, but underexpressed in a disruption of PDR1. These observations provide strong experimental support for our former proposal that the PDR5 gene is a target for regulation by the PDR1 gene product.

Anti-Bacterial Agents↗

PubMed1992

[Proliferation kinetic studies in the syngeneic liver transplantation mode in the rat. Effect of re-arterialization on the transplant].

Successful orthotopic liver transplantation (OLT) can be achieved in the rat. We used bromo-2-deoxyuridine (BrdU) as a proliferation marker to document morphological differences between OLT with and without rearterialization. Animals with portal anastomosis alone had a significantly increased proliferation rate of hepatocytes, Kupffer cells, and bile duct epithelial cells, as indicated by strong staining with BrdU, 8 days post-transplant compared to animals with rearterialization. Regeneration of ischemically damaged liver parenchymal cells may account for this observation. Thus, OLT with rearterialization appears to be the more physiological transplant model.

Anastomosis, Surgical↗

PubMed1992

Clinical and polymyographic investigation of spasmodic torticollis.

Polymyographic recordings were used to identify the most dystonic muscles suitable for local injection with botulinum toxin in 100 patients with spasmodic torticollis (TS). Rotating TS (72% of the patients) was due to dystonic activity of the splenius muscle ipsilateral to and/or the sternocleidomastoid muscle contralateral to the side of chin deviation. One-third of these patients had also dystonic activation of the contralateral splenius muscle and, rarely, the contralateral trapezius muscle. Ten patients had laterocollis due to dystonic activation of all recorded muscles on one side of the neck. Nine patients had retrocollis due to activity of both splenius muscles and rarely additional activity in both trapezius muscles. The type of dystonic muscle activity was found to be tonic, phasic or tremulous. Besides the evaluation of spontaneous dystonic EMG activity further examination during the "geste antagoniste" or the muscle activity during rotating head movements can provide additional information. It is concluded that polymyography may provide a rationale for identifying the dystonic muscles underlying the different forms of TS. It may prove to be helpful for the successful therapy with botulinum toxin and may be useful in differentiating tremulous torticollis from other types of head tremor.

Adolescent↗

PubMed1992

Treatment of spasmodic torticollis with local injections of botulinum toxin. One-year follow-up in 37 patients.

Thirty-seven patients with spasmodic torticollis (cervical dystonia) who received repeated local injections of botulinum toxin have been followed up for a mean period of 12.3 (10-29) months, during which time 138 treatment sessions were performed. Mean doses per muscle averaged 320 mouse units (mu; range 160-1000 mu botulinum toxin A prepared by CAMR, Porton Down, UK). Eighty-six per cent of patients experienced significant improvement of posture and 84% of those with pain had relief following the first injection. Muscular patterns of recurrent torticollis were relatively constant and in most patients efficacy was maintained with subsequent injections, while 15% of all follow-up sessions failed. Only 2 of 37 patients were consistent nonresponders; 22% and 10% of all sessions were complicated by transient dysphagia and weakness of neck muscles, respectively. It is concluded that local botulinum toxin injections can be a safe and efficaceous long-term treatment of spasmodic torticollis and that optimal doses should be between 200 and 400 mu/muscle.

Botulinum Toxins↗

PubMed1992

[Judgement of prognosis in multiple trauma of patients with blunt abdominal injuries based on different useful trauma scores].

The use of trauma indices for characterisation of polytraumatized patients concerning prognoses is being discussed for nearly 30 years. In the seventies a couple of trauma indices were developed. Some of them are in use all over the world. In the Department for Trauma surgery in the General Hospital of Salzburg three of the common trauma scores were analysed and compared with one another: Injury Severity Score (Baker), Polytraumascore of the University Clinic of Hannover, Trauma index (Schreinlechner and Eber). As a rule most studies presently published are based on the Injury Severity Score. In a retrospective analysis of 62 polytraumatized patients we could show that the Trauma index of Schreinlechner and Eber is more practicable for classification of polytrauma because it shows a good correlation of score, fatality and prognoses.

Abdominal Injuries↗

PubMed1992

Localization of the cholesterol oxidase in Rhodococcus erythropolis IMET 7185 studied by immunoelectron microscopy.

Rhodococcus erythropolis IMET 7185 produces an inducible cholesterol oxidase (COD) which can easily be extracted by treatment of cells with 0.1% Triton X-100. The yield of the enzyme was 3.3 U/g wet wt from induced cells, which is about 5 times more than from non-induced cells. A study of the location of COD on intact cells and on ultrathin sections by means of immunogold electron microscopy revealed the following distribution, which corresponds with the biochemical results: (1) COD, which is extractable by the detergent, was localized in a distance up to 80 nm above the cell surface. It belongs to a surface layer, which only became visible after lysine/glutaraldehyde treatment and staining with ruthenium red, indicating a high carbohydrate content. (2) Non-extractable COD was found on the cell surface in a shorter distance to the cell wall as well as within the cell wall, in the cytoplasmic membrane and in the peripheral cytoplasm. In the latter clusters of gold particles on some places suggest the presence of larger amounts of insoluble enzyme.

Blotting, Western↗

PubMed1992

Signal transduction in murine B16 melanoma cells.

The relationship between lung colonization and signal transduction was investigated for six B16 melanoma variants. A range of experimental metastatic potential (as determined by lung colonization), forskolin-stimulated cyclic AMP accumulation and FCS-stimulated protein kinase C activity was found. The major findings were that: (1) cells with the highest agonist-stimulated cyclic AMP production were those with the highest level of membrane-associated protein kinase C activity; (2) clones which differed in protein kinase C levels and distribution did so in the presence but not in the absence of foetal calf serum; and (3) no simple relationship was seen between either signal transduction system and lung colonization for all six variants. Altered ras expression was also excluded as an explanation for the differences in signal transduction and lung colonization potential which were observed. We conclude that differences in signal transduction in vitro between these cells do not relate simply to lung colonization potential in vivo.

Animals↗

PubMed1992

Intracellular regulation of cell adhesion to extracellular matrix components in murine B16 melanoma cells.

The involvement of signal transduction systems in the initial attachment of two murine B16 melanoma clones of differing metastatic potential to extracellular matrix components was examined to learn more of the early events in cell-matrix interaction. Clones of high and low metastatic capacity attached similarly in the absence of any stimulators, exhibiting a two phase time course of attachment with 100% attachment by 60 min. A slight difference in attachment characteristics between the clones was seen in response to phorbol ester stimulation, which significantly inhibited attachment of the low metastatic clone but which had no effect on the highly metastatic clone. Total protein kinase C activity and distribution was similar for both clones. Attachment of both clones was severely reduced, however, if intracellular calcium was elevated or intracellular calmodulin inhibited. This study suggests that signal transduction mechanisms are involved in melanoma cell attachment to matrix proteins and offers an approach to pharmacological manipulation of these cell-matrix interactions which may be relevant to reducing metastatic spread.

Animals↗

PubMed1992

Flow cytometric analysis of lymphocyte phenotypes in bronchoalveolar lavage fluid: comparison of a two-color technique with a standard immunoperoxidase assay.

Characterization of lymphocytes in bronchoalveolar fluid has provided insight into the pathogenesis of many pulmonary diseases. Identification of lymphocyte phenotypes has become highly successful due to development of specific monoclonal antibodies and reliable methods for detecting labeled cells such as flow cytometry (FCM) and immunocytochemistry. FCM permits rapid screening of many cells, but this analysis may be confounded by heterogeneity in the size and granularity of the cells being evaluated. Such heterogeneity may lead to exclusion of cells of interest and inclusion of unwanted cells. Often peripheral blood leukocytes are used to define the gate for lung lymphocytes, but this gate may be inappropriate due to considerable variation in size and granularity of cells in bronchoalveolar lavage (BAL) fluid. Here we report an alternative method for generating a gate which employed fluorescence and side scatter signals to analyze lymphocyte subsets in BAL fluid by FCM. This gating technique avoids the pitfalls inherent in using the conventional lymphocyte gate to analyze lung cells. To validate this approach, we compared the results generated by this gate and those from the conventional forward/side light scatter gate to results derived from an immunocytochemical technique (ABC) that has been extensively employed in our laboratory to identify lymphocyte subsets in blood and lavage fluid. FCM tended to underestimate the proportions of T-cell subsets compared with ABC when the conventional gate was used. Counting only cells that stained with fluorescein-conjugated anti-CD45 antibody and that had side scatter properties of lymphocytes, however, resulted in excellent agreement between FCM and ABC. It appears that the CD45+/side scatter gate includes the vast majority of lymphocytes in BAL fluid while excluding most of the nonlymphoid cells that contaminate the conventional gate. It was this latter group of cells, and erythrocytes in particular, that led to the artificially low values for lymphocyte phenotypes in BAL fluid by FCM when the conventional lymphocyte gate was used. Although erythrocytes in BAL fluid may be eliminated by hypotonic lysis, this may also result in contamination of the conventional lymphocyte gate with nuclear debris and particulates from macrophages. Despite these advantages, the fluorescence/side scatter gate may not always be optimal for the evaluation of T lymphocytes if BAL fluid contains CD45+, nonlymphoid cells with low side light scatter. In these instances, additional antibodies such as anti-CD14 and anti-CD11 may be employed to determine the size of contaminant monocytic cells and neutrophils.(ABSTRACT TRUNCATED AT 400 WORDS)

Bronchoalveolar Lavage Fluid↗

PubMed1992

MELAS syndrome with mitochondrial tRNA(Leu)(UUR) mutation: correlation of clinical state, nerve conduction, and muscle 31P magnetic resonance spectroscopy during treatment with nicotinamide and riboflavin.

We report a patient with mitochondrial encephalomyopathy, lactic acidosis, and strokelike episodes treated with riboflavin and nicotinamide for 18 months, during which time previously frequent encephalopathic spells ceased. To confirm clinical benefit, we withdrew treatment and monitored response with muscle 31P magnetic resonance spectroscopy (MRS) and sural nerve conduction studies. Of three prospectively chosen MRS variables, two changed coincidentally with clinical end points; phosphocreatine (PCr)/adenosine triphosphate recovery rates fell in parallel with sural nerve sensory amplitudes, and a drop in muscle bioenergetic efficiency (relationship of inorganic phosphate/PCr to the accelerating force of contracting muscle) coincided with development of encephalopathy. Investigations revealed a deficiency of respiratory complex I and mutation of the mitochondrial tRNA(Leu)(UUR). We suggest that a defective cellular energy state in mitochondrial disease may be partially treatable and that changes seen in appropriate muscle spectroscopy studies may parallel improvement in brain and peripheral nerve function.

Adult↗

PubMed1992

Regional differences in retinoid release from embryonic neural tissue detected by an in vitro reporter assay.

Retinoic acid and related retinoids have been suggested to contribute to the pattern of cell differentiation during vertebrate embryonic development. To identify cell groups that release morphogenetically active retinoids, we have developed a reporter assay that makes use of a retinoic acid inducible response element (RARE) to drive lacZ or luciferase reporter genes in stably transfected cell lines. This reporter gene assay allows detection of retinoids released from embryonic tissues over a range equivalent to that induced by femtomole amounts of retinoic acid. We have used this assay first to determine whether the floor plate, a cell group that has polarizing properties in neural tube and limb bud differentiation, is a local source of retinoids within the spinal cord. We have also examined whether the effects of exogenously administered retinoic acid on anteroposterior patterning of cells in the developing central nervous system correlate with differences in retinoid release from anterior and posterior neural tissue. We find that the release of morphogenetically active retinoids from the floor plate is only about 1.5-fold that of the dorsal spinal cord, which does not have neural tube or limb polarizing activity. These results suggest that the spatial distribution of retinoid release from spinal cord tissues differs from that of the neural and limb polarizing activity. This assay has also shown that retinoids are released from the embryonic spinal cord at much greater levels than from the forebrain. This result, together with previous observations that the development of forebrain structures is suppressed by low concentrations of retinoic acid, suggest that the normal development of forebrain structures is dependent on the maintenance of low concentrations of retinoids in anterior regions of the embryonic axis. This assay has also provided initial evidence that other embryonic tissues with polarizing properties in vivo release retinoids in vitro.

Animals↗

PubMed1992

Commentary: the public health consequences of restricted induced abortion--lessons from Romania.

The question of whether abortion should be legal is currently being decided in many countries. Although much of the discussion has focused on ethical issues, the public health consequences should not be overlooked and should be addressed realistically and responsibly. Nowhere are the public health manifestations of restricted abortion more apparent than in Romania. The pronatalist policies of the Ceaucescu regime resulted in the highest maternal mortality rate in Europe (approximately 150 maternal deaths per 100,000 live births) and in thousands of unwanted children in institutions.

Abortion, Illegal↗