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Biomedical subjects

G Pfleiderer

Publications and source records attributed to G Pfleiderer.

At least 19 recordsLinked to original sources

[The anesthesia of anesthesia].

Viewed from a cultural-ethical perspective, anesthesiology can be understood as a comprehensive concept of medicine in general. As such it contains two dilemmas: very often pain must be inflicted in order to alleviate pain and this can only be done by somebody who is himself relatively free of pain. The necessary apathy or anesthesia of the anesthetist is correlated with a general twentieth century-type of perception: the cool observer. Nevertheless, it is also a modern variation of the original religious constellation of the priest in relationship to the sick person. Curing occurs by representation. The weak self of the sick person is able to take over the strong self, represented by the therapist. In twentieth century art and literature this process of self-therapy by representation was often illustrated. On the background of a phenomenological philosophy that process can be understood as the regaining of a balance between body and soul. In the psalms of the biblical Book of Job there a variety of fundamental forms of pain which may be helpful even in this secular age.

Anesthesia↗

Limited proteolysis of lactate dehydrogenase from porcine heart with trypsin: characterization and reactivation of the fragments.

The ternary complex formed by native lactate dehydrogenase (LDH) from porcine heart, NAD+ and sulfite, was digested with trypsin over a period of 12-16 h3. After removal of the ligands and residual native lactate dehydrogenase by ion exchange chromatography dimers were obtained which were almost inactive. The dimers were lacking a hexapeptide at the N-terminus; however, the secondary structure was the same as that of native lactate dehydrogenase. The circular dichroism spectra showed a dependence on temperature which suggested an equilibrium of two different structural states. The reaction of antibodies against native porcine heart LDH with the dimers restored the catalytic activity, and subsequently the dimers behaved similarly to the native enzyme. Addition of 1 M phosphate or NAD-sulfite to the dimers restored 80-90% of the catalytic activity. It could be demonstrated that the behavior of the reactivated dimers, in contrast to that of the inactive dimers, was similar to the behavior of native lactate dehydrogenase. For instance, ultracentrifugal analysis showed that dimers reactivated with NAD-SO3- were associated to give tetramers. The reaction of antibodies against native LDH with the dimers reactivated with NAD-SO3- demonstrated that the native LDH and the dimers have the same surface determinants.

Amino Acid Sequence↗

Glucose dehydrogenase from Bacillus subtilis expressed in Escherichia coli. I: Purification, characterization and comparison with glucose dehydrogenase from Bacillus megaterium.

Escherichia coli containing the Bacillus subtilis glucose dehydrogenase gene on a plasmid (prL7) was used to produce the enzyme in high quantities. Gluc-DH-S was purified from the cell extract by (NH4)2SO4-precipitation, ion-exchange chromatography and Triazine-dye chromatography to a specific activity of 375 U/mg. The enzyme was apparently homogenous on SDS-PAGE with a subunit molecular mass of 31.5 kDa. Investigation of Gluc-DH-S was performed for comparison with the corresponding properties of Gluc-DH-M. The limiting Michaelis constant at pH 8.0 for NAD+ is Ka = 0.11 mM and for D-glucose Kb = 8.7 mM. The dissociation constant for NAD+ is Kia = 17.1 mM. Similar to Gluc-DH-M, Gluc-DH-S is inactivated by dissociation under weak alkaline conditions at pH 9.0. Complete reactivation is attained by readjustment to pH 6.5. Ultraviolet absorption, fluorescence and CD-spectra of native Gluc-DH-S, as well as fluorescence- and CD-backbone-spectra of the dissociated enzyme were nearly identical to the corresponding spectra of Gluc-DH-M. The aromatic CD-spectrum of dissociated Gluc-DH-S was different, representing a residual ellipticity of tryptophyl moieties in the 290-310 nm region. Density gradient centrifugation proved that this behaviour is due to the formation of inactive dimers in equilibrium with monomers after dissociation. In comparison to Gluc-DH-M, the kinetics of inactivation as well as the time-dependent change of fluorescence intensity at pH 9.0 of Gluc-DH-S showed a higher velocity and a changed course of the dissociation process.

Bacillus megaterium↗

The differentiation of normal and transformed human fibroblasts in vitro is influenced by electromagnetic fields.

We studied the effect of symmetric, biphasic sinusoidal electromagnetic fields (EMF) (20 Hz, 6 mT) on the differentiation of normal human skin fibroblasts (HH-8), normal human lung fibroblasts (WI38), and SV40-transformed human lung fibroblasts (WI38SV40) in in vitro cultures. Cells were exposed up to 21 days for 2 x 6 h per day to EMF. Normal mitotic human skin and lung fibroblasts could be induced to differentiate into postmitotic cells upon exposure to EMF. Concomitantly, the synthesis of total collagen as well as total cellular protein increased significantly by a factor of 5-13 in EMF-induced postmitotic cells. As analyzed by two-dimensional gel electrophoresis of [35S]methionine-labeled polypeptides, EMF-induced postmitotic cells express the same differentiation-dependent and cell type-specific marker proteins as their spontaneously arising counterparts. In SV40-transformed human lung fibroblasts (cell line WI38SV40) the exposure to EMF induced the differentiation of mitotic WI38SV40 cells into postmitotic and degenerating cells in subpopulations of WI38SV40 cell cultures. Other subpopulations of WI38SV40 cells did not show any effect of EMF on cell proliferation and differentiation. These results indicate that long-term EMF exposure of fibroblasts in vitro induces the differentiation of mitotic to postmitotic cells that are characterized by differentiation-specific proteins and differentiation-dependent enhanced metabolic activities.

Cell Differentiation↗

Acidic and basic forms of glutathione S-transferases from human placenta and comparison with human kidney glutathione S-transferase.

Glutathione S-transferase (GSH-transferase) was purified from human placenta and kidney by affinity chromatography on S-glutathione-carbamidomethyl-epsilon-aminolysyl-Sepharose CL 4B and gel filtration chromatography on Sephades G-75. Electrophoretically pure enzyme with the specific activities of 50.7 and 55.9 U/mg, respectively, were obtained. In addition to the known acidic isoenzyme from human placenta (isoelectric point, pI, 4.5), we describe here for the first time the presence of 6 basic forms with pI values between 8.0 and 9.0. The kidney GSH-transferase contained 2 acidic forms with isoelectric points at 4.6 and 4.65, and 6 basic forms with pI values between 8.7 and 9.4. The basic and acidic isoenzymes from placenta were separated by ion exchange chromatography on Sephadex DEAE A-25. The acidic form accounted for 36% of the total GSH-transferase activity from placenta. Antibodies against the kidney enzyme were raised in rabbit. Total cross-reactivity of placental GSH-transferase with antikidney-GSH-transferase antibodies was obtained, suggesting that the kidney and placental enzymes are immunologically closely related.

Adult↗

An integrated prediction of secondary, tertiary and quaternary structure of glucose dehydrogenase.

Based on homology of partial sequences, on physico-chemical evidence and on studies using chemical modification, we came to the tentative conclusion that tetrameric glucose dehydrogenases from Bacillus megaterium and B. subtilis should have a structure closely related to that of lactate dehydrogenase. The overall homology of primary structures was found to be very low, however, and independent predictions of secondary structure produced a clearly different pattern of beta-strands and alpha-helices. We nevertheless tried a manual prediction based on the hydrophobic nature of internal beta-sheet and on the amphiphilic character of external helices. This treatment led to the identification of analogues of all the beta-strands present in lactate dehydrogenase with the exception of beta C. Six amphiphilic helices were identified corresponding to alpha B, alpha C, alpha D, alpha 1F, alpha 2F and alpha 3G in lactate dehydrogenase. Conserved functional residues were found at analogous positions. The Q and R intersubunit contacts could be identified and partial proteolysis was found to occur on the outer surface of the tetramer. The structure was found to explain the better binding of NADP as compared to NAD+ and offered a rationalization of the role of the essential lysine at position 201.

Amino Acid Sequence↗

The amount of Tamm-Horsfall-protein in the human kidney, related to its daily excretion.

1. The amount of Tamm-Horsfall-protein in the human kidney was determined using zonal-immunoelectrophoresis. 2. A value of 115 micrograms Tamm-Horsfall-protein per gram kidney-tissue was found (1.3 micrograms Tamm-Horsfall-protein per mg protein), representing a total amount of about 40 mg Tamm-Horsfall-protein in both kidneys. 3. As the well documented daily excretion of Tamm-Horsfall-protein is 40 mg, i.e. in the range of its content in the kidneys, it is inferred that the total amount of Tamm-Horsfall-protein must be synthesized de novo each day.

Humans↗

[Isoenzymes].

Isoenzymes, especially non-allelic forms, are enzyme variants with different primary structure, apparently adapted during phylogeny to distinct physiological conditions. They are ideal research objects either for studies of structure-function relationship and morphogenesis in vertebrates or as diagnostic indicators for pathological processes.

Animals↗

Colour development of immunogold-labelled antibodies for light microscopy.

We describe an improvement of the immunogold technique, which is based on the colour development of silver-intensified immunogold signals. This method (referred to as the coloured-SIG technique) was found to be as sensitive as the silver-intensified immunogold method and more sensitive (in two of the three tested systems) than immunoenzymatic procedures, such as the peroxidase/antiperoxidase technique or the avidin-biotin system. The coloured SIG-method results in either a magenta-red or a cyan-blue final reaction product. Therefore, this new improvement of the immunogold technique should be useful in double-staining methods, immunoblot methods and conventional immunostaining methods.

Antibodies↗

[Alkaline phosphatase in human lymphocytes. I. Cytochemical detection of alkaline phosphatase in normal human peripheral blood lymphocytes].

The present paper deals with a sensitive cytochemical method of identifying alkaline phosphatase (AP) in rosette-forming lymphocytes gained from the peripheral blood of healthy human beings. The percentage of AP-positive lymphocytes amounts to 5%, with all cells comprising B- and O-lymphocyte population and with T-lymphocytes being negative. In a group of healthy test persons, recently, however, having undergone various inflammatory processes or virus diseases, the number of AP-positive lymphocytes is significantly higher, from 41-73% in B- and O-lymphocytes and from 6-38% in T-lymphocytes. This observation indicates that AP in lymphocytes may have a clinical significance in reactive lymphoproliferative processes, which must be elucidated by further investigations.

Adult↗

[Alkaline phosphatase in human lymphocytes. II. A method for ultracytochemical detection of alkaline phosphatase in lymphocytes].

For the ultracytochemical identification of alkaline phosphatase in lymphocytes gained from the peripheral blood of healthy individuals a sensitive method is described which allows the low enzyme activity of these cells to be determined. This was possible because the authors succeeded in stabilizing lead ions in the alkaline medium by forming a complex directly between tris-(hydroxymethyl) aminomethan and lead (II) citrate. AP localized ultrachemically in lymphocytes in particular formations similar to phosphasomes of neutrophilic granulocytes. In those lymphocytes stimulated by lipopolysaccharides a high enzyme activity could be observed and, in addition to phosphasomes, the product of response can also be found in canal-like structures of the endoplasmatic reticulum. These findings contribute to clarify the ultrastructural localization of alkaline phosphatase in lymphocytes and may be regarded as an aid in discovering the importance of the enzyme in the biology of lymphocytes or in its activation, respectively.

Alkaline Phosphatase↗

Complete amino acid sequence of glucose dehydrogenase from Bacillus megaterium.

The amino acid sequence of glucose dehydrogenase from Bacillus megaterium has been determined. The enzyme consists of 4 identical subunits, each containing 262 amino acid residues. Its structure was established using manual Edman degradation procedures after modification of the enzyme in the native form with reagents specific to the amino acids histidine, tyrosine, tryptophan and lysine in order to identify residues involved in catalysis or located in the subunit binding area.

Amino Acid Sequence↗

Alkaline phosphatase isoenzymes in human kidney and urine, immunohistochemical, immunological and biochemical characterization.

Human kidney contains two antigenetically distinct isoenzymes of alkaline phosphatase (AP): a liver type and an intestinal type. The intestinal type AP is a minor component (1%-4%) of the total AP activity: it is found only in the cytoplasm. Both isoenzymes are located, found by an immunohistochemical technique, in the proximal convoluted tubules. This histochemical result eliminates the possibility that the low intestinal AP content in the kidney might only originate from blood vessels, where the intestinal isoenzyme was also found. The renal isoenzymes contribute to urinary AP. Intestinal type AP in urine of healthy persons, 10%-40% of the total AP activity, was found after high speed centrifugation predominantly in the supernatant (100,000 g), the liver type mainly in the sediment. Biochemical characterization revealed that intestinal type AP in kidney and urine are identical and differ from the isoenzyme of intestinal mucosa only slightly in their electrophoretic mobility.

Alkaline Phosphatase↗

Synthesis and properties of 5-membered heterocyclic disulfides: application for enzyme modification.

Synthesis of the disulfides 2,2'-dithiodithiazole, 2,2'-dithiobis(4-methylthiazole), and 5,5'-dithiobis(3-methylisothiazole), which are thiol-specific reagents, is described and the uv-absorption spectra of the disulfides and corresponding thiols and/or thiones are presented. The utility of these and several other reagents for the modification of thiol groups in enzymes has been demonstrated with lactate dehydrogenase and malate dehydrogenase and compared with the frequently applied modification reagents 5,5'-dithiobis(2-nitrobenzoic acid) and 2,2'-dithiodipyridine. The 5-membered heterocyclic disulfides permit the spectrophotometric determination of all sulfhydryl groups of malate dehydrogenase and lactate dehydrogenase without requiring their prior denaturation. Application of these reagents makes possible the determination of SH groups in the pH range less than or equal to 7.

Animals↗

An ultracentrifuge study on the self-association of glucose dehydrogenase from Bacillus megaterium.

The self-association of glucose dehydrogenase (beta-D-glucose:NAD(P) 1-oxidoreductase, EC 1.1.1.47) from Bacillus megaterium was studied by analytical ultracentrifugation. The pH and composition of the buffer used were such that, owing to a reversible partial dissociation of the tetrameric enzyme, enzyme activity was reduced. It was found that under these conditions the protein exists in a monomer/dimer/tetramer association equilibrium.

Bacillus megaterium↗

Placental alkaline phosphatase in tumour tissue and serum.

Using immunochemical techniques, alkaline phosphatase isoenzymes were determined in tissue samples of breast carcinomas and carcinomas of the gastrointestinal tract. In breast carcinomas only 19% of the patients expressed significant placental alkaline phosphatase activity, compared with 78% in gastrointestinal tumours. The intestinal isoenzyme was found in 50% of the breast carcinomas and in nearly all of the other examined tissues. The two isoenzymes usually represent 1% of the total alkaline phosphatase activity, but in a few cases they may constitute between 10 and 90%. In the serum of the patients under examination, elevated total alkaline phosphatase activity was found in only 7%, and elevated placental alkaline phosphatase in 6% of the cases. No cases of elevated serum intestinal alkaline phosphatase were found. We therefore consider that serum placental alkaline phosphatase is a poor tumour marker for a general screening.

Alkaline Phosphatase↗