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Biomedical subjects

K Hofmann

Publications and source records attributed to K Hofmann.

At least 19 recordsLinked to original sources
PubMed1992

Structure, expression, and functional analysis of a Na(+)-dependent glutamate/aspartate transporter from rat brain.

Transport systems specific for L-glutamate and L-aspartate play an important role in the termination of neurotransmitter signals at excitatory synapses. We describe here the structure and function of a 66-kDa glycoprotein that was purified from rat brain and identified as an L-glutamate/L-aspartate transporter (GLAST). A GLAST-specific cDNA clone was isolated from a rat brain cDNA library. The cDNA insert encodes a polypeptide with 543 amino acid residues (59,697 Da). The amino acid sequence of GLAST suggests a distinctive structure and membrane topology, with some conserved motifs also present in prokaryotic glutamate transporters. The transporter function has been verified by amino acid uptake studies in the Xenopus laevis oocyte system. GLAST is specific for L-glutamate and L-aspartate, shows strict dependence on Na+ ions, and is inhibited by DL-threo-3-hydroxy-aspartate. In situ hybridization reveals a strikingly high density of GLAST mRNA in the Purkinje cell layer of cerebellum, presumably in the Bergmann glia cells, and a less dense distribution throughout the cerebrum. These data suggest that GLAST may be involved in the regulation of neurotransmitter concentration in central nervous system.

Amino Acid Sequence↗
PubMed1992

An expression vector system providing plasmid stability and conditional suicide of plasmid-containing cells.

A cloning vector system was constructed on the basis of the pBR322 derivative pEG1 by introducing the whole parB locus of plasmid R1 cloned behind the promoter of the alkaline phosphatase gene (phoA) of Escherichia coli. The parB locus in combination with the phoA promoter ensures both (i) plasmid stabilization due to the post-segregational killing of plasmid-free cells during growth and (ii) killing of the cells induced by the potential environmental signal phosphate limitation. This vector, therefore, appears to be a model system for increasing the stability of recombinant plasmids and for decreasing the potential risks in the application of recombinant bacteria in industrial fermentations.

Alkaline Phosphatase↗
PubMed1992

Maximizing the expression of a recombinant gene in Escherichia coli by manipulation of induction time using lactose as inducer.

The use of isopropyl-beta-D-thiogalactoside (IPTG) for induction of the lac-promoter in small-scale cultivations is well established. However, for large-scale microbiological processes the cost of this inducer is a severe limitation. Here is described a method by which lactose is used as inducer of the lac promoter with the same efficiency as that of IPTG. It was found that after growth on glucose the time of the addition of lactose is important for the quality of induction. The resulting yield of the recombinant protein increased when lactose was added to the culture if the glucose concentration was rather low. By careful monitoring of the glucose level in the fermentation, using a biosensor, it was possible to add the inducer when the carbon source was nearly depleted. Using Escherichia coli BL21 (pET3), in which was cloned the main antigen coat protein of the foot and mouth disease virus, induction of the gene led to expression of the target protein at a level exceeding 20% of the total cell protein.

Aphthovirus↗
PubMed1992

PROFILEGRAPH: an interactive graphical tool for protein sequence analysis.

The computer program PROFILEGRAPH, a graphical interactive tool for the analysis of amino acid sequences, is described. The main task of the program is to integrate a variety of sliding-window methods into a single user-friendly shell. The program allows the user to combine any amino acid specific parameter with a selection of several possible types of analysis and to plot the resulting graph in one of several windows on the screen. It is also possible to calculate the moment of the amino acid specific parameter for a given secondary structure and to display both the absolute moment value and the moment angle relative to a reference residue. Also included are several utilities that facilitate visual analysis of protein primary structures like, for example, helical-wheel diagrams. It is possible to adapt the majority of published sliding-window analysis procedures for use with PROFILEGRAPH.

Algorithms↗
PubMed1992

Mouse apolipoprotein AI. cDNA-derived primary structure, gene organisation and complete nucleotide sequence.

Apolipoprotein AI, the dominant protein component of serum high density lipoprotein, is intimately involved in cholesterol homeostasis. Apo AI activates the lecithin-cholesterol acyltransferase within the HDL particle and functions as ligand for a putative HDL receptor--two properties, which render this apolipoprotein a key mediator in reversed cholesterol transport. A functional analysis of the apo AI gene demands the isolation of the mouse apo AI gene for expression as transgenes in different mutant forms in the mouse. Here we describe the isolation of a full length apo AI-specific mouse liver cDNA clone with the human cDNA (892 bp) and the derived amino-acid sequence coding a polypeptide of 264 amino-acid residues. It showed a 70.7% homology to the rat and 66% to the human apo AI sequence. With this cDNA as probe the mouse apo AI gene was isolated and its organization analysed. Four exons, three of which are coding sequences, are aligned similarly to the human gene. The gene embraces 1825 bp between the transcription start, and the poly(A)+ tail attached 62 bp downstream of the stop codon. The complete nucleotide sequence of the four exons and three introns of the mouse apo AI gene was determined and its homology compared with that of the rat and human gene. Extensive deletions and a strongly reduced homology of the three introns of the two genes are obvious.

Amino Acid Sequence↗
PubMed1992

Effects of laparotomy on systemic macrophage function.

Surgical trauma induces immunosuppression that may adversely influence survival. This study examined the effect of laparotomy on two different macrophage populations, peritoneal macrophages (PM phi) and Kupffer cells. Female, 6- to 8-week old, CFW/C3H-HeN mice (n = 160) were randomly allocated to one of three study groups: control, ether anesthetic only, or ether anesthetic and laparotomy. On postoperative days 1 and 3, PM phis and Kupffer cells were harvested and assayed for superoxide anion production (O2-), percent macrophage phagocytosis of Candida albicans (CAP), percent C. albicans killed by macrophages (CAK), percent major histocompatibility complex (MHC)-class II antigen expression, and antigen presentation. Macrophages isolated on postoperative day 1 were also cocultured with 100 units/10(6) cells/ml interferon-gamma (IFN-gamma). Laparotomy significantly impaired microbicidal activity (O2-, percent CAP, and percent CAK) and antigen presentation on postoperative day 1. On postoperative day 3, O2- and antigen presentation were increased significantly (p less than 0.05) over control values, indicating a rebound phenomenon. Kupffer cell microbicidal function was unchanged on postoperative days 1 and 3. The initial immune impairment (PM phis: O2-, CAP, and CAK) was abrogated by IFN-gamma treatment. In immunosuppressed hosts after injury, administration of macrophage-activating factors such as IFN-gamma could be of therapeutic benefit.

Animals↗
PubMed1992

[Ultrasonic diagnostics of the external nose].

Twenty megahertz ultrasound B-scanners achieve an axial resolution of 80 microns. The range of 7 mm in soft tissue suffices for imaging of dermal and cartilaginous structures with anatomical precision, and the form, position and diameter of the cartilaginous structures of the nose can be assessed. Distinct echoes within the cartilage indicate inflammation or trauma. The position, scarring and rate of resorption of implants can be observed non-invasively.

Adult↗
PubMed1991

Method for long-term preservation of thin-layer polyacrylamide gels by producing a gelatine coating.

Thin-layer polyacrylamide gels can be preserved and stored for unlimited periods by covering them with a gelatine coating. The method is inexpensive and simple. After air-drying, the gel is immersed in an aqueous 10% solution of highly viscous gelatine between 55 and 60 degrees C. The coated gel is dried by hanging it in air. The method was checked successfully with gels of different thicknesses (0.15-0.50 mm) and after using different staining methods, e.g., with silver, Coomassie Brilliant Blue and pseudoperoxidase.

Acrylic Resins↗
PubMed1991

Impaired macrophage function in severe protein-energy malnutrition.

Protein-energy malnutrition induces immunosuppression that predisposes to sepsis, but the mechanisms are unclear. This study examines the role of the macrophage in host defense in the malnourished state. Swiss-Webster mice (N = 300) were randomly allocated to control (24% casein) or low-protein (2.5% casein) diets for 8 weeks. We studied the ability of two populations of macrophages, peritoneal macrophages, and Kupffer cells to produce superoxide anion after in vivo administration of endotoxin or mycobacterium (bacille Calmette-Guérin). Phorbol diester and Candida albicans were used as stimuli. In another group of mice, we evaluated the ability of interferon gamma to up-regulate superoxide anion release and Candida phagocytosis and killing. Mice under protein-energy malnutrition demonstrated decreased mean body weights, serum protein levels, and cell yields. Superoxide anion production in resident and activated (lipopolysaccharide, interferon gamma, bacille Calmette-Guérin infection) peritoneal macrophages was significantly reduced in the malnourished group. Candida phagocytosis and killing were also both depressed in malnourished mice. Kupffer cells failed to generate superoxide anion in all groups. We conclude that severe protein-energy malnutrition significantly impairs macrophage function, which could diminish response to acute and chronic septic challenges. Interferon gamma up regulated peritoneal macrophage and Kupffer cell microbicidal function, which suggests a therapeutic role for this lymphokine in the malnourished septic host.

Animals↗
PubMed1990

In vivo 13C nuclear magnetic resonance investigations of choline metabolism in rabbit brain.

The metabolism of choline in rabbit brain was studied by the noninvasive approach of in vivo 13C NMR spectroscopy. 13C-Enriched precursors were introduced into the brain. Surgery of the head skin was avoided through controlled localization of the surface coil. For long-term accumulation studies in brain, repeated subcutaneous injections proved to be advantageous over other forms of application. The resorption kinetics was calculated to be zero order which suggests slow delivery from the subcutaneous depots. Choline metabolism was studied by two approaches: [N-13CH3]choline and S-[13CH3]methionine were administered separately to adult and myelinating rabbits (Days 5 to 32), respectively, over 4 weeks. [N-13CH3]Choline and the 13CH3 group of methionine were incorporated into lecithin and sphingomyelin of brain myelin. In vivo kinetic studies of the turnover of these labeled structures were carried out. Choline and methionine are readily transported through the blood-brain barrier and utilized by the myelinating brain for the biosynthesis of its phospholipids.

Animals↗
PubMed1990

[Effect of heat on the solubility and the electrophoretic behavior of myoglobin].

The heat stability of beef myoglobin was investigated by means of its solubility and intensity of protein bands separated by isoelectric focusing. A method for the sensitive determination of myoglobin in the extracts was developed based on its ability to form a red dye by the oxidative coupling of 2,4-dichlorophenol and 4-aminoantipyrine in the presence of hydrogen peroxide. By this method, myoglobin could be determined to a concentration of 0.01 mg/ml. For the detection of electro-focused myoglobins, o-dianisidine has proved to be the most sensitive compound. The myoglobin content of the extracts, according to the intensity of the myoglobin bands in the electrophoretic gel, decreased with increasing temperature and heating-time. However, the position of the bands was not changed by the heat treatment.

Animals↗
PubMed1990

Labile disulfide bonds in human placental insulin receptor.

The disulfide crosslinking pattern of human placental insulin receptor was investigated using selective reduction with tributylphosphine followed by alkylation with N-[3H]ethylmaleimide. Insulin receptor contains a single sulfhydryl group in each beta subunit whose alkylation with N-[3H]ethylmaleimide inhibits receptor autophosphorylation. Alkylation is partially inhibited by ATP or the nonhydrolyzable substrate analog adenosine 5'-[beta,gamma-imido]triphosphate when the nucleotides are added as Mn2+ complexes. Neither insulin nor 6 M guanidinium chloride renders additional sulfhydryl groups accessible to alkylation. When the receptor is reduced under drastic conditions with tributylphosphine in guanidinium chloride, 32 of the 37 sulfhydryl groups in the receptor's alpha subunit can be alkylated with N-[3H]ethylmaleimide. Surprisingly only three of the 10 cysteines in the beta subunit become titratable under identical conditions. By using highly selective reducing conditions, we were able to determine quantitatively the maximum number of disulfide bridges that link the two alpha beta halves to form the tetrameric structure and those that couple the alpha to the beta subunits. Liberation of two sulfhydryl groups in the alpha and one in the beta subunit resulted in formation of alpha beta dimers. Free beta subunit was formed when an additional disulfide bond was reduced. It is remarkable that the tetrameric structure of this highly complex receptor molecule, which contains a large number of cysteine residues, is maintained by such a small number of disulfide bonds. Three models of the arrangement of the labile disulfide bonds, consistent with these findings, are proposed.

Adenosine Triphosphate↗
PubMed1989

Hepatocytes produce nitrogen oxides from L-arginine in response to inflammatory products of Kupffer cells.

A metabolic pathway by which L-arginine (L-arg) is converted to the biologically active compound NO. has recently been described in macrophages (M phi) and endothelial cells. This report demonstrates that transferable products from activated Kupffer cells (KC) induce the conversion of large quantities of L-arg to nitrogen oxides within hepatocytes (HC). In M phi and endothelial cells, citrulline and NO2-/NO3- are the stable endproducts of this metabolic pathway. In contrast, HC L-arg metabolism resulted in significantly greater production of NO2-/NO3- than citrulline. The generation of NO. within HC was associated with a concurrent decrease in total protein synthesis.

Animals↗
PubMed1989

Kupffer cell cytotoxicity to hepatocytes in coculture requires L-arginine.

Activated macrophages convert L-arginine to citrulline and unstable nitrogen oxides that have cytotoxic properties. We recently have shown that the inhibition of protein synthesis in Kupffer cell (KC):hepatocyte (HC) coculture, following exposure to gram-negative bacterial endotoxin (lipopolysaccharide), is due to the metabolism of L-arginine by this cytotoxic pathway. Although this finding supports a role for activated KCs and the L-arginine-dependent mechanism in the HC dysfunction seen in sepsis, it and previous studies have failed to demonstrate direct damage to HCs by adjacent KCs. The current study was undertaken to determine if KCs exposed to lipopolysaccharide could directly damage HCs and, if so, whether the damage was dependent on the metabolism of L-arginine. By using the release of aspartate aminotransferase as a marker of HC damage, it was found that a significant aspartate aminotransferase release by KC:HC cocultures in response to lipopolysaccharide occurred only if L-arginine was present. In addition, requirements for significant aspartate aminotransferase release included KC:HC ratios of 7.5:1 or greater and L-arginine concentrations of 1 mmol or more. Although the KC-induced damage was mild, these results show that in vitro HC damage in KC:HC coculture does require the metabolism of L-arginine and supports a hypothesis that toxic L-arginine metabolites may contribute to liver cell damage in patients with sepsis.

Animals↗
PubMed1989

Cyclic AMP, fructose-2,6-bisphosphate and catabolite inactivation of enzymes in the hydrocarbon-assimilating yeast Candida maltosa.

The inactivation of fructose-1,6-bisphosphatase, isocitrate lyase and cytoplasmic malate dehydrogenase in Candida maltosa was found to occur after the addition of glucose to starved cells. The concentration of cyclic AMP and fructose-2,6-bisphosphate increased drastically within 30 s when glucose was added to the intact cells of this yeast. From these results it was concluded that catabolite inactivation, with participation of cyclic AMP and fructose-2,6-bisphosphate, is an important control mechanism of the gluconeogenetic sequence in the n-alkane-assimilating yeast Candida maltosa, as described for Saccharomyces cerevisiae.

Adolescent↗