Biomedical subjects

B Hamprecht

Publications and source records attributed to B Hamprecht.

At least 19 recordsLinked to original sources

PubMed2005

End-to-end distribution function of stiff polymers for all persistence lengths.

We set up recursion relations for calculating all even moments of the end-to-end distance of Porod-Kratky wormlike chains in D dimensions. From these moments we derive a simple analytic expression for the end-to-end distribution in three dimensions valid for all peristence lengths. It is in excellent agreement with Monte Carlo data for stiff chains and approaches the Gaussian random-walk distributions for low stiffness.

Journal Article↗

PubMed2001

AMP deaminase in rat brain: localization in neurons and ependymal cells.

The purine nucleotide cycle enzyme AMP deaminase (AMPD) catalyzes the irreversible hydrolytic deamination of AMP. The physiological function of the purine nucleotide cycle in the brain is unknown. In situ hybridization and immunocytochemical studies were performed to identify the regional and cellular expression of AMPD in rat brain with the goal of elucidating the neural function of the purine nucleotide cycle. AMPD messenger RNA was detected in ventricular ependymal cells and cells of the choroid plexus and in neurons of distinct brain areas. Although only low antibody titers were obtained by immunization with the purified sheep brain AMPD, immunization of mice with synthetic lipopeptide vaccines containing oligopeptides derived from a known partial complementary DNA sequence of the enzyme yielded an antiserum suitable for immunocytochemistry. Immunostaining of cells in culture showed that neurons but not astroglial cells express appreciable amounts of the enzyme. Results of immunocytochemical staining performed on rat brain slices were in accord with the localization of AMPD messenger RNA, thus confirming the expression of AMPD in neurons of the brain stem, hippocampus, cerebellar nuclei and mesencephalic nuclei, as well as in ventricular ependymal cells and their cilia.

AMP Deaminase↗

PubMed2001

Primary cultures as a model for studying ependymal functions: glycogen metabolism in ependymal cells.

Ependymal cells form a single-layered, ciliated epithelium at the interface between the cerebrospinal fluid and the brain parenchyma. Although their morphology has been studied in detail, ependymal functions remain largely speculative. We have established and characterized a previously described cell culture model to investigate ependymal glycogen metabolism. During growth in minimal medium lacking many non-essential amino acids including L-glutamate, but containing glucose at physiological concentration, the cells contained negligible amounts of glycogen (7+/-3 nmol glucosyl residues/mg protein) despite the presence of insulin. However, during a period of 24 h, the cells accumulated glycogen to very high levels after transferal to a medium containing insulin, glucose at a 5-fold higher concentration, and all proteinogenic amino acids except L-asparagine and L-serine (990+/-112 nmol glucosyl residues/mg protein). Omission of insulin resulted in a 50% reduction in glycogen accumulation. Upon glucose deprivation, glycogen was degraded with a half-life of 21 min. The ependymal primary cultures contained 80+/-5 mU glycogen phosphorylase (Pho)/mg protein and stained positively with antibodies raised against this enzyme. Astroglial cultures built up less glycogen and had less Pho activity under identical conditions. Ependymal glycogen was mobilized by noradrenaline and serotonin. Our results indicate that ependymal cells maintain glycogen as a functional energy store, subject to rapid turnover dependent on the availability of energy substrates and the presence of appropriate signal molecules. Thus ependymocytes appear to be active players in the multitude of processes resulting in normal brain function, and ependymal primary cultures are suggested as a suitable model for studying the role of ependymal cells in these processes.

Animals↗

PubMed2001

Catalase in astroglia-rich primary cultures from rat brain: immunocytochemical localization and inactivation during the disposal of hydrogen peroxide.

The expression of catalase in cells of astroglia-rich primary cultures derived from the brains of newborn rats was investigated by double-labelling immunocytochemical staining. Strong catalase immunoreactivity was found in cells positive for glial fibrillary acidic protein and galactocerebroside, cellular markers for astroglial and oligodendroglial cells, respectively. The cells of these cultures dispose of exogenously applied hydrogen peroxide (initial concentration 200 microM) quickly with first order kinetics. In contrast, after inhibition of glutathione peroxidases by mercaptosuccinate the rate of the catalase-dependent disposal of H(2)O(2) declined with time and after about 10 min the extracellular concentration of H(2)O(2) remained almost constant at a concentration of about 100 microM. Catalase activity after 10 min of incubation under these conditions was no longer detectable. In contrast, in the absence of mercaptosuccinate catalase activity was maintained during H(2)O(2) disposal. These results demonstrate that in astroglia-rich cultures catalase is strongly expressed in the predominant astroglial cells and in the minor population of oligodendroglial cells and that the enzyme is rapidly inactivated during the disposal of H(2)O(2), if the glutathione system of the cells is compromised.

Animals↗

PubMed2001

Distribution of key enzymes of branched-chain amino acid metabolism in glial and neuronal cells in culture.

Transamination of branched-chain amino acids (BCAAs) catalyzed by the branched chain aminotransferase isoenzymes (BCATs) is believed to play an important role in nitrogen shuttling and excitatory neurotransmitter glutamate metabolism in brain. Recently, we have shown that the mitochondrial isoenzyme (BCATm) is the predominant form found in cultured astrocytes. In this study we used immunocytochemistry to examine the distribution of BCAT isoenzymes in cultured rat neurons and microglial cells. The cytoplasm of neurons displayed intense staining for the cytosolic isoenzyme (BCATc), whereas BCATm staining was not detectable in neurons. In contrast, microglial cells expressed BCATm in high concentration. BCATc appeared to be absent in this cell type. The second and committed step in the BCAA catabolic pathway is oxidative decarboxylation of the alpha-keto acid products of BCAT catalyzed by the branched-chain alpha-keto acid dehydrogenase (BCKD) enzyme complex. Because the presence of BCKD should provide an index of the ability of a cell to oxidize BCAA, we have also immunocytochemically localized BCKD in neuron and glial cell cultures from rat brain. Our results suggest ubiquitous expression of this BCKD enzyme complex in cultured brain cells. BCKD immunoreactivity was detected in neurons and in astroglial and microglial cells. Therefore, the expression of BCAT isoenzymes shows cell-specific localization, which is consistent with the operation of an intercellular nitrogen shuttle between neurons and astroglia. On the other hand, the ubiquitous expression of BCKD suggests that BCAA oxidation can probably take place in all types of brain cells and is most likely regulated by the activity state of BCKD rather than by its cell-specific localization.

Amino Acids, Branched-Chain↗

PubMed2000

Glycogen is mobilized during the disposal of peroxides by cultured astroglial cells from rat brain.

Regeneration of reduced nicotinamide adenine dinucleotide phosphate (NADPH) is essential for the activity of glutathione redox cycling during cellular peroxide detoxification. In order to test for a function of astroglial glycogen to serve as endogenous precursor for glucose-6-phosphate, the substrate for the regeneration of NADPH by the pentose phosphate pathway, the content of glycogen in astroglia-rich primary cultures derived from the brains of newborn rats was determined after application of peroxides. In the presence of hydrogen peroxide or cumene hydroperoxide in concentrations of 200 microM glycogen was mobilized with a half-life of 16 min in incubation medium containing 20 mM glucose, whereas in the absence of peroxides the glycogen content decreased more slowly with a half-life of 42 min. After 30 min of incubation with or without peroxides 30 and 73%, respectively, of the initial glycogen content was found. The degree of glycogen mobilization was reduced by lowering the initial concentration of the peroxides. These results demonstrate that in astroglial cells (i) glucosyl residues of glycogen are mobilized after application of peroxides despite the presence of exogenous glucose, and (ii) that the demand for glucose-6-phosphate as substrate for NADPH regeneration via the pentose phosphate pathway can, at least partially, be met by mobilization of glycogen.

Animals↗

PubMed2000

Isozyme pattern of glycogen phosphorylase in the rat nervous system and rat astroglia-rich primary cultures: electrophoretic and polymerase chain reaction studies.

Of the three isozymes of glycogen phosphorylase (GP) known, the brain (B) and muscle (M) isoforms have been reported to occur in brain. We investigated the regional and cellular occurrence of the three isozymes in various parts of the rat nervous system, fetal brain and astroglia-rich primary cultures by means of electrophoresis of native proteins with subsequent activity stain and by reverse transcriptase polymerase chain reaction. In the cortex, cerebellum, olfactory bulb, brainstem, spinal cord and dorsal root ganglia, both mRNA and enzyme protein were found for the B and M isozymes. In addition, the liver (L) isoform mRNA was detected in fetal brain and cultured astrocytes. Our studies indicate that there is no regional difference in distribution pattern between brain regions, spinal cord and dorsal root ganglia. In immature brain and cultured glial cells, the additional presence of the L isozyme is possible. These results support the idea that astrocytes express two or even three GP isozymes simultaneously.

Animals↗

PubMed2000

Immunocytochemical localization of beta-methylcrotonyl-CoA carboxylase in astroglial cells and neurons in culture.

Astroglia-rich primary cultures and brain slices rapidly metabolize branched-chain amino acids (BCAAs), in particular leucine, as energy substrates. To allocate the capacity to degrade leucine oxidatively in neural cells, we have purified beta-methylcrotonyl-CoA carboxylase (beta-MCC) from rat liver as one of the enzymes unique for the irreversible catabolic pathway of leucine. Polyclonal antibodies raised against beta-MCC specifically cross-reacted with both enzyme subunits in liver and brain homogenates. Immunocytochemical examination of astroglia-rich rat primary cultures demonstrated the presence of beta-MCC in astroglial cells, where the enzyme was found to be located in the mitochondria, the same organelle that the mitochondrial isoform of the BCA(A) aminotransferase (BCAT) is located in. This colocalization of the two enzymes supports the hypothesis that mitochondrial BCAT is the isoenzyme that in brain energy metabolism prepares the carbon skeleton of leucine for irreversible degradation in astrocytes. Analysis of neuron-rich primary cultures revealed also that the majority of neurons contained beta-MCC. The presence of beta-MCC in most neurons demonstrates their ability to degrade the alpha-ketoisocaproate that could be provided by neighboring astrocytes or could be generated locally from leucine by the action of the cytosolic isoform of BCAT that is known to occur in neurons.

Animals↗

PubMed2000

Synthesis and release of L-serine by rat astroglia-rich primary cultures.

L-serine is known to have important functions in the mammalian CNS other than being a constituent of proteins. It is the metabolic precursor of the neuroactive substances D-serine and glycine, serves as a building block for phospholipid biosynthesis and is likely to be a neurotrophic factor. Based on the observation that rat astroglia-rich primary cultures release L-serine into their culture medium, the biosynthesis and release of L-serine in these cultures has been investigated. Release of L-serine is due to both biosynthesis from glucose and to proteolysis. Amino groups for L-serine synthesis originate from transamination of amino acids that are either taken up from the extracellular space or produced intracellularly by proteolysis. The enzymes of the "phosphorylated pathway" of serine biosynthesis, i.e., 3-phosphoglycerate dehydrogenase, phosphoserine aminotransferase and phosphoserine phosphatase are present in rat brain as well as in rat astroglia-rich primary cultures and their specific activities have been determined. The presence of these enzymes indicates the operation of the "phosphorylated pathway" of L-serine biosynthesis in brain.

Amino Acids↗

PubMed1999

The regeneration of reduced glutathione in rat forebrain mitochondria identifies metabolic pathways providing the NADPH required.

Metabolic pathways underlying the regeneration of reduced glutathione were investigated in acutely isolated metabolically active mitochondria from rat forebrain. The application of hydrogen peroxide to the organelles was accompanied by a transient increase in glutathione disulfide. The recovery of reduced glutathione was significantly improved in the presence of alternatively succinate, malate, citrate, isocitrate, or beta-hydroxybutyrate. Inhibition of succinate dehydrogenase by malonate abolished the beneficial effect of succinate on the reduction of glutathione disulfide but did not influence the effect of isocitrate. Fluorocitrate, an inhibitor of aconitase, blocked the effect exerted by citrate but did not inhibit the effects of malate or beta-hydroxybutyrate. Uncoupling of the respiratory chain by carbonyl cyanide m-chlorophenylhydrazone prevented the beneficial effect of beta-hydroxybutyrate but did not abolish the improved reduction of mitochondrial glutathione disulfide in the presence of malate and isocitrate. These results suggest that NADP+-dependent isocitrate dehydrogenase as well as malic enzyme and nicotinamide nucleotide transhydrogenase contribute to the regeneration of NADPH required for the reduction of glutathione disulfide in brain mitochondria.

Animals↗

PubMed1999

Synthesis of the antioxidant glutathione in neurons: supply by astrocytes of CysGly as precursor for neuronal glutathione.

Deficiency of the antioxidant glutathione in brain appears to be connected with several diseases characterized by neuronal loss. To study neuronal glutathione metabolism and metabolic interactions between neurons and astrocytes in this respect, neuron-rich primary cultures and transient cocultures of neurons and astroglial cells were used. Coincubation of neurons with astroglial cells resulted within 24 hr of incubation in a neuronal glutathione content twice that of neurons incubated in the absence of astroglial cells. In cultured neurons, the availability of cysteine limited the cellular level of glutathione. During a 4 hr incubation in a minimal medium lacking all amino acids except cysteine, the amount of neuronal glutathione was doubled. Besides cysteine, also the dipeptides CysGly and gammaGluCys were able to serve as glutathione precursors and caused a concentration-dependent increase in glutathione content. Concentrations giving half-maximal effects were 5, 5, and 200 microM for cysteine, CysGly, and gammaGluCys, respectively. In the transient cocultures, the astroglia-mediated increase in neuronal glutathione was suppressed by acivicin, an inhibitor of the astroglial ectoenzyme gamma-glutamyl transpeptidase, which generates CysGly from glutathione. These data suggest the following metabolic interaction in glutathione metabolism of brain cells: the ectoenzyme gamma-glutamyl transpeptidase uses as substrate the glutathione released by astrocytes to generate the dipeptide CysGly that is subsequently used by neurons as precursor for glutathione synthesis.

Animals↗

PubMed1999

N-acetylcysteine, but not methionine or 2-oxothiazolidine-4-carboxylate, serves as cysteine donor for the synthesis of glutathione in cultured neurons derived from embryonal rat brain.

The ability of neurons to metabolize sulfur-containing compounds to cysteine was investigated using as indicator the glutathione content in neuron-rich primary cultures derived from the brains of embryonal rats. The-glutathione content of these cultures was doubled during a 4-h incubation in a minimal medium containing cysteine, glutamine and glycine. In contrast, absence of cysteine or replacement of cysteine by methionine or 2-oxothiazolidine-4-carboxylate failed to increase the glutathione content of cultured neurons. Besides cysteine, N-acetylcysteine (NAC) also caused in the millimolar range, a concentration-dependent increase in the neuronal glutathione content during a 4-h incubation. These data suggest that neurons in culture, contain an acylase activity which allows them to generate from extracellular NAC as precursor intracellular cysteine in concentrations sufficient for glutathione synthesis. In contrast, generation of cysteine from 2-oxothiazolidine-4-carboxylate by the reaction of 5-oxoprolinase or from methionine by the transsulfuration pathway appears not to take place in these cultured neurons.

Acetylcysteine↗

PubMed1999

Rapid uptake and degradation of glycine by astroglial cells in culture: synthesis and release of serine and lactate.

Free glycine is known to have vital functions in the mammalian brain, where it serves mainly as both neurotransmitter and neuromodulator. Despite its importance, little is known about the metabolic pathways of glycine synthesis and degradation in the central nervous system. In this study, the pathway of glycine metabolism in astroglia-rich primary cultures from rat brain was examined. The cells were allowed to degrade glycine in the presence of [U-(14)C]glycine, [U-(13)C]glycine or [(15)N]glycine. The resulting intra- and extracellular metabolites were analyzed both by high-performance liquid chromatography and by (13)C/(15)N nuclear magnetic resonance spectroscopy. Glycine was rapidly consumed in a process obeying first-order kinetics. The initial glycine consumption rate was 0.47 nmol per mg protein. The half-life of glycine radiolabel in the incubation medium was shorter than that of glycine mass. This suggests that glycine is produced from endogenous sources and released simultaneously with glycine uptake and metabolism. As the main metabolites of the glycine carbon skeleton in astroglia-rich primary cultures from rat brain, serine and lactate were released during glycine consumption. The main metabolite containing the glycine amino nitrogen was glutamine. To establish a metabolic pathway from glycine to serine in neural tissue, homogenates of rat brain and of neural primary cultures were assayed for their content of serine hydroxymethyltransferase (SHMT) and glycine cleavage system (GCS). SHMT activity was present in homogenates of rat brain as well as of astroglia-rich and neuron-rich primary cultures, whereas GCS activity was detectable only in homogenates of rat brain and astroglia-rich primary culture. Of the two known SHMT isoenzymes, only the mitochondrial form was found in rat brain homogenate. It is proposed that, in neural tissue, glycine is metabolized by the combined action of SHMT and the GCS. Owing to the absence of the GCS from neurons, astrocytes appear to be the only site of this part of glycine metabolism in brain. However, neurons are able to utilize as energy source the lactate formed by astroglial cells in this metabolic pathway.

Animals↗

PubMed1999

Application and modulation of a permanent hydrogen peroxide-induced oxidative stress to cultured astroglial cells.

Hydrogen peroxide is often applied to cultured brain cells to investigate functions of these cells under oxidative stress. However, this peroxide might be quickly detoxified by cultured neural cells. At least cultured astroglial cells dispose of exogenous H(2)O(2) with a half-time in the minute range [R. Dringen, B. Hamprecht, Involvement of glutathione peroxidase and catalase in the disposal of exogenous hydrogen peroxide by cultured astroglial cells, Brain. Res. 759 (1997) pp. 67-75]. Therefore, application of hydrogen peroxide to astroglial cells leads only to a period of transient oxidative stress which depends on the ability of the cells to detoxify the peroxide. In order to apply a permanent H(2)O(2)-induced oxidative stress to astroglial cells the generation of H(2)O(2) by the coupled reactions of xanthine oxidase (XO) and superoxide dismutase (SOD) was studied and this system was applied to cultured astroglial cells. In the presence of astroglial cells, an almost constant steady state concentration of H(2)O(2) in the range up to 100 microM was generated, which depended on the activity of the XO applied. These steady state levels of H(2)O(2) were: (i) elevated by application of additional XO, (ii) slowly reduced by application of the XO inhibitor allopurinol, and (iii) immediately reduced to zero by application of catalase or a combination of allopurinol plus catalase. In conclusion, the method presented allows the application of an almost constant exogenous H(2)O(2) stress to cultured cells.

Allopurinol↗

PubMed1999

The glutathione system of peroxide detoxification is less efficient in neurons than in astroglial cells.

The ability of neurons to detoxify exogenously applied peroxides was analyzed using neuron-rich primary cultures derived from embryonic rat brain. Incubation of neurons with H2O2 at an initial concentration of 100 microM (300 nmol/3 ml) led to a decrease in the concentration of the peroxide, which depended strongly on the seeding density of the neurons. When 3 x 10(6) viable cells were seeded per dish, the half-time for the clearance by neurons of H2O2 from the incubation buffer was 15.1 min. Immediately after application of 100 microM H2O2 to neurons, glutathione was quickly oxidized. After incubation for 2.5 min, GSSG accounted for 48% of the total glutathione. Subsequent removal of H2O2 caused an almost complete regeneration of the original ratio of GSH to GSSG within 2.5 min. Compared with confluent astroglial cultures, neuron-rich cultures cleared H2O2 more slowly from the incubation buffer. However, if the differences in protein content were taken into consideration, the ability of the cells to dispose of H2O2 was identical in the two culture types. The clearance rate by neurons for H2O2 was strongly reduced in the presence of the catalase inhibitor 3-aminotriazol, a situation contrasting with that in astroglial cultures. This indicates that for the rapid clearance of H2O2 by neurons, both glutathione peroxidase and catalase are essential and that the glutathione system cannot functionally compensate for the loss of the catalase reaction. In addition, the protein-normalized ability of neuronal cultures to detoxify exogenous cumene hydroperoxide, an alkyl hydroperoxide that is reduced exclusively via the glutathione system, was lower than that of astroglial cells by a factor of 3. These results demonstrate that the glutathione system of peroxide detoxification in neurons is less efficient than that of astroglial cells.

Animals↗

PubMed1999

The detoxification of cumene hydroperoxide by the glutathione system of cultured astroglial cells hinges on hexose availability for the regeneration of NADPH.

The ability of astroglia-rich primary cultures derived from the brains of newborn rats to detoxify exogenously applied cumene hydroperoxide (CHP) was analyzed as a model to study glutathione-mediated peroxide detoxification by astrocytes. Under the conditions used, 200 microM CHP disappeared from the incubation buffer with a half-time of approximately 10 min. The half-time of CHP in the incubation buffer was found strongly elevated (a) in cultures depleted of glutathione by a preincubation with buthionine sulfoximine, an inhibitor of glutathione synthesis, (b) in the presence of mercaptosuccinate, an inhibitor of glutathione peroxidase, and (c) in the absence of glucose, a precursor for the regeneration of NADPH. The involvement of glutathione peroxidase in the clearance of CHP was confirmed by the rapid increase in the level of GSSG after application of CHP. The restoration of the initial high ratio of GSH to GSSG depended on the presence of glucose during the incubation. The high capacity of astroglial cells to clear CHP and to restore the initial ratio of GSH to GSSG was fully maintained when glucose was replaced by mannose. In addition, fructose and galactose at least partially substituted for glucose, whereas exogenous isocitrate and malate were at best marginally able to replace glucose during peroxide detoxification and regeneration of GSH. These results demonstrate that CHP is detoxified rapidly by astroglial cells via the glutathione system. This metabolic process strongly depends on the availability of glucose or mannose as hydride donors for the regeneration of the NADPH that is required for the reduction of GSSG by glutathione reductase.

Animals↗

PubMed1999

Purification of glutathione reductase from bovine brain, generation of an antiserum, and immunocytochemical localization of the enzyme in neural cells.

Glutathione reductase (GR) is an essential enzyme for the glutathione-mediated detoxification of peroxides because it catalyzes the reduction of glutathione disulfide. GR was purified from bovine brain 5,000-fold with a specific activity of 145 U/mg of protein. The homogeneity of the enzyme was proven by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining of the gel. The purified GR from bovine brain is a dimer of two subunits that have an apparent molecular mass of 55 kDa. The purified GR was used to generate a rabbit antiserum with the intention to localize GR in brain cells. The antiserum was useful for the detection of GR by double-labeling immunocytochemical staining in astroglia-rich and neuron-rich primary cultures from rat brain. In homogenates of these cultures, no significant difference in the specific activities of GR was determined. However, not all cell types present in these cultures showed identical staining intensity for GR. In astroglia-rich primary cultures, strong GR immunoreactivity was found in cells positive for the cellular markers galactocerebroside and C3b (antibody Ox42), indicating that oligodendroglial and microglial cells, respectively, contain GR. In contrast, only weak immunoreactivity for GR was found in cells positive for glial fibrillary acidic protein. In neuron-rich primary cultures, GAP43-positive cells stained with the antiserum against GR. These data demonstrate that, in cultures of neural cells, neurons, oligodendroglial cells, and microglial cells express high levels of GR.

Animals↗

PubMed1998

Discrimination of two amino acid transport activities in 4F2 heavy chain- expressing Xenopus laevis oocytes.

Expression of the type II membrane proteins of the rbAT/4F2hc family in Xenopus laevis oocytes results in the induction of amino acid transport activity. To elucidate the mechanism of action, amino acid transport was investigated in oocytes expressing the surface antigen 4F2hc. Leucine transport was mediated by a Na+-independent and a Na+-dependent transport mechanism. Both systems could be further discriminated by their stereochemical constraints. Isoleucine, with a branch at the beta-position, shared only the Na+-independent transport system with leucine. Both transport systems were sensitive to inhibition by arginine, but only the Na+-independent system was sensitive to inhibition by 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid. When compared with known transport systems the two transport activities could be described as similar to, but not identical with, mammalian systems b0,+ and y+L. The Na+-independent b0,+-like transport system was found both in rbAT and 4F2hc expressing oocytes, indicating that both proteins act in a similar way.

Amino Acid Transport Systems↗