Search PubMed⌕ Search

Biomedical subjects

G Drews

Publications and source records attributed to G Drews.

At least 37 records · Page 2Linked to original sources
PubMed1998

Distribution of protein phosphatases type 1 and 2A in RINm5F cells.

In the insulin producing cell line RINm5F distribution of serine/threonine specific protein phosphatases type 1 (PP1) and 2A (PP2A) was studied. Using different agents which inhibit or stimulate PP1 and PP2A we found that in membrane and nuclear fractions phosphatase activity was inhibited by okadaic acid (OA), protamine, heparin, and inhibitor-2 in a concentration-dependent manner. C2-ceramide had no effect. In the cytosolic fraction the inhibitory effect of okadaic acid was tenfold higher. Protamine stimulated phosphatase activity at low concentrations and became inhibitory at higher concentrations. Inhibitor-2 and heparin caused a decrease in phosphatase activity whereas C2-ceramide led to a slight activation. The data suggest that in membrane and nuclear fractions of RINmSF cells predominantly PP1 is present, whereas in the cytosol PP1 as well as PP2A can be detected.

Animals↗
PubMed1998

Ion channels involved in insulin release are activated by osmotic swelling of pancreatic B-cells.

Measurements of the membrane potential showed that osmotic swelling (-80 mosmol/l) of pancreatic B-cells led to a transient hyperpolarization followed by a more sustained depolarization of the cell membrane. Cell swelling triggers a transient activation of the K+ATP current and of an inward current, carried by Cl-. This current was inhibited by DIDS, D600, and by omission of extracellular Ca2+. The depolarization opens voltage dependent L-type Ca2+ channels, thereby increasing the intracellular Ca2+ activity ([Ca2+]i). This effect was blunted by D600 or abolished by omission of Ca2+. Moreover, osmotic swelling transiently increased the amplitude of the Ca2+ currents. Replacement of NaCl by d-mannitol proved that the observed effects are due to an increase in cell volume and not to a reduction of extracellular Na+ or Cl-. Our results suggest that regulatory volume decrease is achieved by activation of K+ and Cl- currents. The Cl- current is responsible for the previously described depolarization and increase in insulin release induced by osmotic cell swelling.

Adenosine Triphosphate↗
PubMed1998

Complex I of Rhodobacter capsulatus and its role in reverted electron transport.

The activities of NAD+-photoreduction and NADH/decyl-ubiquinone reductase in membrane preparations of Rhodobacter capsulatus changed to the same extent under different conditions. These results indicated that NADH:ubiquinone oxidoreductase (complex I) catalyzes the electron transport in the downhill direction (respiratory chain) and in the uphill direction (reverted electron flow). This conclusion was confirmed by the characterization of a complex-I-deficient mutant of R. capsulatus. The mutant was not able to reduce NAD+ in the light. Since this mutant was not able to grow photoautotrophically, we concluded that complex I is the enzyme that catalyzes the reverted electron flow to NAD+ to provide reduction equivalents for CO2 fixation. Complex I is not essential for the reverted electron flow to nitrogenase since the mutant grew under nitrogen-fixing conditions. As shown by immunological means, NuoE, a subunit of complex I from R. capsulatus having an extended C-terminus, was modified depending on the nitrogen source present in the growth medium. When the organism used N2 instead of NH4+, a smaller NuoE polypeptide was synthesized. The complex-I-deficient mutant was not able to modify NuoE. The function of the modification is discussed.

Electron Transport↗
PubMed1998

The phosphorylation of light-harvesting polypeptides LHIalpha (B870) and LHIIalpha (B800-850) of Rhodobacter capsulatus B10 was higher under chemotrophic oxic than under phototrophic anoxic growth conditions.

In Rhodobacter capsulatus B10 (wild type) both alpha subunits of the light-harvesting complexes are phosphorylated during photosynthetic membrane synthesis. During the process of insertion of these polypeptides, there is a dephosphorylation resulting in intracytoplasmic membranes in which no radioactive phosphate could be detected. Moreover, we show that their phosphate-specific contents depends on the growth conditions, the highest being observed under oxic conditions. When photosynthetic membrane synthesis was induced under light and anaerobiosis, a decrease in the phosphate-specific contents ensued. An inverse relationship exists between specific phosphorylation levels and the degree of membrane differentiation. The phosphorylation is thus a transient phenomenon characteristic of the photosynthetic membrane synthesis governed by the external redox conditions constituting an additional post-transcriptional level of regulation.

Autoradiography↗
PubMed1998

[Effect of forced organ cooling on microperfusion of donor livers].

UNLABELLED: With regard to the factors of graft damage the role of rapid cooling during cold in situ perfusion should not be underestimated. The aim of this study was to analyse the effect of rapid cooling on microperfusion of rat livers in an in vitro model. MATERIAL AND METHODS: During harvest of 14 rat livers the organs' core temperature was monitored. Two groups were formed: (1) cold UW perfusion. (2) cold UW perfusion under additional graft cooling by iced water. Thereafter, isolated livers were perfused (4 degrees C. 1 h, 5 mmHg) monitoring the portocaval resistance, and liver enzyme release and pH levels in the perfusate. Finally, portal angiography of each liver was performed. RESULTS: Rapid in situ cooling resulted in a decrease of the plateau temperature of the organ by 6.5 degrees C (p < 0.01). The pH slope in the perfusate and the enzyme release were diminished obviously, but not significantly. On the other hand, the portocaval resistance was increased by more than 20% (p < 0.05). Portal angiographies assessed by a special score expressed a marked deterioration of microperfusion in peripheral and subcapsular regions. CONCLUSIONS: This study shows that rapid cooling results in worsened microperfusion of liver grafts. The effect of rapid cooling on function and histomorphology of transplanted livers should be analysed in further investigations.

Animals↗
PubMed1997

Protein and gene structure of the NADH-binding fragment of Rhodobacter capsulatus NADH:ubiquinone oxidoreductase.

Membranes of aerobically grown Rhodobacter capsulatus contain only one type of NADH:ubiquinone oxidoreductase which is homologous to the proton-translocating complex I. The K(m) value of the enzyme for NADH was determined to be 8 microM. After solubilization of the membranes with an alkylglucoside detergent, two fragments of complex I with molecular masses of 110 kDa and 140 kDa were isolated by chromatographic steps in the presence of detergent. Both fragments contain at least two polypeptides with apparent molecular masses of 46 kDa and 42 kDa. FMN was identified as cofactor in the preparations. Degenerative oligonucleotide primers were used to amplify a part of the sequence coding for the NADH-binding subunit of complex I by PCR. With the PCR product as probe, a genomic fragment was cloned and sequenced containing the genes encoding the two purified polypeptides and additional reading frames. The two genes are named nuoE and nuoF and are homologous to nqo2 and nqo1 of Paracoccus denitrificans. However, NuoE contains a C-terminal extension of 149 amino acids compared with Nqo2. NuoE and NuoF have molecular masses of 41259 Da and 47133 Da and contain the NADH-, FMN- and FeS-cluster-binding motifs.

Amino Acid Sequence↗
PubMed1997

Imidazole antimycotics affect the activity of various ion channels and insulin secretion in mouse pancreatic B-cells.

In the present paper the effects of antimycotics with imidazole structure on the activity of various ion currents of mouse pancreatic B-cells and insulin secretion from isolated islets have been studied. Clotrimazole (0.1-10 microM, bath solution without albumin) reversibly inhibited the whole-cell K + ATP current studied with the patch-clamp technique and concomitantly depolarized the membrane potential. Two other structurally related compounds, econazole and ketoconazole, exhibited similar effects on the whole-cell K + ATP current. Clotrimazole also inhibited the current through single K + ATP channels measured in the inside-out configuration. According to these results it seems unlikely that a cytoplasmic factor is involved in the action of clotrimazole on K + ATP currents. Clotrimazole (10 microM) also reduced the current through voltage-dependent Ca2+ and K+ channels and altered inactivation kinetics. Moreover, clotrimazole reversibly abolished a recently described inward current which is induced by hypotonic cell swelling. The results show that clotrimazole altered the activity of all ion currents in B-cells investigated in this study. Clotrimazole (3-100 microM, solution with albumin) irreversibly inhibited insulin secretion from isolated islets. With econazole and ketoconazole similar effects on hormone release were observed. The changes in the activity and kinetics of voltage-dependent Ca2+ and K+ currents are likely to contribute to the observed inhibition of insulin secretion. However, we cannot entirely rule out that imidazole antimycotics also interfere with a step in stimulus-secretion coupling distal to changes in membrane potential.

Animals↗
PubMed1997

Structure of the puf operon of the obligately aerobic, bacteriochlorophyll alpha-containing bacterium Roseobacter denitrificans OCh114 and its expression in a Rhodobacter capsulatus puf puc deletion mutant.

Roseobacter denitrificans (Erythrobacter species strain OCh114) synthesizes bacteriochlorophyll a (BChl) and the photosynthetic apparatus only in the presence of oxygen and is unable to carry out primary photosynthetic reactions and to grow photosynthetically under anoxic conditions. The puf operon of R. denitrificans has the same five genes in the same order as in many photosynthetic bacteria, i.e., pufBALMC. PufC, the tetraheme subunit of the reaction center (RC), consists of 352 amino acids (Mr, 39,043); 20 and 34% of the total amino acids are identical to those of PufC of Chloroflexus aurantiacus and Rubrivivax gelatinosus, respectively. The N-terminal hydrophobic domain is probably responsible for anchoring the subunit in the membrane. Four heme-binding domains are homologous to those of PufC in several purple bacteria. Sequences similar to pufQ and pufX of Rhodobacter capsulatus were not detected on the chromosome of R. denitrificans. The puf operon of R. denitrificans was expressed in trans in Escherichia coli, and all gene products were synthesized. The Roseobacter puf operon was also expressed in R. capsulatus CK11, a puf puc double-deletion mutant. For the first time, an RC/light-harvesting complex I core complex was heterologously synthesized. The strongest expression of the R. denitrificans puf operon was observed under the control of the R. capsulatus puf promoter, in the presence of pufQ and pufX and in the absence of pufC. Charge recombination between the primary donor P+ and the primary ubiquinone Q(A)- was observed in the transconjugant, showing that the M and L subunits of the RC were correctly assembled. The transconjugants did not grow photosynthetically under anoxic conditions.

Amino Acid Sequence↗
PubMed1996

Effects of the nitric oxide synthase inhibitor N omega nitro-L-arginine methyl ester on electrical activity and ion channels of mouse pancreatic B cells.

The effects of the nitric oxide (NO) synthase inhibitor N omega-nitro-L-arginine methyl ester (L-NAME) were studied on electrical activity, K+ATP currents and voltage-dependent K+ and Ca2+ channel currents in mouse pancreatic B cells. In the presence of 15 mM glucose, L-NAME (> 5 mM) depolarized the B cell membrane and electrical activity became continuous. The depolarization caused by L-NAME was not reversed by the addition of a membrane permeant cyclic GMP analogue. L-NAME inhibited the whole-cell K+ATP current reversibly by reducing the single channel current conductance and open probability. The results are consistent with the assumption that L-NAME depolarizes the B cell membrane by a direct action on the K+ATP channel. L-NAME also influenced the whole-cell current through voltage-dependent K+ and Ca2+ channels.

Animals↗
PubMed1996

Origin of the two carbonyl oxygens of bacteriochlorophyll a. Demonstration of two different pathways for the formation of ring E in Rhodobacter sphaeroides and Roseobacter denitrificans, and a common hydratase mechanism for 3-acetyl group formation.

A respiring culture of Rhodobacter sphaeroides, grown in the dark under defined aerobic conditions, produced cells capable of immediately commencing adaptation to photosynthetic growth on exposure to light and further reduction of oxygen tension. Adaptation was complete after 12 h and the bacteriochlorophyll a content increased 10-20-fold. This adaptation was performed in the presence of either H2(18)O or 18O2. The extracted bacteriochlorophyll a was examined by mass spectrometry to determine the origin of both the 3-acetyl adn 13(1)-oxo oxygen atoms: both were derived from water. The derivation of the 13(1)-oxo group from water in R. sphaeroides indicates that the formation of isocyclic ring E from the 13-propionic acid methylester side chain of Mg(2+)-protoporphyrin IX monomethylester is an anaerobic process involving a hydratase. This is very different to the situation in higher plants and green algae where the formation of isocyclic ring E is an aerobic process in which the 13(1)-oxo group is derived from molecular oxygen via an oxygenase. In contrast to adapting R. sphaeroides cells, the 13(1)-oxo group of bacteriochlorophyll a in growing cells of the obligate aerobic chemotrophic bacterium Roseobacter denitrificans, was labelled by 18O2 and is, therefore, derived from molecular oxygen like in higher plants and green algae; however, the 3-acetyl group was not labelled by 18O2. Thus, while the 13(1)-oxo group has different origins in R. sphaeroides and R. denitrificans, the 3-acetyl group arises in both bacteria by enzymic hydration of the vinyl group of a chlorophyll a derivative.

Bacteria↗
PubMed1996

Promoter analysis of the catalase-peroxidase gene (cpeA) from Rhodobacter capsulatus.

The expression of the Rhodobacter capsulatus catalase-peroxidase (cpeA) was studied by in-frame fusions of the upstream region of the cpeA gene to a promoter-less lacZ gene. The transcription of the cpeA gene is about 20-50-fold higher under aerobic-dark than under anaerobic-light conditions. The promoter was localized within a 69-bp upstream DNA region. The transcription start site, determined by primer extension, is 28 bases upstream from the initiation codon, confirming the postulated promoter localized by deletion analysis. Deletion of the part of the upstream region specifically responsible for oxygen regulation resulted in constitutive expression of the cpeA gene.

Amino Acid Sequence↗
PubMed1996

The phosphatase inhibitor okadaic acid blocks KCl-depolarization-induced rise of cytosolic calcium of rat insulinoma cells (RINm5F).

It has been shown that okadaic acid (OA) diminishes insulin secretion of rat pancreatic islets in response to glucose, glyceraldehyde and KCl. Glucose, glyceraldehyde and KCl cause release of insulin by depolarization and subsequent opening of L-type calcium channels. Calcium entry into cells is thought to be related to protein phosphorylation. To evaluate whether or not OA mediated inhibition of insulin secretion in response to depolarization might be due to an interference with calcium uptake, we studied its effect on KCl (30 mM)-induced increases of cytosolic calcium and discharge of insulin in the insulin secreting clonal tumor cell line RINm5F. OA inhibited KCl-stimulated insulin release in concentrations > or = 1 microM. In intact RINm5F cells similar concentrations of OA decreased the activity of protein phosphates PP-1/PP-2A and inhibited the depolarization-induced rise of cytosolic calcium ([Ca2+]i). The latter action could also be achieved with the protein phosphatase inhibitor calyculin A, whereas the OA analogue 1-nor-okadaone, which is without effect on phosphatases, did not affect [Ca2+]i or insulin release. It is concluded that depression of depolarization-induced insulin secretion by OA is due to inhibition of calcium entry along voltage dependent calcium channels. The data also suggest that in RINm5F cells protein phosphatases PP-1/PP-2A are related to the function of voltage-dependent calcium channels.

Animals↗
PubMed1996

Formation of the light-harvesting complex I (B870) of anoxygenic phototrophic purple bacteria.

The light-harvesting (LH) complex I (B870) of anoxygenic photosynthetic purple bacteria is the oligomeric form of its subunit B820 consisting of the low-molecular-weight polypeptides alpha, beta, bacteriochlorophyll (BChl), and carotenoids in the stoichiometric ratio [alpha1 beta1 (BChl2) Crt1-2]n. LHI surrounds the photochemical reaction center (RC). The major absorption band of the LHI complex is species-specific and is found at 870-890 nm; those of the subunit and the monomeric BChl a (dissolved in methanol) absorb at 820 and 770 nm, respectively. The isolated LHI complex can be reversibly dissociated to the B820 subunit or to the polypeptides and pigments by addition of detergents. Reconstitution of the B820 or the functional B870 complex is still possible after partial truncation of the N- or C-terminal regions of the alpha- or beta-polypeptide or of the beta-polypeptide only. The minimal structural requirements for reconstitution of a spectrally wild-type form after truncation of the polypeptides and/or modifications of the BChl molecule are described. The insertion of the LHIalpha- and LHIbeta-polypeptides into the membrane and the in vivo assembly of LHI, studied in a cell-free system and in whole cells of Rhodobacter capsulatus, depend on the primary structures of both polypeptides, BChl, the chaperones DnaK and GroEL, membrane-bound proteins, and energized membranes. Exchanges, deletions, or insertions of amino acyl residues, especially in the conserved region of the N-terminus of the LHIalpha-polypeptide, prevent or reduce the efficiency and stability of the LHI assembly. Therefore, reconstitution of LHI in a detergent micelle does not exactly reproduce the formation of the LHI complex in the photosynthetic membrane in vivo. The N-terminal domains play a crucial role in the formation of the oligomeric protein scaffold and of the pigment array. Facultatively phototrophic bacteria such as Rhodospirillum (Rsp.) rubrum or Rhodobacter (Rba.) capsulatus can adjust to changes in oxygen tension, light intensity, temperature, and substrates to grow under chemotrophic or phototrophic conditions. The photosynthetic apparatus (PSA), localized mainly on intracytoplasmic membranes (ICM), is usually synthesized only under low oxygen partial pressure. The cellular amount and composition of the PSA are modified upon changing light intensity in relation to cell growth (Drews and Golecki 1995). The morphogenesis of cellular structures like ICM is quite different from self-assembly. Self-assembly is a reversible process of aggregation of the constituents of a complex structure without protein synthesis and is driven by weak or strong forces in the interactions of the constituents. Morphogenesis results from the interplay of numerous gene products and the cellular organization and is always dependent upon pre-existent structures (Harold 1995). The morphogenesis of the photosynthetic membrane in purple bacteria has been studied in its different steps. The regulation at the transcriptional and post-transcriptional levels in purple bacteria, and the structure and morphogenesis of the ICM have been described recently (Armstrong 1995; Bauer 1995; Biel 1995; Drews and Golecki 1995; Klug 1995). In this mini-review, I will focus on the minimal requirements for the in vitro assembly of light-harvesting (LH) complex I (B870) from its constituents in detergent micelles and compare the results with observations on the complex process of targeting and import of LHI polypeptides into the membrane and assembly of B870.

Amino Acid Sequence↗
PubMed1995

NO synthase activity does not influence electrical activity of mouse pancreatic B-cells.

The membrane potential of mouse pancreatic B-cells was recorded with intracellular microelectrodes. At a low concentration the NO synthase inhibitor N omega-nitro-L-arginine methyl ester (L-NAME, 100 mumol/l) did not alter electrical activity induced by 15 mmol/l glucose. At 5 mmol/l L-NAME depolarized B-cells similarly to L-arginine. The depolarization of the B-cell membrane induced by 5 mmol/l L-arginine in the presence of 15 mmol/l glucose was not inhibited but further increased by the addition of 15 mmol/l L-NAME. D-arginine which is not used as a substrate by the NO synthase also depolarized the B-cells. However, higher concentrations were necessary than for L-arginine pointing to a preferential transport of the L-enantiomer. It is concluded that the electrical activity of pancreatic B-cells is not influenced by the NO synthase and that the enzyme is not involved in the arginine-induced depolarization of B-cells.

Amino Acid Oxidoreductases↗
PubMed1995

Effects of H2O2 on membrane potential and [Ca2+]i of cultured rat arterial smooth muscle cells.

The effect of 1 mmol/l H2O2 was studied on the membrane potential and [Ca2+]i with microelectrodes and the fura-2 technique, respectively. H2O2 induced a biphasic increase in [Ca2+]i with a fast transient peak and a subsequent plateau. H2O2 also led to a biphasic hyperpolarization of the cells with a similar time course. This was followed by a slight depolarization after wash-out of H2O2. External Ca2+ free solutions and treatment with the Ca2+ ionophore A23187 (1 mumol/l) abolished the effect of H2O2 on [Ca2+]i and almost entirely reduced the effect on the membrane potential. Phenylephrine (10 mumol/l) or A23187 also induced very similar biphasic hyperpolarizations of the membrane as H2O2 which were fully reversible after wash-out. It is concluded that H2O2 hyperpolarizes the membrane by opening of Ca2+ dependent K+ channels.

Animals↗
PubMed1995

Glucagon-like peptide-1 modulates Ca2+ current but not K+ATP current in intact mouse pancreatic B-cells.

The influence of GLP-1 on electrical activity and ion currents of mouse pancreatic B-cells was studied with intracellular microelectrodes and the whole-cell configuration of the patch-clamp technique. In the presence of 15 mmol/l glucose 5, 50 and 100 nmol/l GLP-1 slightly increased electrical activity. This effect may be caused by the slowing of Ca2+ channel inactivation observed with GLP-1. Thus, changes in Ca2+ channel kinetics are suggested to contribute to the insulinotropic action of the hormone. The most prominent effect of GLP-1 on the membrane potential was the conversion of irregular electrical activity into regular oscillations of the membrane potential. At the threshold concentration for insulin secretion (7 mmol/l glucose) GLP-1 did not alter the membrane potential. Accordingly, in patch-clamp experiments GLP-1 had no effect on the whole-cell K+ATP current.

Adenosine Triphosphate↗