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Biomedical subjects

G Drews

Publications and source records attributed to G Drews.

At least 73 records · Page 4Linked to original sources
PubMed1991

Mechanisms of the stimulation of insulin release by oxytocin in normal mouse islets.

Oxytocin (OT) produced a dose-dependent increase in somatostatin, glucagon and insulin release by isolated mouse islets. A small effect on somatostatin release was observed with 0.1 nM-OT, but 1-10 nM-OT was required to affect A- and B- cells significantly. The effects of OT on somatostatin and glucagon release were similar in the presence of 3 mM- and 10 mM-glucose. No change in insulin release was produced by OT in 3 mM-glucose, but a stimulation was still observed in the presence of a maximally effective concentration of glucose (30 mM). The increase in insulin release produced by OT (in 15 mM-glucose) was accompanied by small accelerations of 86Rb and 45Ca efflux from islet cells. Omission of extracellular Ca2+ accentuated the effect of OT on 86Rb efflux, attenuated that on 45Ca efflux, and abolished that on release. OT never inhibited 86Rb efflux. It did not affect the resting potential of B-cells, but slightly increased the Ca2(+)-dependent electrical activity induced by 15 mM-glucose. OT did not affect cyclic AMP levels, but increased inositol phosphate levels in islet cells. It is suggested that the amplification of glucose-induced insulin release that OT produces is due to a stimulation of phosphoinositide metabolism, and presumably an activation of protein kinase C, rather than to a change in cyclic AMP levels or a direct action on the membrane potential. Since OT is present in the pancreas, it is possible that it exerts a neuropeptidergic control of the islet function.

Animals↗
PubMed1991

Analysis of the Rhodobacter capsulatus puc operon: the pucC gene plays a central role in the regulation of LHII (B800-850 complex) expression.

Formation of the light harvesting complex B800-850 (LHII) of Rhodobacter capsulatus requires the expression of more than the three known genes specific for that complex (pucA, pucB and pucE) encoding the alpha, beta and gamma subunits of LHII, respectively. In this work evidence is presented that the product of the gene pucC, which is located downstream from pucA, is essential for high-level transcription of the pucBACDE operon and formation of LHII. Plasmids were constructed containing deletions in one or several puc genes and transferred to a pucC::Tn5 mutant in which the puc operon is not expressed. It was found that the LHII- phenotype of the mutant was due to the missing PucC protein and that all known puc genes are located in one operon. To dissect the pucC, pucD and pucE genes from pucB and pucA and independently regulate them, they were placed under control of the nifHDK promoter. Only under nitrogen-fixing growth conditions was the LHII- pucC::Tn5 mutant complemented by this construction. It is concluded that expression of pucC is essential for formation of the LHII complex in R.capsulatus. Analysis of the pucD and pucE genes led to the conclusion that the products of these genes stabilize the B800-850 complex.

Blotting, Northern↗
PubMed1991

Adrenaline inhibition of insulin release: role of the repolarization of the B cell membrane.

Activation of alpha 2-adrenergic receptors affects several signalling pathways in pancreatic B cells. However, since adrenaline can inhibit insulin release by interfering with a late step of the secretory process, the functional significance of the earlier effects is unclear. In this study, normal mouse islets were used to determine whether the repolarization of the B cell membrane caused by adrenaline contributes to the inhibition of insulin release. The decrease in 86Rb efflux and the repolarization of the B cell membrane produced by adrenaline were attenuated by tolbutamide, which depolarizes by blocking ATP-sensitive K+ channels, and by arginine, which depolarizes because of its transport in a charged form. It is also known that adrenaline does not affect the membrane potential and 86Rb efflux in B cells depolarized by high K+. These three depolarizing conditions similarly shifted to the right the concentration dependence of adrenaline inhibition of insulin release: the effect of 1 nM and 10 nM adrenaline was reduced, but high concentrations of adrenaline still inhibited insulin release nearly completely under all conditions. In contrast, increasing insulin release by cytochalasin B did not alter the inhibitory potency of adrenaline. It is concluded that the repolarization of the B cell membrane and the ensuing decrease in Ca2+ influx play a significant role in the inhibition of insulin release by low concentrations of adrenaline. When high concentrations are used, a more distal effect becomes predominant.

Animals↗
PubMed1991

Adrenaline inhibition of insulin release: role of cyclic AMP.

Catecholamines inhibit adenylate cyclase in pancreatic B-cells, but the importance of the resulting fall in cAMP concentration for the decrease in insulin release remains controversial. Adrenaline caused a dose-dependent inhibition (EC50 = 5.7 nM) of insulin release by mouse islets incubated in a medium containing 15 mM glucose. Supplementation of the medium with 500 microM dibutyryl-cAMP or 1 microM forskolin potentiated the effect of glucose on release and attenuated the inhibition by 1 and 10 nM adrenaline; the EC50 value was increased 2-fold. The inhibitory action of 100 nM or 1 microM adrenaline was, however, not affected. This apparent change in adrenaline potency was not simply due to the larger rate of release since it was not observed when the effect of glucose was potentiated by cytochalasin-B. However, when the same rate of insulin release as that produced by 15 mM glucose alone was achieved by combining 10 mM glucose and 250 microM dibutyryl-cAMP, the inhibitory potency of adrenaline was unaffected. Intracellular microelectrodes were used to determine whether the changes in B-cell membrane potential brought about by adrenaline are mediated by a fall in cAMP levels. Addition of dibutyryl-cAMP or forskolin to a medium containing 10 or 15 mM glucose increased the Ca(2+)-dependent electrical activity triggered by the sugar. However, this did not prevent adrenaline from hyperpolarizing the membrane transiently and causing a steady-state decrease in the intensity of the electrical activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylyl Cyclase Inhibitors↗
PubMed1991

Organization of the genes coding for the reaction-centre L and M subunits and B870 antenna polypeptides alpha and beta from the aerobic photosynthetic bacterium Erythrobacter species OCH114.

In the aerobic photosynthetic bacterium Erythrobacter species OCH114 the structural genes coding for the light-harvesting (LH) complex B870 and the reaction-centre (RC) polypeptides (the gene products of the pufB, pufA, pufL and pufM genes) are mapped on a 2.728 kbp EcoRI fragment. Sequencing of this fragment revealed that the deduced amino acid sequences contain 50 (B870 beta), 52 (B850 alpha), 283 (RCL) and 331 (RCM) residues with the corresponding molecular weights of 5592, 5814, 31364, and 37671, respectively. In the corresponding mRNA a 'hairpin' structure (delta G degrees = -26.6 kcal) is predicted to be located immediately downstream of pufA. The RC and LH polypeptides are highly homologous to those of the purple photosynthetic bacteria Rhodobacter capsulatus, Rhodobacter sphaeroides and Rhodopseudomonas viridis. Directly downstream of pufM there is an open reading frame (ORF) of unknown size. Partial sequencing indicates that this ORF is highly homologous to the cytochrome subunit of the photosynthetic reaction centre from R. viridis. In the puf operon no pufQ or pufX genes could be found, but the bchA gene is located upstream of that operon. Plasmid pESS8.9 containing the 2.728 kbp EcoRI fragment reconstituted a photoinactive mutant of Erythrobacter species OCH114. Comparative analysis of the DNA region upstream of the puf operon and of bacteriochlorophyll (Bchl) synthesis indicated that Bchl synthesis and puf gene expression are regulated differently in Erythrobacter and purple bacteria, respectively.

Amino Acid Sequence↗
PubMed1991

The influence of temperature on the effects of acetylcholine and adrenaline on the membrane potential and 86Rb efflux in mouse pancreatic B-cells.

Pancreatic mouse islets were used to evaluate the influence of temperature on the B-cell response to acetylcholine and adrenaline. At 20 degrees C, the rate of 86Rb efflux from islet cells was lower, the membrane potential of B-cells was slightly less negative, and glucose-induced electrical activity was characterized by longer slow waves than at 37 degrees C. At 20 degrees C, the acceleration of 86Rb efflux produced by 1 microM-ACh was only reduced by 25%, but its reversibility was slower. Acetylcholine rapidly depolarized the B-cell membrane and increased electrical activity regardless of the temperature. However, this increase was characterized by the appearance of short slow waves of high frequency at 37 degrees C and by continuous spiking at 20 degrees C. Adrenaline (1 microM) inhibited 86Rb efflux at 37 and 20 degrees C, but the amplitude of the inhibition was decreased and its time course and reversibility were altered at the lower temperature. Adrenaline repolarized the B-cell membrane and abolished glucose-induced electrical activity for a longer period at 20 degrees C than at 37 degrees C. In conclusion, no marked decrease in signal transduction occurs at 20 degrees C. This suggests that the difficulty of identifying the currents induced by acetylcholine and adrenaline in patch-clamp experiments performed at room temperature is probably due to the small magnitude of these currents.

Acetylcholine↗
PubMed1991

Incorporation of light-harvesting complex I alpha and beta polypeptides into the intracytoplasmic membrane of Rhodobacter capsulatus.

The light-harvesting complex I (LHI) of Rhodobacter capsulatus is an oligomer of basic subunits each consisting of the two different pigment-binding polypeptides LHI alpha and LHI beta, encoded by the pufA (LHI alpha) and pufB (LHI beta) genes. Pulse-labeling experiments showed that in the presence of the LHI alpha polypeptide, the LHI beta polypeptide was inserted earlier into the intracytoplasmic membrane than was the LHI alpha polypeptide. Each of the pufA and pufB genes was deleted to test whether the LHI alpha and beta polypeptides, respectively, are inserted into the intracytoplasmic membrane independently of the LHI partner polypeptide. Neither deletion mutant strain formed the LHI antenna, but a functional reaction center complex was present. Pulse-labeling experiments indicated that the LHI beta polypeptide was inserted into the intracytoplasmic membrane with the same kinetics and in the same amounts regardless of whether the LHI alpha polypeptide was present. However, the LHI beta polypeptide did not accumulate in the membrane in the absence of the LHI alpha protein but was degraded linearly within about 12 min. In contrast to the LHI beta protein, only trace amounts of the LHI alpha polypeptide were inserted into or attached to the membrane if the LHI beta polypeptide was not synthesized.

Bacterial Proteins↗
PubMed1990

Mechanisms of the stimulation of insulin release by arginine-vasopressin in normal mouse islets.

The mechanisms by which arginine-vasopressin (AVP) affects pancreatic B-cell function were studied in normal mouse islets. AVP produced a dose-dependent (0.1-1000 nM; EC50 approximately 1-2 nM) amplification of glucose-induced insulin release. This amplification was of slow onset and reversibility. AVP was ineffective when the concentration of glucose was less than 7 mM, but was still very effective in 30 mM glucose. The increase in insulin release produced by AVP was accompanied by small accelerations of 86Rb and 45Ca efflux from islet cells. Omission of extracellular Ca2+ accentuated the effect of AVP on 86Rb efflux, attenuated that on 45Ca efflux, and abolished that on release. Under no condition did AVP inhibit 86Rb efflux. AVP did not significantly affect cAMP levels, but increased inositol phosphate levels in islet cells, even in the absence of extracellular Ca2+. AVP did not affect the membrane potential in unstimulated B-cells and augmented glucose-induced electrical activity only slightly. This was not due to a direct action on ATP-sensitive K+ channels as revealed by patch-clamp recordings (whole cell and outside-out patches). In conclusion, AVP is not an initiator of insulin release, but it potently amplifies glucose-induced insulin release in normal mouse B-cells. This effect involves a stimulation of phosphoinositide metabolism, and presumably an activation of protein kinase C, rather than a change in cAMP levels or a direct control of the membrane potential.

Acetylcholine↗
PubMed1990

Effects on the formation of antenna complex B870 of Rhodobacter capsulatus by exchange of charged amino acids in the N-terminal domain of the alpha and beta pigment-binding proteins.

The N-terminal domains of the alpha and beta polypeptides of the B870 antenna complex of Rhodobacter capsulatus are oppositely charged. In both polypeptides two charged amino acids are located close to the N-terminus, and two of them are close to the hydrophobic central domain. To test the hypothesis that charged amino acids in the N-terminus have a function for insertion and assembly of pigment-binding polypeptides, charged amino acids were replaced by amino acids of opposite charge. The results show that an exchange of amino acid positions 3 and 6 in alpha (Lys----Glu) or 2 and 5 in beta (Asp----Lys, Arg) has little effect under semiaerobic conditions on the formation of B870 but the additional exchange of positions 14 and 15 in alpha (Arg----Glu, Asp) and/or 13 and 14 in beta (Asp, Glu----Arg) inhibits strongly under semiaerobic dark and anaerobic light conditions the stable incorporation of the polypeptides into the membrane and the formation of the B870 complex. The mutant U43(pTXAB5) is able to grow without any antenna.

Amino Acid Sequence↗
PubMed1990

Cloning and sequencing of the hemA gene of Rhodobacter capsulatus and isolation of a delta-aminolevulinic acid-dependent mutant strain.

The Rhodobacter capsulatus hemA gene, coding for the enzyme delta-aminolevulinic acid synthase (ALAS), was isolated from a genome bank by hybridization with a hemT probe from Rhodobacter sphaeroides. Subcloning of the initial 3.9 kb HindIII fragment allowed the isolation of a 2.5 kb HindIII-BglII fragment which was able to complement the delta-aminolevulinic acid-requiring (ALA-requiring) Escherichia coli mutant SHSP19. DNA sequencing revealed an open reading frame coding for a protein with 401 amino acids which displayed similarity to the amino acid sequences of other known ALASs. However, no resemblance was seen to the HemA protein of E. coli K12. Based on the sequence data, an ALA-requiring mutant strain of R. capsulatus was constructed by site-directed insertion mutagenesis. Introduction of a plasmid, containing the hemA gene of R. capsulatus on the 3.9 kb HindIII fragment, restored ALA-independent growth of the mutant indicating that there is only one gene for ALA biosynthesis in R. capsulatus. Transfer of the R' factor pRPS404 and hybridization analysis revealed that the ALAS gene is not located within the major photosynthetic gene cluster.

5-Aminolevulinate Synthetase↗
PubMed1990

A negatively charged N terminus in the alpha polypeptide inhibits formation of light-harvesting complex I in Rhodobacter capsulatus.

Light-harvesting complex I (LHI) of Rhodobacter capsulatus contains bacteriochlorophyll and carotenoids which are noncovalently bound to two different apoproteins (alpha and beta polypeptides) carrying oppositely charged N-terminal ends. The contribution of these charged segments to the assembly of LHI was studied with mutants having oppositely charged amino acids in the alpha or beta polypeptide. The influence of these mutations on the insertion and assembly process of the LHI complex was investigated by means of spectroscopic analysis of isolated intracytoplasmic membranes and pulse-chase experiments. Exchange of four positively charged amino acids to negatively charged amino acids on the N-terminal domain of the alpha subunit inhibited completely the assembly of the LHI complex. Although this mutant has no antenna, the reaction center is active and the cells were able to grow anaerobically in the light. Conversely, mutation of the four negatively charged amino acids of the N-terminal segment of the beta polypeptide did not prevent the assembly of the LHI complex, although the stability of the complex and the size of the photosynthetic unit were affected. The presence of the mutated beta polypeptide was confirmed by protein sequencing.

Amino Acid Sequence↗
PubMed1990

Galanin and epinephrine act on distinct receptors to inhibit insulin release by the same mechanisms including an increase in K+ permeability of the B-cell membrane.

The mechanisms by which galanin and epinephrine affect pancreatic B-cell function were studied in normal mouse islets. In the presence of 15 mM glucose and 2.5 mM Ca2+, galanin (50 nM) and epinephrine (100 nM) hyperpolarized the B-cell membrane and suppressed electrical activity only transiently. These changes were accompanied by a decrease in 86Rb+ efflux from islet cells and nearly complete inhibition of insulin release. Both agents also decreased 86Rb+ efflux in the absence of Ca2+. Low concentrations (10-15 microM) of diazoxide, an activator of ATP-sensitive K+ channels, mimicked some effects of galanin and epinephrine. However, insulin release was more markedly inhibited by galanin or epinephrine than by diazoxide when electrical activity was similarly decreased, and diazoxide had no effect on 86Rb+ efflux in the absence of Ca2+. When the permeability to K+ was increased by 100 microM diazoxide and the hyperpolarization reversed by high extracellular K+, galanin and epinephrine still inhibited insulin release, but did not affect the membrane potential or 86Rb+ efflux. Galanin and epinephrine decreased glucose utilization and oxidation in islet cells by about 10%, whereas diazoxide had no effect. Blockade of alpha 2-adrenoceptors by yohimbine suppressed the effects of epinephrine, but not those of galanin. It is concluded that activation of galanin and alpha2-adrenergic receptors inhibits insulin release by the same mechanisms. These may involve an increase in K+ permeability of the B-cell membrane by opening ATP-sensitive K+ channels and an additional effect independent of the membrane potential.

Animals↗
PubMed1989

Effects of a synthetic cationic polymer on sodium and potassium currents of frog nerve fibres.

(1) Voltage-clamped nerve fibres of the frog Rana esculenta were treated with the synthetic cationic polymer hexadimethrine bromide (HDM) [Polybrene]. (2) 1 microM HDM irreversibly reduced INa to about 65% of its original size. The time to peak remained almost unaffected, while the decaying phase of the currents (for pulse potentials less than -10 mV) became accelerated. The size of INa was hardly reduced further even with a 1,000-fold higher concentration of HDM. (3) The cationic polymer HDM was without effect on the voltage-dependence of activation and there was only a small shift (-4.5 mV) of the voltage dependence of steady-state inactivation. (4) Variation of [Ca2+] in the Ringer solution shifted the voltage-dependence of activation and of steady-state inactivation to the same extent before and after treatment with HDM. By contrast, 0.5 mM La3+ markedly decreased the Ca2+-induced shifts. (5) The decrease of the size of INa by increasing [Ca2+] was strongly reduced by HDM, as well as by the presence of La3+. (6) These results suggest, that HDM is bound irreversibly to Ca2+ and La3+ binding sites of the membrane that affect the size of INa. These sites are different from Ca2+ (or La3+) binding sites acting on the gating mechanism. (7) The potassium currents were also irreversibly reduced by HDM-treatment. This effect, however, was different for inward and outward currents; for inward currents the IK remaining was about 20% of its original size, while for outward currents IK was reduced only to about 65%. The time course of activation of IK was not affected by HDM-treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Genes downstream from pucB and pucA are essential for formation of the B800-850 complex of Rhodobacter capsulatus.

The formation of the light-harvesting complex B800-850 (LH-II) of Rhodobacter capsulatus requires, in addition to the synthesis of the polypeptides alpha and beta (the gene products of pucA and pucB), the synthesis of bacteriochlorophyll and carotenoids and the expression of at least one gene localized downstream from the pucBA operon. This was concluded from the observation that a Tn5 insertion downstream from pucBA inhibited the formation of the LH-II complex and the formation of the pucBA mRNA. The Tn5 insertion point was mapped and found to be over 500 base pairs (bp) downstream from the end of the pucA gene, suggesting the presence of additional puc genes. A region of about 3,000 bp including the pucB and pucA genes and DNA downstream from pucA was sequenced and found to contain three open reading frames (ORFs C, D, and E). The polypeptide deduced from the first ORF (C) contains 403 amino acids with strongly hydrophobic stretches and one large and three small hydrophilic domains carrying many charged residues. The other two ORFs contain 113 (D) and 118 (E) codons. The amino acid sequences of the N terminus and two tryptic peptides of an alkaline-soluble Mr-14,000 subunit of the isolated LH-II complex were identical with the deduced amino acid sequence of ORF E.

Amino Acid Sequence↗
PubMed1988

Effects of aconitine and batrachotoxin on Na currents and gating currents in the frog node of Ranvier.

The effects of aconitine and batrachotoxin (BTX) on Na currents and gating currents were studied on voltage clamped frog nodes of Ranvier. The relation between steady-state values of charge movement and test pulse potential (Q-E) was compared with the fraction of open channels at a given potential (F-E). In aconitine-treated nodes the half potential, Emid, of the F-E curve was -87 mV and the slope factor, k, was 6.6 mV. Compared to normal nodes aconitine shifted the F-E curve by -53 mV along the voltage axis without significantly altering the slope. The parameters of the Qon-E curve of aconitine-treated nodes were Emid = -63 mV and k = 18.8 mV compared with Emid = -30 mV and k = 17 mV in a normal node. For the Qoff-E curve Emid was -76 mV, fairly close to Emid of the F-E curve, but the slope remained much smaller than that of the F-E curve. Thus, in aconitine-treated nodes, the normalized Qon-E and Qoff-E curves stay below the F-E curve over almost the whole potential range, a situation different from that in untreated or chloramine-T-treated nodes (Drews 1987). Similar results were obtained with BTX. The F-E curve of BTX-treated fibres had a half potential Emid = -85.1 mV and a slope factor k = 4.7 mV. In contrast to aconitine-treated nodes, the F-E curve showed a secondary rise at E greater than -50 mV.(ABSTRACT TRUNCATED AT 250 WORDS)

Aconitine↗
PubMed1988

Modification of sodium and gating currents by amino group specific cross-linking and monofunctional reagents.

To test the possible role of lysine residues in Na channel function the effects of several imidoesters on Na and gating currents were studied in voltage-clamped single frog nerve fibers. Mono- and bisimidoesters were used. These reagents modify amino groups exclusively and do not change the net charge. The three bisimidoesters used easily introduce cross-links between neighboring amino groups. Their structure is almost identical; only the length of the spacers between the two amino-reactive groups is different. An irreversible reduction of Na currents and gating currents was observed with the longest (dimethyl suberimidate [DMS]) and the shortest (dimethyl adipimidate [DMA]) of the cross-linkers used. Of the three cross-linking reagents only the shortest made Na current inactivation slow and incomplete. The steady-state inactivation curve, h infinity (E), was shifted by greater than 25 mV in the hyperpolarizing direction by each of the reagents. The voltage dependence of activation, however, remained unchanged. Furthermore, the effects of two different monoimidoesters (ethyl acetimidate [EAI] and isethionyl acetimidate [IAI]) on gating currents were tested. EAI can penetrate a membrane, whereas IAI is membrane impermeant. IAI was almost without effect, whereas EAI caused a considerable reduction of the gating currents. EAI and DMS reduced the Qoff/Qon ratio without affecting the decay of the Na currents. The results show that lysine residues are critically involved in Na channel gating.

Animals↗