bluuebunny/crossref_metadata_2025 huggingface dataset:
Background and tandem‐pore potassium channels in magnocellular neurosecretory cells of the rat supraoptic nucleus
Authors: Han Jaehee , Gnatenco Carmen , Sladek Celia D. , Kim Donghee
Published: February 2003

Magnocellular neurosecretory cells (MNCs) were isolated from the supraoptic nucleus of rat hypothalamus, and properties of K+ channels that may regulate the resting membrane potential and the excitability of MNCs were studied. MNCs showed large transient outward currents, typical of vasopressin‐ and oxytocin‐releasing neurons. K+ channels in MNCs were identified by recording K+ channels that were open at rest in cell‐attached and inside‐out patches in symmetrical 150 mm KCl. Eight different K+ channels were identified and could be distinguished unambiguously by their single‐channel kinetics and voltage‐dependent rectification. Two K+ channels could be considered functional correlates of TASK‐1 and TASK‐3, as judged by their single‐channel kinetics and high sensitivity to pHo. Three K+ channels showed properties similar to TREK‐type tandem‐pore K+ channels (TREK‐1, TREK‐2 and a novel TREK), as judged by their activation by membrane stretch, intracellular acidosis and arachidonic acid. One K+ channel was activated by application of pressure, arachidonic acid and alkaline pHi, and showed single‐channel kinetics indistinguishable from those of TRAAK. One K+ channel showed strong inward rectification and single‐channel conductance similar to those of a classical inward rectifier, IRK3. Finally, a K+ channel whose cloned counterpart has not yet been identified was highly sensitive to extracellular pH near the physiological range similar to those of TASK channels, and was the most active among all K+ channels. Our results show that in MNCs at rest, eight different types of K+ channels can be found and six of them belong to the tandem‐pore K+ channel family. Various physiological and pathophysiological conditions may modulate these K+ channels and regulate the excitability of MNCs.
Low doses of ethanol and a neuroactive steroid positively interact to modulate rat GABAA receptor function
Authors: Akk Gustav , Steinbach Joe Henry
Published: February 2003

Fast inhibitory responses in the central nervous system are mediated by the GABAA receptor. The activation and function of the GABAA receptor can be modulated by a variety of compounds including benzodiazepines, barbiturates and neuroactive steroids. Modulation of the GABAA receptor function by ethanol has been observed in some but not all studies. We have studied the effect of ethanol at concentrations corresponding to light intoxication on the function of the recombinant GABAA receptor containing α1β2γ2 subunits. The experiments were performed both in the absence and presence of low, subthreshold concentrations of a neuroactive steroid. The results demonstrate that, in the presence of the steroid, 0.05 % (9 mm) ethanol potentiates the GABAA receptor function by increasing the channel mean open duration. No effect was observed on the channel closed time durations. The data suggest that ethanol influences channel closing with no effect on the affinity of the receptor for GABA or the channel opening rate constant.
Ubiquitin‐proteasome‐dependent muscle proteolysis responds slowly to insulin release and refeeding in starved rats
Authors: Kee Anthony J. , Combaret Lydie , Tilignac Thomas , Souweine Bertrand , Aurousseau Eveline , Dalle Michel , Taillandier Daniel , Attaix Didier
Published: February 2003

The central role of the ubiquitin‐proteasome system in the loss of skeletal muscle protein in many wasting conditions has been well established. However, it is unclear what factors are responsible for the suppression of this system during periods of protein gain. Thus, the aim of these studies was to examine the short‐term effects of insulin release and nutrients on skeletal muscle protein turnover in young rats starved for 48 h, and then infused intravenously with amino acids (AA), or fed an oral diet. Forty‐eight hours of starvation (i.e. prolonged starvation in young rats) decreased muscle protein synthesis and increased proteasome‐dependent proteolysis. Four‐hour AA infusion and 4 h of refeeding increased plasma insulin release and AA concentrations, and stimulated muscle protein synthesis, but had no effect on either total or proteasome‐dependent proteolysis, despite decreased plasma corticosterone concentrations. Both muscle proteasome‐dependent proteolysis and the rate of ubiquitination of muscle proteins were not suppressed until 10 h of refeeding. The temporal response of these two measurements correlated with the normalised expression of the 14‐kDa E2 (a critical enzyme in substrate ubiquitination in muscle) and the expression of the MSS1 subunit of the 19S regulatory complex of the 26S proteasome. In contrast, the starvation‐induced increase in mRNA levels for 20S proteasome subunits was normalised by refeeding within 24 h in muscle, and 6 h in jejunum, respectively. In conclusion, unlike protein synthesis, skeletal muscle proteasome‐dependent proteolysis is not acutely responsive in vivo to insulin, AA, and/or nutrient intake in refed starved rats. This suggests that distinct and perhaps independent mechanisms are responsible for the nutrient‐dependent regulation of protein synthesis and ubiquitin‐proteasome‐dependent proteolysis following a prolonged period of catabolism. Furthermore, factors other than the expression of ubiquitin‐proteasome pathway components appear to be responsible for the suppression of skeletal muscle proteasome‐dependent proteolysis by nutrition.
Strong, reliable and precise synaptic connections between thalamic relay cells and neurones of the nucleus reticularis in juvenile rats
Authors: Gentet Luc J. , Ulrich Daniel
Published: February 2003

The thalamic reticular nucleus (nRT) is composed entirely of GABAergic inhibitory neurones that receive input from pyramidal cortical neurones and excitatory relay cells of the ventrobasal complex of the thalamus (VB). It plays a major role in the synchrony of thalamic networks, yet the synaptic connections it receives from VB cells have never been fully physiologically characterised. Here, whole‐cell current‐clamp recordings were obtained from 22 synaptically connected VB‐nRT cell pairs in slices of juvenile (P14‐20) rats. At 34‐36 °C, single presynaptic APs evoked unitary EPSPs in nRT cells with a peak amplitude of 7.4 ± 1.5 mV (mean ±s.e.m.) and a decay time constant of 15.1 ± 0.9 ms. Only four out of 22 pairs showed transmission failures at a mean rate of 6.8 ± 1.1 %. An NMDA receptor (NMDAR)‐mediated component was significant at rest and subsequent EPSPs in a train were depressed. Only one out of 14 pairs tested was reciprocally connected; the observed IPSPs in the VB cell had a peak amplitude of 0.8 mV and were completely abolished in the presence of 10 μm bicuculline. Thus, synaptic connections from VB cells to nRT neurones are mainly ‘drivers’, while a small subset of cells form closed disynaptic loops.
Group I metabotropic glutamate receptors activate a calcium‐sensitive transient receptor potential‐like conductance in rat hippocampus
Authors: Gee Christine E. , Benquet Pascal , Gerber Urs
Published: February 2003

In CA3 pyramidal neurons from organotypic slice cultures, activation of Gq‐coupled group I metabotropic glutamate receptors (mGluRs) induces a non‐selective cationic conductance that enhances excitability. We have found that this response shares several properties with conductances that are mediated by the transient receptor potential (TRP) family of ion channels, including inhibition by La3+, 2‐aminoethoxydiphenylborane (2APB), cis‐N‐(2‐phenylcyclopentyl)azacyclotridec‐1‐en‐2‐amine (MDL 12,330A) and a doubly rectifying current‐voltage relationship. Stimulation of mGluR1 and mGluR5 converged to activate the TRP‐like conductance in a synergistic manner, and activation of either subtype alone produced only a fraction of the normal response. Activation of the cationic current required elevated intracellular Ca2+. Chelating intracellular Ca2+ or blocking Ca2+ entry through voltage‐gated Ca2+ channels attenuated responses to the activation of mGluRs. Conversely, raising intracellular Ca2+ potentiated mGluR activation of the TRP‐like conductance. Under control conditions, blocking G protein activation using intracellular GDPβS with or without N‐(2, 6‐dimethylphenylcarbamoylmethyl) triethylammonium chloride (QX‐314) prevented mGluR‐mediated activation of the TRP‐like conductance. Following G protein blockade, however, the coupling between mGluRs 1 and/or 5 and the TRP‐like conductance was rescued by increasing intracellular Ca2+. This suggests that a G protein‐independent signalling pathway is also activated by group I mGluRs. Such a pathway may represent an alternative transduction mechanism to maintain metabotropic responses under conditions where G proteins are functionally uncoupled from their cognate receptors.
Involvement of intramuscular interstitial cells in nitrergic inhibition in the mouse gastric antrum
Authors: Suzuki H. , Ward S. M. , Bayguinov Y. R. , Edwards F. R. , Hirst G. D. S.
Published: February 2003

Intracellular recordings were made from isolated bundles of the circular muscle layer of mouse gastric antrum and the responses evoked by stimulating intrinsic nerve fibres were examined. Transmural nerve stimulation evoked a fast inhibitory junction potential (fast‐IJP) which was followed initially by a smaller amplitude long lasting inhibitory junction potential (slow‐IJP) and a period of excitation. The excitatory component of the response was abolished by atropine, suggesting that it resulted from the release of acetylcholine and activation of muscarinic receptors. Fast‐IJPs were selectively reduced in amplitude by apamin and slow‐IJPs were abolished by Nω‐nitro‐l‐arginine. Slow‐IJPs were associated with a drop in membrane noise, suggesting that inhibition resulted from a reduced discharge of unitary potentials by intramuscular interstitial cells of Cajal (ICCIM). The chloride channel blocker, anthracene‐9‐carboxylic acid, reduced the discharge of membrane noise in a manner similar to that detected during the slow‐IJP. When recordings were made from the antrum of W/WV mice, which lack ICCIM, the cholinergic and nitrergic components were absent, with only fast‐IJPs being detected. The observations suggest that neurally released nitric oxide selectively targets ICCIM causing a hyperpolarization by suppressing the discharge of unitary potentials.
Mossy fibre synaptic NMDA receptors trigger non‐hebbian long‐term potentiation at entorhino‐CA3 synapses in the rat
Authors: Tsukamoto Masako , Yasui Takuya , Yamada Maki K. , Nishiyama Nobuyoshi , Matsuki Norio , Ikegaya Yuji
Published: February 2003

Hippocampal CA3 pyramidal cells receive two independent afferents from the enthorinal cortex, i.e. a direct input via the temporoammonic pathway (TA, perforant path) and an indirect input via the mossy fibres (MF) of dentate granule cells. In spite of past suggestions that the TA is assigned an important role in exciting the pyramidal cells, little is known about their physiological properties. By surgically making an incision through the sulcus hippocampi and a small part of the dentate molecular layer, we succeeded in isolating TA‐mediated monosynaptic responses in CA3 stratum lacunosum‐moleculare. The TA‐CA3 synaptic transmission was completely blocked by a combination of d,l‐2‐amino‐5‐phosphonopentanoic acid (AP5) and 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX), NMDA and non‐NMDA receptor antagonists, respectively, and displayed paired‐pulse facilitation and NMDA receptor‐dependent long‐term potentiation, which are all typical of glutamatergic synapses. We next addressed the heterosynaptic interaction between TA‐CA3 and MF‐CA3 synapses. The TA‐CA3 transmission was partially attenuated by single‐pulse MF pre‐stimulation at inter‐pulse intervals of up to 70 ms. However, surprisingly, burst stimulation of the MF alone induced long‐lasting facilitation of TA‐CA3 synaptic efficacy. This non‐Hebbian form of synaptic plasticity was efficiently prevented by local application of AP5 into the MF synapse‐rich area. Therefore, MF‐activated NMDA receptors are responsible for the heterosynaptic modification of TA‐CA3 transmission, and thereby, the history of MF activity may be etched into TA‐CA3 synaptic strength. Our findings predict a novel form of spatiotemporal information processing in the hippocampus, i.e. a use‐dependent intersynaptic memory transfer.
Glutamate modulation of GABA transport in retinal horizontal cells of the skate
Authors: Kreitzer Matthew A. , Andersen Kristen A. , Malchow Robert Paul
Published: February 2003

Transport of the amino acid GABA into neurons and glia plays a key role in regulating the effects of GABA in the vertebrate retina. We have examined the modulation of GABA‐elicited transport currents of retinal horizontal cells by glutamate, the likely neurotransmitter of vertebrate photoreceptors. Enzymatically isolated external horizontal cells of skate were examined using whole‐cell voltage‐clamp techniques. GABA (1 mm) elicited an inward current that was completely suppressed by the GABA transport inhibitors tiagabine (10 μm) and SKF89976‐A (100 μm), but was unaffected by 100 μm picrotoxin. Prior application of 100 μm glutamate significantly reduced the GABA‐elicited current. Glutamate depressed the GABA dose‐response curve without shifting the curve laterally or altering the voltage dependence of the current. The ionotropic glutamate receptor agonists kainate and AMPA also reduced the GABA‐elicited current, and the effects of glutamate and kainate were abolished by the ionotropic glutamate receptor antagonist 6‐cyano‐7‐nitroquinoxaline. NMDA neither elicited a current nor modified the GABA‐induced current, and metabotropic glutamate analogues were also without effect. Inhibition of the GABA‐elicited current by glutamate and kainate was reduced when extracellular calcium was removed and when recording pipettes contained high concentrations of the calcium chelator BAPTA. Caffeine (5 mm) and thapsigargin (2 nm), agents known to alter intracellular calcium levels, also reduced the GABA‐elicited current, but increases in calcium induced by depolarization alone did not. Our data suggest that glutamate regulates GABA transport in retinal horizontal cells through a calcium‐dependent process, and imply a close physical relationship between calcium‐permeable glutamate receptors and GABA transporters in these cells.
The role of neuropeptide Y in the ovine fetal cardiovascular response to reduced oxygenation
Authors: Sanhueza Emilia M. , Johansen‐Bibby Anja A. , Fletcher Andrew J. W. , Riquelme Raquel A. , Daniels Alejandro J. , Serón‐Ferré Maria , Gaete Cristián R. , Carrasco Jorge E. , Llanos Aníbal J. , Giussani Dino A.
Published: February 2003

This study investigated the role of neuropeptide Y (NPY) in mediating cardiovascular responses to reduced oxygenation in the late gestation ovine fetus by: (1) comparing the effects on the cardiovascular system of an exogenous infusion of NPY with those elicited by moderate or severe reductions in fetal oxygenation; and (2) determining the effect of fetal i.v. treatment with a selective NPY‐Y1 receptor antagonist on the fetal cardiovascular responses to acute moderate hypoxaemia. Under general anaesthesia, 14 sheep fetuses (0.8‐0.9 of gestation) were surgically prepared with vascular and amniotic catheters. In 5 of these fetuses, a Transonic flow probe was also implanted around a femoral artery. Following at least 5 days of recovery, one group of fetuses (n= 9) was subjected to a 30 min treatment period with exogenous NPY (17 μg kg−1 bolus plus 0.85 μg kg−1 min−1 infusion). In this group, fetal blood pressure and heart rate were monitored continuously and the distribution of the fetal combined ventricular output was assessed via injection of radiolabelled microspheres before and during treatment. The second group of fetuses instrumented with the femoral flow probe (n= 5) were subjected to a 3 h experiment consisting of 1 h of normoxia, 1 h of hypoxaemia, and 1 h of recovery during a slow i.v. infusion of vehicle. One or two days later, the acute hypoxaemia protocol was repeated during fetal i.v. treatment with a selective NPY‐Y1 receptor antagonist (50 μg kg−1bolus + 1.5μg kg−1 min−1 infusion). In these fetuses, fetal arterial blood pressure, heart rate and femoral vascular resistance were recorded continuously. The results show that fetal treatment with exogenous NPY mimics the fetal cardiovascular responses to asphyxia, and that treatment of the sheep fetus with a selective NPY‐Y1 receptor antagonist does not affect the fetal cardiovascular response to acute moderate hypoxaemia. These results support a greater role for NPY in mediating the fetal cardiovascular responses to acute asphyxia than to acute moderate hypoxaemia.
Exercise induces transient transcriptional activation of the PGC‐1α gene in human skeletal muscle
Authors: Pilegaard Henriette , Saltin Bengt , Neufer P. Darrell
Published: February 2003

Endurance exercise training induces mitochondrial biogenesis in skeletal muscle. The peroxisome proliferator activated receptor co‐activator 1α (PGC‐1α) has recently been identified as a nuclear factor critical for coordinating the activation of genes required for mitochondrial biogenesis in cell culture and rodent skeletal muscle. To determine whether PGC‐1α transcription is regulated by acute exercise and exercise training in human skeletal muscle, seven male subjects performed 4 weeks of one‐legged knee extensor exercise training. At the end of training, subjects completed 3 h of two‐legged knee extensor exercise. Biopsies were obtained from the vastus lateralis muscle of both the untrained and trained legs before exercise and after 0, 2, 6 and 24 h of recovery. Time to exhaustion (2 min maximum resistance), as well as hexokinase II (HKII), citrate synthase and 3‐hydroxyacyl‐CoA dehydrogenase mRNA, were higher in the trained than the untrained leg prior to exercise. Exercise induced a marked transient increase (P < 0.05) in PGC‐1α transcription (10‐ to > 40‐fold) and mRNA content (7‐ to 10‐fold), peaking within 2 h after exercise. Activation of PGC‐1α was greater in the trained leg despite the lower relative workload. Interestingly, exercise did not affect nuclear respiratory factor 1 (NRF‐1) mRNA, a gene induced by PGC‐1α in cell culture. HKII, mitochondrial transcription factor A, peroxisome proliferator activated receptor α, and calcineurin Aα and Aβ mRNA were elevated (≈2‐ to 6‐fold; P < 0.05) at 6 h of recovery in the untrained leg but did not change in the trained leg. The present data demonstrate that exercise induces a dramatic transient increase in PGC‐1α transcription and mRNA content in human skeletal muscle. Consistent with its role as a transcriptional coactivator, these findings suggest that PGC‐1α may coordinate the activation of metabolic genes in human muscle in response to exercise.
Glycinergic mIPSCs in mouse and rat brainstem auditory nuclei: modulation by ruthenium red and the role of calcium stores
Authors: Lim Rebecca , Oleskevich Sharon , Few Alexandra P. , Leao Richardson N. , Walmsley Bruce
Published: February 2003

Spontaneous miniature inhibitory postsynaptic currents (mIPSCs) recorded in central neurons are usually highly variable in amplitude due to many factors such as intrinsic postsynaptic channel fluctuations at each release site, site‐to‐site variability between release sites, electrotonic attenuation due to variable dendritic locations of synapses, and the possibility of synchronous multivesicular release. A detailed knowledge of these factors is essential for the interpretation of mIPSC amplitude distributions and mean quantal size. We have studied glycinergic mIPSCs in two auditory brainstem nuclei, the rat anteroventral cochlear nucleus (AVCN) and the mouse medial nucleus of the trapezoid body (MNTB). Our previous results have demonstrated the location of glycinergic synapses on these neurons to be somatic, thus avoiding electrotonic complications. Spontaneous glycinergic mIPSCs were recorded from AVCN and MNTB neurons in brainstem slices, in the presence of TTX to block action potentials, and 6‐cyano‐7‐nitroquinoxaline‐2, 3‐dione, (±)‐2‐amino‐5‐phosphonopentanoic acid and bicuculline to block glutamatergic and GABAergic synaptic currents. Ruthenium red (RuR), which was used to increase the frequency of mIPSCs, significantly changed the shape of most (90 %) mIPSC amplitude distributions by increasing the proportion of large‐amplitude mIPSCs. The possibility was investigated (following previous evidence at GABAergic synapses) that large‐amplitude glycinergic mIPSCs are due to synchronous multivesicular release initiated by presynaptic calcium sparks from ryanodine‐sensitive calcium stores. Interval analysis of mIPSCs indicated that the number of potentially undetected (asynchrony < 0.5 ms) multivesicular mIPSCs was low in comparison with the number of large‐amplitude mIPSCs. Ryanodine, thapsigargin and calcium‐free perfusate did not reduce the frequency of large‐amplitude mIPSCs (> 150 pA), arguing against a significant role for presynaptic calcium stores. Our results support previous evidence suggesting that RuR increases miniature postsynaptic current (mSC) frequency by a mechanism that does not involve presynaptic calcium stores. Our results also indicate that at glycinergic synapses in the AVCN and MNTB, site‐to‐site variability in mIPSC amplitude, rather than multivesicular release, is a major factor underlying the large range of amplitudes of glycinergic mIPSCs.
Modulation of inhibitory autapses and synapses on rat CA1 interneurones by GABAa receptor ligands
Authors: Pawelzik H. , Hughes D. I. , Thomson A. M.
Published: February 2003

To determine whether autaptic inhibition plays a functional role in the adult hippocampus, the action potential afterhyperpolarisations (spike AHPs) of CA1 interneurones were investigated in 25 basket, three bistratified and eight axo‐axonic cells. The spike AHPs showed two minima in all regular‐spiking (5), burst‐firing (3) and in many fast‐spiking cells (17:28). The fast component had a time‐to‐peak (TTP) of 1.2 ± 0.5 ms, the slower TTP was very variable (range of 3.3‐103 ms). The AHP width at half‐amplitude (HW) was 12.5 ± 5.7 ms in fast‐spiking, 29.3 ± 18 ms in regular‐spiking and 99.7 ± 42 ms in burst‐firing cells. Axo‐axonic cells never establish autapses, and the fast‐spiking variety showed narrow (HW: 3.9 ± 0.7 ms) spike AHPs with only one AHP minimum (TTP: 0.9 ± 0.1 ms). When challenged with GABAA receptor modulators, spike AHPs in basket and bistratified cells were enhanced by zolpidem (HW by 18.4 ± 6.2 % in 10:15 cells tested), diazepam (45.2 ± 0.5 %, 6:7), etomidate (43.9 ± 36 %, 6:8) and pentobarbitone sodium (41 %, 1:1), and were depressed by bicuculline (‐41 ± 5.7 %, 5:8) and picrotoxin (‐54 %, 1:1), and the enhancement produced by zolpidem was reduced by flumazenil (‐31 ± 13 %, relative to the AHP HW during exposure to zolpidem, 3:4). Neuronal excitability was modulated in parallel. The spike AHPs of three axo‐axonic cells tested showed no sensitivity to etomidate, pentobarbitone or diazepam. Interneurone‐to‐interneurone inhibitory postsynaptic potentials (IPSPs), studied with dual intracellular recordings, had time courses resembling those of the spike AHPs. The IPSP HW was 13.4 ± 2.8 ms in fast‐spiking (n= 16) and 28.7 ± 5.8 ms in regular‐spiking/burst‐firing cells (n= 6), and the benzodiazepine1‐selective modulator zolpidem strongly enhanced these IPSPs (45 ± 28 %, n= 5). Interneurones with spike AHPs affected by the GABAA receptor ligands exhibited 3.8 ± 1.9 close autaptic appositions. In three basket cells studied at the ultrastructural level 6 of 6, 1 of 2 and 1 of 2 close appositions were confirmed as autapses. Therefore, in the hippocampus autaptic connections contribute to spike AHPs in many interneurones. These autapses influence neuronal firing and responses to GABAA receptor ligands.
Temperature and redox state dependence of native Kv2.1 currents in rat pancreatic β‐cells
Authors: MacDonald Patrick E. , Salapatek Anne Marie F. , Wheeler Michael B.
Published: February 2003

In pancreatic β‐cells, voltage‐dependent K+ (Kv) channels repolarise glucose‐stimulated action potentials. Kv channels are therefore negative regulators of Ca2+ entry and insulin secretion. We have recently demonstrated that Kv2.1 mediates the majority of β‐cell voltage‐dependent outward K+ current and now investigate the function of native β‐cell Kv2.1 channels at near‐physiological temperatures (32‐35 °C). While β‐cell voltage‐dependent outward K+ currents inactivated little at room temperature, both fast‐inactivation (111.5 ± 14.3 ms) and slow‐inactivation (1.21 ± 0.12 s) was observed at 32‐35 °C. Kv2.1 mediates the fast‐inactivating current observed at 32‐35 °C, since it could be selectively ablated by expression of a dominant‐negative Kv2.1 construct (Kv2.1N). The surprising ability of Kv2.1N to selectively remove the fast‐inactivating component, together with its sensitivity to tetraethylammonium (TEA), demonstrate that this component is not mediated by the classically fast‐inactivating and TEA‐resistant channels such as Kv1.4 and 4.2. Increasing the intracellular redox state by elevating the cytosolic NADPH/NADP+ ratio from 1/10 to 10/1 increased the rates of both fast‐ and slow‐inactivation. In addition, increasing the intracellular redox state also increased the relative contribution of the fast‐inactivation component from 38.8 ± 2.1 % to 55.9 ± 1.8 %. The present study suggests that, in β‐cells, Kv2.1 channels mediate a fast‐inactivating K+ current at physiological temperatures and may be regulated by the metabolic generation of NADPH.
Branching of
Escherichia coli
Cells Arises from Multiple Sites of Inert Peptidoglycan
Authors: de Pedro Miguel A. , Young Kevin D. , Höltje Joachim-Volker , Schwarz Heinz
Published: January 2003

Some strains of
Escherichia coli
defective for
dacA
, the gene coding for penicillin-binding protein 5, exhibit a strong branching phenotype when cell division is blocked. Since such branch formation implies a differentiation of polar caps at ectopic locations in the cell envelope, we analyzed murein segregation and observed a strong correlation between areas of inert murein and these morphological anomalies. In particular, the tips of branches exhibited the same properties as those described for polar caps of wild-type cells, i.e., the synthesis and turnover of murein were inhibited. Also, the mobility of cell envelope proteins was apparently constrained in areas with morphological defects. Polar regions of branching cells and sacculi had aberrant morphologies with a very high frequency. Of special interest was that areas of inert murein at polar caps were often split by areas of active synthesis, a situation unlike that observed in wild-type cells. These observations suggest that in
dacA
mutants, branches and other morphological anomalies may arise from split polar caps or by de novo generation of new poles built around inert peptidoglycan patches in the side walls of the cell.
Molecular Analysis of the Gene Encoding a Novel Cold-Adapted Chitinase (ChiB) from a Marine Bacterium,
Alteromonas
sp. Strain O-7
Authors: Orikoshi Hideyuki , Baba Nao , Nakayama Shigenari , Kashu Hiroshi , Miyamoto Katsushiro , Yasuda Masahide , Inamori Yoshihiko , Tsujibo Hiroshi
Published: January 2003

The chitinase B (ChiB) secreted by
Alteromonas
sp. strain O-7 was purified, and the corresponding gene (
chiB
) was cloned and sequenced. The open reading frame of the
chiB
gene encodes a protein of 850 amino acids with a calculated molecular mass of 90,223 Da. ChiB is a modular enzyme consisting of two reiterated domains and a catalytic domain belonging to chitinase family 18. The reiterated domains are composed of chitin-binding domain (ChtBD) type 3 and two fibronectin type III (Fn3)-like domains. Expression plasmids coding for ChiB or deletion derivatives thereof were constructed in
Escherichia coli
. Deletion analysis showed that the ChtBD of ChiB plays an important role in efficient hydrolysis of insoluble chitin. The optimum pH and temperature of ChiB were 6.0 and 30°C, respectively. The enzyme showed relatively high catalysis, even at low temperatures close to 0°C, and remarkable thermal lability compared to ChiA and ChiC, which are the mesophilic chitinases of the same strain. The
k
cat
/
K
m
value for the ChiB reaction at 10°C was about 4.7 times higher than that of ChiC. These results suggest that ChiB is a cold-adapted enzyme. The RNA transcript of
chiB
was induced by 1% GlcNAc, and along with a rise in temperature, the RNA transcript showed a tendency to decrease. Thus, among the ChiA, ChiB, and ChiC chitinases, production of ChiB may be advantageous for the strain, allowing it to easily acquire nutrients from chitin and to survive in cold environments.
YfiK from
Escherichia coli
Promotes Export of
O
-Acetylserine and Cysteine
Authors: Franke Isabel , Resch Armin , Daßler Tobias , Maier Thomas , Böck August
Published: January 2003

yfiK
was discovered as a gene augmenting cysteine production when it was overexpressed in an industrial
Escherichia coli
production strain. The gene product is an integral membrane protein with about six predicted transmembrane helices; it belongs to the RhtB family of export proteins. YfiK overproduction from a plasmid leads to drastic and parallel secretion of
O
-acetylserine and cysteine into the medium but only when the organism possesses a serine transacetylase that is feedback insensitive to cysteine. Externally provided
O
-acetylserine obviated this requirement for cysteine secretion both in the
yfiK
-carrying transformant and in the wild type. A
ΔyfiK
mutant did not show any phenotype, and it exported
O
-acetylserine and cysteine when transformed with a plasmid carrying
ydeD,
a previously characterized, alternate
O
-acetylserine/cysteine exporter. Since a
ydeD-yfiK
double mutant showed the same pattern, it appears that YfiK and YdeD act independently. The necessity for the cell to regulate the size of the internal pool of
O
-acetylserine via synthesis of exporter proteins could be connected to the fact that this compound (when supplied externally) inhibits growth. Overexpression of either
ydeD
or
yfiK
leads to alleviation of this inhibition paralled by increased resistance to azaserine, which is an analog of
O
-acetylserine.
In Vivo Assay for Low-Activity Mutant Forms of
Escherichia coli
Ribonucleotide Reductase
Authors: Ekberg Monica , Birgander Pernilla , Sjöberg Britt-Marie
Published: January 2003

Ribonucleotide reductase (RNR) catalyzes the essential production of deoxyribonucleotides in all living cells. In this study we have established a sensitive in vivo assay to study the activity of RNR in aerobic
Escherichia coli
cells. The method is based on the complementation of a chromosomally encoded nonfunctional RNR with plasmid-encoded RNR. This assay can be used to determine in vivo activity of RNR mutants with activities beyond the detection limits of traditional in vitro assays.
E. coli
RNR is composed of two homodimeric proteins, R1 and R2. The R2 protein contains a stable tyrosyl radical essential for the catalysis that takes place at the R1 active site. The three-dimensional structures of both proteins, phylogenetic studies, and site-directed mutagenesis experiments show that the radical is transferred from the R2 protein to the active site in the R1 protein via a radical transfer pathway composed of at least nine conserved amino acid residues. Using the new assay we determined the in vivo activity of mutants affecting the radical transfer pathway in RNR and identified some residual radical transfer activity in two mutant R2 constructs (D237N and W48Y) that had previously been classified as negative for enzyme activity. In addition, we show that the R2 mutant Y356W is completely inactive, in sharp contrast to what has previously been observed for the corresponding mutation in the mouse R2 enzyme.
Interactions between Phage-Shock Proteins in
Escherichia coli
Authors: Adams Hendrik , Teertstra Wieke , Demmers Jeroen , Boesten Rolf , Tommassen Jan
Published: January 2003

Expression of the
pspABCDE
operon of
Escherichia coli
is induced upon infection by filamentous phage and by many other stress conditions, including defects in protein export. Expression of the operon requires the alternative sigma factor σ
54
and the transcriptional activator PspF. In addition, PspA plays a negative regulatory role, and the integral-membrane proteins PspB and PspC play a positive one. In this study, we investigated whether the suggested protein-protein interactions implicated in this complex regulatory network can indeed be demonstrated. Antisera were raised against PspB, PspC, and PspD, which revealed, in Western blotting experiments, that PspC forms stable sodium dodecyl sulfate-resistant dimers and that the hypothetical
pspD
gene is indeed expressed in vivo. Fractionation experiments showed that PspD localizes as a peripherally bound inner membrane protein. Cross-linking studies with intact cells revealed specific interactions of PspA with PspB and PspC, but not with PspD. Furthermore, affinity-chromatography suggested that PspB could bind PspA only in the presence of PspC. These data indicate that regulation of the
psp
operon is mediated via protein-protein interactions.
CDP-2,3-Di
-O-
Geranylgeranyl-
sn
-Glycerol:
l
-Serine
O
-Archaetidyltransferase (Archaetidylserine Synthase) in the Methanogenic Archaeon
Methanothermobacter thermautotrophicus
Authors: Morii Hiroyuki , Koga Yosuke
Published: January 2003

CDP-2,3-di
-O-
geranylgeranyl-
sn
-glycerol:
l
-serine
O
-archaetidyltransferase (archaetidylserine synthase) activity in cell extracts of
Methanothermobacter thermautotrophicus
cells was characterized. The enzyme catalyzed the formation of unsaturated archaetidylserine from CDP-unsaturated archaeol and
l
-serine. The identity of the reaction products was confirmed by thin-layer chromatography, fast atom bombardment-mass spectrum analysis, and chemical degradation. The enzyme showed maximal activity in the presence of 10 mM Mn
2+
and 1% Triton X-100. Among various synthetic substrate analogs, both enantiomers of CDP-unsaturated archaeols with ether-linked geranylgeranyl chains and CDP-saturated archaeol with ether-linked phytanyl chains were similarly active toward the archaetidylserine synthase. The activity on the ester analog of the substrate was two to three times higher than that on the corresponding ether-type substrate. The activity of
d
-serine with the enzyme was 30% of that observed for
l
-serine. A trace amount of an acid-labile, unsaturated archaetidylserine intermediate was detected in the cells by a pulse-labeling experiment. A gene (MT1027) in
M. thermautotrophicus
genome annotated as the gene encoding phosphatidylserine synthase was found to be homologous to
Bacillus subtilis pssA
but not to
Escherichia coli pssA.
The substrate specificity of phosphatidylserine synthase from
B. subtilis
was quite similar to that observed for the
M. thermautotrophicus
archaetidylserine synthase, while the
E. coli
enzyme had a strong preference for CDP-1,2-diacyl-
sn
-glycerol. It was concluded that
M. thermautotrophicus
archaetidylserine synthase belongs to subclass II phosphatidylserine synthase (
B. subtilis
type) on the basis of not only homology but also substrate specificity and some enzymatic properties. The possibility that a gene encoding the subclass II phosphatidylserine synthase might be transferred from a bacterium to an ancestor of methanogens is discussed.
Effect of Intracellular pH on Rotational Speed of Bacterial Flagellar Motors
Authors: Minamino Tohru , Imae Yasuo , Oosawa Fumio , Kobayashi Yuji , Oosawa Kenji
Published: January 2003

Weak acids such as acetate and benzoate, which partially collapse the transmembrane proton gradient, not only mediate pH taxis but also impair the motility of
Escherichia coli
and
Salmonella
at an external pH of 5.5. In this study, we examined in more detail the effect of weak acids on motility at various external pH values. A change of external pH over the range 5.0 to 7.8 hardly affected the swimming speed of
E. coli
cells in the absence of 34 mM potassium acetate. In contrast, the cells decreased their swimming speed significantly as external pH was shifted from pH 7.0 to 5.0 in the presence of 34 mM acetate. The total proton motive force of
E. coli
cells was not changed greatly by the presence of acetate. We measured the rotational rate of tethered
E. coli
cells as a function of external pH. Rotational speed decreased rapidly as the external pH was decreased, and at pH 5.0, the motor stopped completely. When the external pH was returned to 7.0, the motor restarted rotating at almost its original level, indicating that high intracellular proton (H
+
) concentration does not irreversibly abolish flagellar motor function. Both the swimming speeds and rotation rates of tethered cells of
Salmonella
also decreased considerably when the external pH was shifted from pH 7.0 to 5.5 in the presence of 20 mM benzoate. We propose that the increase in the intracellular proton concentration interferes with the release of protons from the torque-generating units, resulting in slowing or stopping of the motors.
Analysis of Residues Determining Specificity ofVibrio choleraeTonB1 for Its Receptors
Authors: Mey Alexandra R. , Payne Shelley M.
Published: January 2003

In gram-negative organisms, high-affinity transport of iron substrates requires energy transduction to specific outer membrane receptors by the TonB-ExbB-ExbD complex.Vibrio choleraeencodes two TonB proteins, one of which, TonB1, recognizes only a subset ofV.choleraeTonB-dependent receptors and does not facilitate transport throughEscherichia colireceptors. To investigate the receptor specificity exhibited byV.choleraeTonB1, chimeras were created betweenV.choleraeTonB1 andE.coliTonB. The activities of the chimeric TonB proteins in iron utilization assays demonstrated that the C-terminal one-third of either TonB confers the receptor specificities associated with the full-length TonB. Single-amino-acid substitutions near the C terminus ofV.choleraeTonB1 were identified that allowed TonB1 to recognizeE.colireceptors and at least oneV.choleraeTonB2-dependent receptor. This indicates that the very C-terminal end ofV.choleraeTonB1 determines receptor specificity. The regions of the TonB-dependent receptors involved in specificity for a particular TonB protein were investigated in experiments involving domain switching betweenV.choleraeandE.colireceptors exhibiting different TonB specificities. Switching the conserved TonB box heptapeptides at the N termini of these receptors did not alter their TonB specificities. However, replacing the amino acid immediately preceding the TonB box inE.colireceptors with an aromatic residue allowed these receptors to useV.choleraeTonB1. Further, site-directed mutagenesis of the TonB box −1 residue in aV.choleraeTonB2-dependent receptor demonstrated that a large hydrophobic amino acid in this position promotes recognition ofV.choleraeTonB1. These data suggest that the TonB box −1 position controls productive interactions withV.choleraeTonB1.
Molecular Genetic Analysis of a Group AStreptococcusOperon Encoding Serum Opacity Factor and a Novel Fibronectin-Binding Protein, SfbX
Authors: Jeng Arthur , Sakota Varja , Li Zhongya , Datta Vivekananda , Beall Bernard , Nizet Victor
Published: January 2003

The group AStreptococcus(GAS)sofgene encodes the serum opacity factor protein, which is capable of opacifying mammalian sera and binding at least two host proteins, fibronectin and fibrinogen. Thesofgene exists in approximately 50% of clinical isolates, and there is a classical association of so-called nephritogenic strains with the opacity factor-positive phenotype. In both a typeemm49strain and a typeemm12strain, the sequences upstream of the 5′ end ofsofand downstream of the putative terminator were determined to be nearly identical to a region in the M type 1 genome approximately 10 kb upstream of theemm1gene. This close genetic linkage is likely reflected in the strict correlation of opacity factor phenotype with specificemmgenotypes. A new fibronectin-binding protein gene,sfbX, was discovered immediately downstream ofsofinemm12andemm49strains and in several othersof-positive strains. ThesofandsfbXgenes were found to be expressed on the same transcription unit, which was correlated with the putative promoter and rho-independant terminator sequences that flank these two genes. ThesfbXgenes from differentemmtypes are predicted to encode ∼650-residue surface-bound proteins sharing 89 to 92% sequence identity. SfbX residues approximately 1 to 480 are not highly similar to those of other known proteins, with the closest match being theStaphylococcus aureuscoagulase protein. The remaining portions of these proteins (residues 481 to 650) contain four putative fibronectin-binding repeats highly similar to those of other streptococcal fibronectin-binding proteins and a potential LP(X)SG cell wall anchor motif. Targeted in-frame allelic-exchange mutagenesis, complementation, and heterologous-expression studies found that serum opacification is encoded bysofalone and thatsfbXencodes a fibronectin-binding function. A recombinant SfbX protein was found to bind immobilized fibronectin and to partially inhibit GAS adherence to fibronectin. ThesfbXgene was found to be present only insof-positive strains, and together these genes could influence the spectrum of tissues colonized bysof-positive GAS.
In Vitro and In Vivo Functional Activity ofChlamydiaMurA, a UDP-N-Acetylglucosamine Enolpyruvyl Transferase Involved in Peptidoglycan Synthesis and Fosfomycin Resistance
Authors: McCoy Andrea J. , Sandlin Robin C. , Maurelli Anthony T.
Published: January 2003

Organisms ofChlamydiaspp. are obligate intracellular, gram-negative bacteria with a dimorphic developmental cycle that takes place entirely within a membrane-bound vacuole termed an inclusion. The chlamydial anomaly refers to the fact that cell wall-active antibiotics inhibitChlamydiagrowth and peptidoglycan (PG) synthesis genes are present in the genome, yet there is no biochemical evidence for synthesis of PG. In this work, we undertook a genetics-based approach to reevaluate the chlamydial anomaly by characterizing MurA, a UDP-N-acetylglucosamine enolpyruvyl transferase that catalyzes the first committed step of PG synthesis. ThemurAgene fromChlamydiatrachomatisserovar L2 was cloned and placed under the control of the arabinose-inducible, glucose-repressiblearapromoter and transformed intoEscherichia coli. After transduction of a lethal ΔmurAmutation into the strain, viability of theE. colistrain became dependent upon expression of theC. trachomatis murA.DNA sequence analysis ofmurAfromC. trachomatispredicted a cysteine-to-aspartate change in a key residue within the active site of MurA. InE. coli, the same mutation has previously been shown to cause resistance to fosfomycin, a potent antibiotic that specifically targets MurA. In vitro activity of the chlamydial MurA was resistant to high levels of fosfomycin. Growth ofC. trachomatiswas also resistant to fosfomycin. Moreover, fosfomycin resistance was imparted to theE. colistrain expressing the chlamydialmurA. Conversion ofC. trachomatiselementary bodies to reticulate bodies and cell division are correlated with expression ofmurAmRNA. mRNA frommurB, the second enzymatic reaction in the PG pathway, was also detected duringC. trachomatisinfection. Our findings, as well as work from other groups, suggest that a functional PG pathway exists inChlamydiaspp. We propose that chlamydial PG is essential for progression through the developmental cycle as well as for cell division. Elucidating the existence of PG inChlamydiaspp. is of significance for the development of novel antibiotics targeting the chlamydial cell wall.
QscR, a LysR-Type Transcriptional Regulator and CbbR Homolog, Is Involved in Regulation of the Serine Cycle Genes in
Methylobacterium extorquens
AM1
Authors: Kalyuzhnaya Marina G. , Lidstrom Mary E.
Published: January 2003

A new gene,
qscR
, encoding a LysR-type transcriptional regulator that is a homolog of CbbR, has been characterized from the facultative methylotroph
Methylobacterium extorquens
AM1 and shown to be the major regulator of the serine cycle, the specific C
1
assimilation pathway. The
qscR
mutant was shown to be unable to grow on C
1
compounds, and it lacked the activity of serine-glyoxylate aminotransferase, a key enzyme of the serine cycle. Activities of other serine cycle enzymes were decreased during growth on C
1
compounds compared to the activities found in wild-type
M. extorquens
AM1. Promoter fusion assays, as well as reverse transcription-PCR assays, have indicated that the serine cycle genes belong to three separate transcriptional units,
sga-hpr-mtdA-fch
,
mtkA-mtkB-ppc-mcl
, and
gly
. Gel retardation assays involving the purified QscR have demonstrated the specific binding of QscR to the DNA regions upstream of
sga
,
mtkA
,
gly
, and
qscR
. We conclude that QscR acts as a positive transcriptional regulator of most of the serine cycle enzymes and also as an autorepressor.
Roles of NhaA, NhaB, and NhaD Na
+
/H
+
Antiporters in Survival of
Vibrio cholerae
in a Saline Environment
Authors: Herz Katia , Vimont Sophie , Padan Etana , Berche Patrick
Published: January 2003

Vibrio cholerae
, the causative agent of cholera, is a normal inhabitant of aquatic environments, where it survives in a wide range of conditions of pH and salinity. In this work, we investigated the role of three Na
+
/H
+
antiporters on the survival of
V. cholerae
in a saline environment. We have previously cloned the Vc-
nhaA
gene encoding the
V. cholerae
homolog of
Escherichia coli
. Here we identified two additional antiporter genes, designated Vc-
nhaB
and Vc
-nhaD
, encoding two putative proteins of 530 and 477 residues, respectively, highly homologous to the respective antiporters of
Vibrio
species and
E. coli.
We showed that both Vc-NhaA and Vc-NhaB confer Na
+
resistance and that Vc-NhaA displays an antiport activity in
E. coli
, which is similar in magnitude, kinetic parameters, and pH regulation to that of
E. coli
NhaA. To determine the roles of the Na
+
/H
+
antiporters in
V. cholerae
, we constructed
nhaA
,
nhaB
, and
nhaD
mutants (single, double, and triple mutants). In contrast to
E. coli
, the inactivation of the three putative antiporter genes (Vc-
nhaABD)
in
V. cholerae
did not alter the bacterial exponential growth in the presence of high Na
+
concentrations and had only a slight effect in the stationary phase. In contrast, a pronounced and similar Li
+
-sensitive phenotype was found with all mutants lacking Vc-
nhaA
during the exponential phase of growth and also with the triple mutant in the stationary phase of growth. By using 2-
n
-nonyl-4-hydroxyquinoline
N
-oxide, a specific inhibitor of the electron-transport-linked Na
+
pump NADH-quinone oxidoreductase (NQR), we determined that in the absence of NQR activity, the Vc-NhaA Na
+
/H
+
antiporter activity becomes essential for the resistance of
V. cholerae
to Na
+
at alkaline pH. Since the ion pump NQR is Na
+
specific, we suggest that its activity masks the Na
+
/H
+
but not the Li
+
/H
+
antiporter activities. Our results indicate that the Na
+
resistance of the human pathogen
V. cholerae
requires a complex molecular system involving multiple antiporters and the NQR pump.