Search PubMed⌕ Search

Biomedical subjects

W C Bowman

Publications and source records attributed to W C Bowman.

At least 19 recordsLinked to original sources
PubMed1998

A bacterial ATP-dependent, enhancer binding protein that activates the housekeeping RNA polymerase.

A commonly accepted view of gene regulation in bacteria that has emerged over the last decade is that promoters are transcriptionally activated by one of two general mechanisms. The major type involves activator proteins that bind to DNA adjacent to where the RNA polymerase (RNAP) holoenzyme binds, usually assisting in recruitment of the RNAP to the promoter. This holoenzyme uses the housekeeping sigma70 or a related factor, which directs the core RNAP to the promoter and assists in melting the DNA near the RNA start site. A second type of mechanism involves the alternative sigma factor (called sigma54 or sigmaN) that directs RNAP to highly conserved promoters. In these cases, an activator protein with an ATPase function oligomerizes at tandem sites far upstream from the promoter. The nitrogen regulatory protein (NtrC) from enteric bacteria has been the model for this family of activators. Activation of the RNAP/sigma54 holoenzyme to form the open complex is mediated by the activator, which is tethered upstream. Hence, this class of protein is sometimes called the enhancer binding protein family or the NtrC class. We describe here a third system that has properties of each of these two types. The NtrC enhancer binding protein from the photosynthetic bacterium, Rhodobacter capsulatus, is shown in vitro to activate the housekeeping RNAP/sigma70 holoenzyme. Transcriptional activation by this NtrC requires ATP binding but not hydrolysis. Oligomerization at distant tandem binding sites on a supercoiled template is also necessary. Mechanistic and evolutionary questions of these systems are discussed.

Adenosine Triphosphate↗
PubMed1998

Translational activation by an NtrC enhancer-binding protein.

The Rhodobacter capsulatus NtrC protein is a bacterial enhancer-binding protein that activates the transcription of at least five genes by a mechanism that does not require the RpoN RNA polymerase sigma factor. The nifR3-ntrB-ntrC operon in R. capsulatus codes for the nitrogen-sensing two component regulators NtrB and NtrC, as well as for NifR3, a protein of unknown function that is highly conserved in both prokaryotes and eukaryotes. Evidence of a unique translational control of NifR3 mediated directly by the NtrC enhancer-binding protein is reported. The nifR3-ntrB-ntrC operon is expressed from a single promoter upstream of nifR3 with the levels of transcript equivalent in wild-type and ntrC mutants under nitrogen-limited or nitrogen-sufficient conditions. LacZ reporter analyses of this operon and immunological quantitation of NifR3 and NtrC demonstrate that, unlike NtrC levels which remain constant, production of NifR3 is at least ten to 40-fold reduced in NtrC- strains. NifR3 is increased at least fivefold upon nitrogen limitation whereas NtrC production is constitutive. Surprisingly, the purified NtrC protein binds cooperatively to the nifR3 promoter region in vitro at two sets of tandem binding sites centered at +1 and -81 nucleotides relative to the transcriptional start site. Deletion analysis demonstrates that the upstream tandem sites are essential for nitrogen and NtrC-dependent production of NifR3 in vivo , but are not necessary for nifR3 transcription. These experiments indicate that NtrC stimulates the translation of the NifR3 messenger RNA while tethered to the promoter DNA. This is in contrast to five other promoters (nifA1, nifA2, glnB, mopA and anfA) in R. capsulatus which are transcriptionally activated by NtrC bound to one set of tandem binding sites that are centered greater than 100 bp upstream of the transcriptional start site.

Bacterial Proteins↗
PubMed1997

Characterization of the Rhodobacter capsulatus housekeeping RNA polymerase. In vitro transcription of photosynthesis and other genes.

To begin to characterize biochemically the transcriptional activation systems in photosynthetic bacteria, the Rhodobacter capsulatus RNA polymerase (RNAP) that contains the sigma70 factor (R. capsulatus RNAP/sigma70) was purified and characterized using two classical sigma70 type promoters, the bacteriophage T7A1 and the RNA I promoters. Transcription from these promoters was sensitive to rifampicin, RNase, and monoclonal antibody 2G10 (directed against the Escherichia coli sigma70 subunit). Specific transcripts were detected in vitro for R. capsulatus cytochrome c2 (cycA) and fructose-inducible (fruB) promoters and genes induced in photosynthesis (puf and puc) and bacteriochlorophyll biosynthesis (bchC). Alignment of these natural promoters activated by R. capsulatus RNAP/sigma70 indicated a preference for the sequence TTGAC at the -35 region for strong in vitro transcription. To test the -35 recognition pattern, the R. capsulatus nifA1 promoter, which exhibits only three of the five consensus nucleotides at the -35 region, was mutated to four and five of the consensus nucleotides. Although the nifA1 wild type promoter showed no transcription, the double mutated promoter exhibited high levels of in vitro transcription by the purified R. capsulatus RNAP/sigma70 enzyme. Similarities and differences between the RNAPs and the promoters of R. capsulatus and E. coli are discussed.

Bacterial Proteins↗
PubMed1996

In vitro reconstitution and characterization of the Rhodobacter capsulatus NtrB and NtrC two-component system.

Enhancer-dependent transcription in enteric bacteria depends upon an activator protein that binds DNA far upstream from the promoter and an alternative sigma factor (sigma 54) that binds with the core RNA polymerase at the promoter. In the photosynthetic bacterium Rhodobacter capsulatus, the NtrB and NtrC proteins (RcNtrB and RcNtrC) are putative members of a two-component system that is novel because the enhancer-binding RcNtrC protein activates transcription of sigma 54-independent promoters. To reconstitute this putative two-component system in vitro, the ReNtrB protein was overexpressed in Escherichia coli and purified as a maltose-binding protein fusion (MBP-RcNtrB). MBP-RcNtrB autophosphorylates in vitro to the same steady state level and with the same stability as the Salmonella typhimurium NtrB (StNtrB) protein but at a lower initial rate. MBP-RcNtrB autophosphorylates the S.typhimurium NtrC (St-NtrC) and RcNtrC proteins in vitro. The enteric NtrC protein is also phosphorylated in vivo by RcNtrB because plasmids that encode either RcNtrB or MBP-Rc-NtrB activate transcription of an NtrC-dependent nifL-lacZ fusion. The rate of phosphotransfer to RcNtrC and autophosphatase activity of phosphorylated RcNtrC (RcNtrC---P) are comparable to the StNtrC protein. However, the RcNtrC protein appears to be a specific RcNtrB P phosphatase since RcNtrC is not phosphorylated by small molecular weight phosphate compounds or by the StNtrB protein. RcNtrC forms a dimer in solution, and RcNtrC - P binds the upstream tandem binding sites of the g1nB promoter 4-fold better than the unphos-phorylated RcNtrC protein, presumably due to oligomerization of RcNtrC -P. Therefore, the R. capsulatus NtrB and NtrC proteins form a two-component system similar to other NtrC-like systems, where specific Rc- NtrB phosphotransfer to the RcNtrC protein results in increased oligoinerization at the enhancer but with subsequent activation of a sigma 54-independent promoter.

Bacterial Proteins↗
PubMed1995

L-arginine-nitric oxide pathway involvement in the nerve-evoked relaxant responses of the 5-HT precontracted chick isolated upper oesophagus.

1. The effects of tetrodotoxin (TTx) and the selective nitric oxide synthase (NOS) inhibitor, NG-nitro-L-arginine methyl ester (L-NAME) on relaxant responses of the 5-HT precontracted chick isolated upper oesophagus to electrical field stimulation (EFS: 25-30V, 5 Hz for 10 s, 1 ms pulse width every 100 s) were investigated; the oesophagus was mounted under 1 g tension in Krebs solution containing 1 microM atropine. Appropriate tissue sections (30 microM thickness) of the chick oesophagus were also processed for NADPH-diaphorase histochemistry. 2. TTx (2 microM) and L-NAME (100-200 microM) inhibited the relaxant responses of the 5-HT precontracted chick oesophagus to EFS in a concentration-dependent manner; L-arginine (0.5-1 mM), but not D-arginine (0.5-1 mM), reversed the inhibition by L-NAME. In the absence of atropine and muscle tone, EFS produced contractile responses of the chick oesophagus that were completely abolished by 1 microM atropine, which also blocked the contractile response to acetylcholine (50 microM). 3. Under light microscopy, NADPH-diaphorase histochemistry confirmed the presence of nitric oxide synthase (NOS)-containing neurones and nerve fibres in the chick oesophagus. 4. The relaxant responses of the 5-HT precontracted chick isolated upper oesophagus to EFS are, therefore, mediated via the stimulation of non-adrenergic non-cholinergic nerves. These are likely to correspond to the histochemically identified NOS-containing neurones involved, presumably, in the synthesis and release of nitric oxide as the relaxant (inhibitory) neurotransmitter in this avian smooth muscle.

Animals↗
PubMed1994

Interactions between suxamethonium and non-depolarizing neuromuscular blocking drugs.

In anaesthetized cats, we have confirmed that previously injected suxamethonium potentiates non-depolarizing neuromuscular blocking drugs whereas, when injected during the block, suxamethonium antagonizes the paralysis. We have attempted to explain these interactions by studying the effects of suxamethonium on extracellularly recorded nerve ending waveforms that correspond to the ionic currents in the mouse triangularis sterni isolated nerve-muscle preparation. The preparations were paralysed with mu-conotoxin (obtained from the cone snail), which is believed to act by selectively blocking sodium channels in muscle, and which therefore should not interfere with currents at the nerve endings. Suxamethonium, in concentrations of 0.5-300 mumol litre-1, produced a concentration-dependent increase in the amplitude of the waveform corresponding to the inward calcium current evoked by a nerve impulse. This effect did not occur in the presence of tubocurarine, suggesting that suxamethonium, which is a nicotinic agonist, may have been acting on a nicotinic receptor on the nerve endings that is coupled to the voltage-operated calcium channels. The inward calcium current is believed to be responsible for neurotransmitter (acetylcholine) release. It is concluded, therefore, that its enhancement by suxamethonium contributes to the ability of this drug to reverse non-depolarizing block. Suxamethonium also exerted complex effects on the waveform corresponding to the outward flowing calcium-activating potassium current at the nerve endings, but no effect was observed in this isolated nerve-muscle preparation that could obviously explain the ability of suxamethonium to potentiate subsequently injected non-depolarizing blocking drugs.

4-Aminopyridine↗
PubMed1993

Prejunctional action of neostigmine on mouse neuromuscular preparations.

We have studied the effects of neostigmine on the mouse diaphragm and triangularis sterni isolated nerve-muscle preparations. Mechanical responses of the muscle, end-plate potentials and miniature end-plate potentials, and extracellularly recorded nerve ending currents were recorded. In the mouse diaphragm nerve-muscle preparations, neostigmine 1 mumol litre-1 continued to produce some antagonism of tubocurarine-induced block after cholinesterase had been inactivated completely by diisopropyl fluorophosphate 22 mumol litre-1. In the mouse triangularis sterni preparation, neostigmine 0.1-1 mumol litre-1 increased the quantal content of the end-plate potential in a concentration-dependent manner. This effect appeared to be sufficient to account for the cholinesterase-independent antagonistic action to tubocurarine under the conditions of the experiments. Neostigmine 1-100 mumol litre-1 depressed the amplitude of the K+ currents of the perineural waveforms in a concentration-dependent manner, and this may account for its ability to increase the quantal content of the end-plate potential. Although inhibition of acetyl-cholinesterase is the main mechanism of action of neostigmine, the drug also exerts an additional direct action on motor nerve endings to block the delayed rectifier K+ channels and enhance transmitter release. This effect occurred at clinically relevant concentrations of neostigmine. Physostigmine and pyridostigmine did not possess this additional action.

Animals↗
PubMed1991

The effects of a 16-N-homopiperidino analogue of vecuronium on neuromuscular transmission in anaesthetized cats, pigs, dogs and monkeys, and in isolated preparations.

Org 9991, a 16-N-homopiperidinium substituted vecuronium analogue, has been tested for neuromuscular blocking activity in anaesthetized cats, pigs, dogs and monkeys, and in isolated nerve-muscle preparations. Org 9991 exhibited non-depolarizing neuromuscular blocking activity of the competitive type, being reversible by neostigmine and showing no endplate channel blocking action in isolated preparations. In cats, 50% vagal block was observed at doses of Org 9991 approximately 10 times those producing 50% neuromuscular block; no ganglion block was seen at these doses. Effects on blood pressure or heart rate at 90% twitch blocking doses were either minor or absent. The potency and time course of action of Org 9991 remained similar in all four species: i.e. 90% block at ca 200-300 micrograms kg-1; onset time ca 1.2-1.9 min; duration 90% ca 4.5-8.9 min. This study suggests that 16-N-homopiperidinium analogues of vecuronium may provide leads in the quest for a potent non-depolarizing replacement for suxamethonium.

Androstanols↗
PubMed1989

Effects of a new neuromuscular blocking agent (Org 9426) in anaesthetized cats and pigs and in isolated nerve-muscle preparations.

The effects of Org 9426 (the 2-morpholino, 3-hydroxy, 16N-allyl pyrrolidino analogue of vecuronium) were studied in anaesthetized cats and pigs and in isolated nerve--muscle preparations using tension and intracellular recording techniques. In isolated preparations, the effects of Org 9426 were antagonized by neostigmine. No contracture of the chick muscle preparation occurred. Org 9426 reduced the amplitude of endplate currents (EPC) in rat and snake muscle, but had no major effects on EPC decay characteristics, indicating a lack of endplate channel blocking action. In anaesthetized animals, no fasciculations were observed and the neuromuscular block was associated with tetanic and train-of-four fade and was antagonized by neostigmine. In anaesthetized cats and pigs, Org 9426 was approximately 20% as potent as vecuronium, its onset of action was twice as rapid as that of vecuronium in the cat and its duration of action was similar to that of vecuronium in both cats and pigs. It blocked the bradycardia produced by vagal stimulation only in doses greater than those necessary to produce neuromuscular block (ratios 7.2 in the cat and 4.4 in the pig--10-14% of the corresponding ratios for vecuronium). Ganglion block was seen only at doses several times those producing vagal block. In general the effects of Org 9426 on the cardiovascular system were slight, a small depressor effect occurring at high doses in the cat. The 17-hydroxy analogue, the potential metabolite of Org 9426, was approximately 20 times less potent than Org 9426 and is thus unlikely to contribute to the neuromuscular block produced by the parent compound.

Action Potentials↗
PubMed1989

A comparison of the neuromuscular blocking and autonomic effects of two new short-acting muscle relaxants with those of succinylcholine in the anesthetized cat and pig.

The actions of two new steroidal nondepolarizing neuromuscular blocking agents, structurally related to vecuronium, have been compared with those of succinylcholine in anesthetized cats and pigs. Both new compounds (Org 7617 and Org 9616) exhibited properties typical of nondepolarizing relaxants in each species. Org 9616 was one-fifth (ED50 cat tibialis 154 micrograms.kg-1) and Org 7617 was one-tenth (ED50 cat tibialis 287 micrograms.kg-1) as potent as vecuronium as a neuromuscular blocking drug. Both compounds produced rapidly developing muscle relaxation in cats that, like that of succinylcholine, was transient in time course (onset/duration of action--tibialis anterior muscle: Org 7617 1.6/3.9 min; Org 9616 1.5/4.3 min; succinylcholine 1.7/5.7 min). In pigs that were used as a predictor of duration of action in humans, both Org 7617 and Org 9616 also produced short-lived neuromuscular blockade. The neuromuscular blocks produced by Org 7617 and Org 9616 were readily reversed by neostigmine. Both compounds blocked the heart rate responses to vagal stimulation at doses higher than those required to produce neuromuscular block. The vagal:neuromuscular blocking ratio for Org 7617 was 10, and for Org 9616 was 17. These compare to approximate published values for vecuronium, atracurium, and pancuronium of 60, 25, and 3, respectively. Ganglion block was only seen at 30-60 times the neuromuscular blocking doses. Both compounds produced a decrease in arterial blood pressure. This was more pronounced with Org 7617. In addition, Org 9616 produced a slight increase in heart rate.(ABSTRACT TRUNCATED AT 250 WORDS)

Anesthesia↗