The contractility of knuckle pads. An in vitro study.

Strips of tissue from knuckle pads contract in response to the myofibroblast stimulant mepyramine in a reversible, repeatable and dose-dependent manner. The significance of this finding is discussed.

Dupuytren's disease treated by palmar fasciectomy and an open palm technique.

The results of long-term follow-up (range 9-19 years) are presented in a continuous series of patients treated for Dupuytren's contracture by one surgeon using the open palm technique. Mean preoperative total range of movement was 48% rising to 96% postoperatively. Mean total range of movement was 92% at follow-up. Survivorship analysis revealed 86% survival at 10 years and 77% survival at 19 years. There was one digital nerve injury and one case of algodystrophy. This technique gives good long-term results without the use of night splintage or physiotherapy.

Control of carbon flux to acetate excretion during growth of Escherichia coli in batch and continuous cultures.

During growth of Escherichia coli ML308 on pyruvate in a continuous culture (turbidostat) or batch culture, flux of carbon into the cells exceeds the amphibolic capacity of the central pathways. This is balanced by diversion of carbon flux to acetate excretion which in turn diminishes the efficiency of carbon conversion to biomass [g] dry wt (mol substrate)-1]. However, restriction of carbon supply in a chemostat diminishes flux to acetate excretion and at a dilution rate (D = mu) of 0.35 h-1 or less, no flux to acetate excretion was sustained thus permitting perfect balance between carbon input on the one hand, and the output to biosynthesis and energy generation on the other. This, in turn, improves the efficiency of carbon conversion to biomass. Inclusion of 3-bromopyruvate (an inhibitor of pyruvate dehydrogenase) at a concentration which diminishes growth rate (mu) to 0.35 h-1 or less also prevented flux to acetate excretion. Furthermore, in a family of fluoroacetate-resistant strains, excessive flux of pyruvate was balanced by diversion of carbon flux to lactate excretion rather than acetate and a higher growth rate (mu = 0.63 h-1) was sustained.

Molecular cloning and over-expression of the glyoxylate bypass operon from Escherichia coli ML308.

A recombinant plasmid carrying an 11 kb restriction-endonuclease-ClaI fragment of genomic DNA from Escherichia coli ML308 was constructed. This plasmid complements an aceA mutation. The plasmid encodes the structural genes of the glyoxylate bypass operon, namely malate synthase A (aceB), isocitrate lyase (aceA) and isocitrate dehydrogenase kinase/phosphatase (aceK), as judged by overexpression of enzyme activities and transcription/translation experiments in vitro. Subcloning confirmed that expression of the aceK gene is essential for growth on acetate.

Acute osteomyelitis of index finger caused by dog bite.

A forty-seven-year-old man was bitten by a dog on his left index finger. Despite initial antibiotic treatment, he developed acute osteomyelitis of the middle phalanx over the next three weeks. Pasteurella multocida and Bacteroides were isolated from the necrotic bone. Subsequently the infection was successfully treated by debridement and Tetracycline.

Control of flux through the citric acid cycle and the glyoxylate bypass in Escherichia coli.

The glyoxylate bypass and citric acid cycle operate concurrently in Escherichia coli when acetate is the sole source of carbon and energy to sustain aerobic growth. The overall carbon balance allows fluxes through the central metabolic pathways (CMPs) to be computed on the assumption that these metabolic pathways are known. Acetate is fluxed via the CMPs to the precursors required for synthesis of new biomass and also to generate the reducing power and ATP required to convert these precursors to biomass. Under these circumstances, a junction is created at isocitrate where isocitrate lyase (ICL) and isocitrate dehydrogenase (ICDH) compete for their common substrate. In general, flux through ICL generates the precursors used for biosynthesis while the larger part of the flux (95%) through ICDH is dedicated to the supply of reducing power and ATP. The system sustains a large intracellular pool of isocitrate to accommodate the rather low affinity of ICL for this substrate. Excessive flux of isocitrate through ICDH is prevented by regulation of ICDH activity: reversible inactivation of ICDH is achieved by a bifunctional kinase/phosphatase, as the phosphorylated form of ICDH has no activity. The kinase/phosphatase responds to two classes of effectors--intermediates of the CMPs generated by flux through ICL and the lower energy forms of ATP and NADPH (ADP, AMP and NADP+) generated when these intermediates are used for biosynthesis. The effect is to adjust flux through ICDH so that the rate of supply of NADPH and ATP is equal to the demands of biosynthesis. Biosynthetic fluxes are limited by the rate of supply of precursors which depends on flux through ICL. Growth rate is most likely limited by the primary flux of acetate to acetyl-CoA or flux through ICL. In the steady state, the flux through ICDH is regulated to be twice the throughput of ICL. The evolution of this complex pattern of control may have depended on alternatives to the citric acid for energy generation.

Regulation of the enzymes at the branchpoint between the citric acid cycle and the glyoxylate bypass in Escherichia coli.

During growth of Escherichia coli on acetate, the glyoxylate bypass supplies the precursors needed for biosynthesis. The glyoxylate bypass enzyme isocitrate lyase competes with the citric acid cycle enzyme isocitrate dehydrogenase for the available isocitrate. We have studied the control of metabolic flux at this branchpoint by examining the regulatory properties of the enzymes concerned. Isocitrate dehydrogenase is controlled by reversible phosphorylation catalysed by a bifunctional kinase/phosphatase whose activities are regulated by isocitrate, biosynthetic precursors and adenine nucleotides. The flux through isocitrate lyase is mainly controlled by the intracellular concentration of isocitrate. The phosphorylation system responds to the availability of energy and precursors and maintains isocitrate at a concentration high enough to sustain the flux through the glyoxylate bypass necessary for biosynthesis.

Pyruvate metabolism and the phosphorylation state of isocitrate dehydrogenase in Escherichia coli.

During growth of Escherichia coli on acetate, isocitrate dehydrogenase (ICDH) is partially inactivated by phosphorylation and is thus rendered rate-limiting in the Krebs cycle so that the intracellular concentration of isocitrate rises which, in turn, permits an increased flux of carbon through the anaplerotic sequence of the glyoxylate bypass. A large number of metabolites stimulate ICDH phosphatase and inhibit ICDH kinase in the wild-type (E. coli ML308) and thus regulate the utilization of isocitrate by the two competing enzymes, ICDH and isocitrate lyase. Addition of pyruvate to acetate grown cultures triggers a rapid dephosphorylation and threefold activation of ICDH, both in the wild-type (ML308) and in mutants lacking pyruvate dehydrogenase (ML308/Pdh-), PEP synthase (ML308/Pps-) or both enzymes (ML308/Pdh-Pps-). Pyruvate stimulates the growth on acetate of those strains with an active PEP synthase but inhibits the growth of those strains that lack this enzyme. When pyruvate is exhausted, ICDH is again inactivated and the growth rate reverts to that characteristic of growth on acetate. Because pyruvate stimulates dephosphorylation of ICDH in strains with differing capabilities for pyruvate metabolism, it seems likely that pyruvate itself is a sufficient signal to activate the dephosphorylation mechanism, but this does not discount the importance of other signals under other circumstances.

Metacarpal lengthening.

Reduction deficits of metacarpals requiring treatment are an uncommon problem. A patient with bilateral short ring fingers is reported and a technique of Z lengthening is described.

The phosphorylation of Escherichia coli isocitrate dehydrogenase in intact cells.

The isocitrate dehydrogenase of Escherichia coli ML308 can be reversibly activated by addition of pyruvate to cells growing on acetate [Bennett & Holms (1975) J. Gen. Microbiol. 87, 37-51]. By using cells pulse-labelled with [32P]Pi we showed that the activation and inactivation of the enzyme in these conditions correlate with its dephosphorylation and rephosphorylation respectively. Incubation of cell extracts prepared during an activation/inactivation cycle with purified isocitrate dehydrogenase phosphatase confirmed that the pyruvate-induced activation of the dehydrogenase goes essentially to completion. The results show that the reversible changes in the activity of the dehydrogenase in cells grown on acetate are solely due to phosphorylation/dephosphorylation. Inactive 32P-labelled isocitrate dehydrogenase was isolated from cells incubated with [32P]Pi in the presence of acetate. Both this material and purified enzyme phosphorylated in vitro were digested with chymotrypsin, and the phosphopeptides were isolated and analysed. Only one phosphopeptide was observed in each case; the results show that the residue phosphorylated in vivo is identical with that phosphorylated by purified isocitrate dehydrogenase kinase in vitro.

Amino acid sequence round the site of phosphorylation in isocitrate dehydrogenase from Escherichia coli ML308.

Isocitrate dehydrogenase from Escherichia coli is regulated by a reversible phosphorylation mechanism. We report here the amino acid sequence round the phosphorylation site; this is the first such sequence to be reported for a bacterial protein kinase. The sequence does not resemble sequences phosphorylated by cyclic AMP-dependent protein kinase.

Isolation of active and inactive forms of isocitrate dehydrogenase from Escherichia coli ML 308.

In Escherichia coli ML308 isocitrate dehydrogenase is partially inactivated during growth on acetate [Bennett, P.M. and Holms, W.H. (1975) J. Gen. Microbiol. 87, 37-51]. The active form of isocitrate dehydrogenase was purified to homogeneity from cells grown on glycerol. The key step in the procedure was chromatography on procion-red-Sepharose, from which the enzyme was specifically eluted with NADP+. Two forms of isocitrate dehydrogenase were purified to homogeneity from cells grown on acetate. One form did not bind to procion-red-Sepharose and was essentially inactive; this form could be resolved from the active form by non-denaturing gel electrophoresis. The other form was specifically eluted from procion-red-Sepharose and was partially active; analysis of this form by non-denaturing gel electrophoresis suggested that it was a mixture of the active and inactive forms. The three forms comigrated on denaturing gel electrophoresis and were identical by the criterion of one-dimensional peptide mapping. Analysis of the active and inactive forms by sedimentation equilibrium centrifugation and non-denaturing gel electrophoresis showed that they differed in charge but not in size. Amino acid analysis and two-dimensional peptide mapping showed that both forms were dimers of identical subunits. The active form of the enzyme contained no detectable alkali-labile phosphate, the inactive form contained 0.8 molecule/subunit and the partially active form contained an intermediate amount. The data suggest that the active and inactive forms of isocitrate dehydrogenase differ only in the presence of one phosphate group per subunit in the latter form; this is consistent with our results from phosphorylation of isocitrate dehydrogenase in vitro (Following paper in this journal). The nature of the partially active form of isocitrate dehydrogenase and the significance of the results are discussed.

Partial purification and properties of isocitrate dehydrogenase kinase/phosphatase from Escherichia coli ML308.

Isocitrate dehydrogenase kinase and isocitrate dehydrogenase phosphatase were purified over 1000-fold from Escherichia coli ML308 by procedure involving fractionation with (NH4)2SO4 and chromatography on DEAE-cellulose, blue-dextran-Sepharose and Sephadex G150. The kinase and phosphatase activities copurified, in agreement with the observation [Laporte, D.C. and Koshland, D.E. (1982) Nature (Lond.) 300, 458-460] that a single protein bears both activities. Isocitrate dehydrogenase kinase catalysed the phosphorylation of homogeneous active isocitrate dehydrogenase with a stoichiometry of just under one phosphate group incorporated per subunit. This almost completely inactivated the dehydrogenase. There was a good correlation between phosphorylation and inactivation. Analysis of a partial acid hydrolysate of phosphorylated isocitrate dehydrogenase showed that the only phosphoamino acid present was phosphoserine. Isocitrate dehydrogenase phosphatase catalysed the release of 32P from 32P-phosphorylated isocitrate dehydrogenase; it required either ADP or ATP for activity. In the presence of ADP, or ATP plus an inhibitor of the kinase, the phosphatase catalysed full reactivation of isocitrate dehydrogenase and there was a good correlation between reactivation and the release of phosphate. In the presence of ATP alone the phosphatase catalysed the release of 32P from phosphorylated isocitrate dehydrogenase but the activity of the dehydrogenase remained low, indicating that the kinase and phosphatase were active simultaneously in these conditions. The active and inactive forms of isocitrate dehydrogenase can be resolved by non-denaturing gel electrophoresis; the two forms of the enzyme were interconverted by phosphorylation and dephosphorylation in vitro. The extent of the interconversion correlated well with the changes in isocitrate dehydrogenase activity.

Antibacterial action of quinolones on Escherichia coli. I. Structure/activity relationship.

Quinolones, a large group of substituted heterocyclic compounds synthesized by ICI (U.K.), showed antibacterial activity both in vivo and in vitro studies. In the present work an attempt was made to evaluate their antibacterial activity in relation to their chemical structure. Active compounds possess carboxylic and nitroside groups. Regarding the pattern of growth inhibition quinolone with carboxylic side group showed a different behaviour than quinolone possessing nitro side group.

Control of the sequential utilization of glucose and fructose by Escherichia coli.

In Escherichia coli (ATCCI5224; ML308), glucose and fructose phosphotransferase systems (PT-systems) are constitutive but activities are increased five and 10-fold respectively by aerobic growth on their respective substrates in defined media. In mixtures, glucose is used preferentially and the fructose PT-system activity is kept at its minimum; but, on glucose exhaustion, it overshoots its steady-state level and growth continues on fructose without lag. Cyclic AMP prevents overshoot. Continuous cultures operating as turbidostats on mixtures of glucose and fructose do not use fructose if sufficient glucose is present to support growth. If less glucose is available, it is all used and sufficient fructose is metabolized concurrently to maintain the growth rate characteristic of glucose. Both PT-systems are inhibited by hexose phosphates. Presence of homologous substrate specifically sensitizes each PT-system to inactivation by N-ethylmaleimide (NEM). Glucose diminishes the ability of fructose to sensitize its PT-system to NEM. This effect parallels the inhibition of fructose utilization by glucose and suggests that glucose denies fructose access to the fructose-specific part of the PT-system.

Reversible inactivation of the isocitrate dehydrogenase of Escherichia coli ML308 during growth on acetate.

During aerobic growth of Escherichia coli ML308 on acetate as sole carbon source, the apparent synthesis of isocitrate dehydrogenase was repressed relative to cultures on other carbon sources, such as glucose, which do not employ the glyoxylate bypass as an anaplerotic sequence. When cells were removed from an acetate medium, or when compounds were added which made the operation of the glyoxylate bypass unnecessary, the activity of isocitrate dehydrogenase rapidly increased 3- to 4-fold but fell again on restoration to an acetate medium. Changes in activity were rapid and, furthermore, could be demonstrated in the absence of protein synthesis. It is thus improbable that the mechanism involved degradation or de novo synthesis of the enzyme protein. Oxaloacetate and glyoxylate showed concerted inhibition of isocitrate dehydrogenase which could be relieved by dialysis. Because extracts of low enzyme activity, derived from acetate-metabolizing cells, could not be stimulated by dialysis or by addition of a wide range of metabolites, it is unlikely that low molecular weight, freely dissociable effectors were responsible for stimulation or inhibition of activity. Control of isocitrate dehydrogenase permitted the efficient utilization of acetate as sole source of carbon and energy but perserved the capacity of the cell to respond rapidly to an improvement in nutritional conditions. A limited survey showed that the mechanism is common but not universal among strains of E. coli and occurs in at least one strain each of Klebsiella aerogenes, Salmonella typhimurium and Serratia marcescens.