The allosteric regulation of pyruvate kinase by fructose-1,6-bisphosphate.

BACKGROUND: Yeast pyruvate kinase (PK) catalyzes the final step in glycolysis. The enzyme therefore represents an important control point and is allosterically activated by fructose-1,6-bisphosphate (FBP). In mammals the enzyme is found as four different isozymes with different regulatory properties: two of these isozymes are produced by alternate splicing. The allosteric regulation of PK is directly related to proliferation of certain cell types, as demonstrated by the expression of an allosterically regulated isozyme in tumor cells. A model for the allosteric transition from the inactive (T) state to the active (R) state has been proposed previously, but until now the FBP-binding site had not been identified. RESULTS: We report here the structures of PK from yeast complexed with a substrate analog and catalytic metal ions in the presence and absence of bound FBP. The allosteric site is located 40 A from the active site and is entirely located in the enzyme regulatory (C) domain. A phosphate-binding site for the allosteric activator is created by residues encoded by a region of the gene corresponding to the alternately spliced exon of mammalian isozymes. FBP activation appears to induce several conformational changes among active-site sidechains through a mechanism that is most likely to involve significant domain motions, as previously hypothesized. CONCLUSIONS: The structure and location of the allosteric activator site agrees with the pattern of alternate genetic splicing of the PK gene in multicellular eukaryotes that distinguishes between a non-regulated isozyme and the regulated fetal isozymes. The conformational differences observed between the active sites of inactive and fully active PK enzymes is in agreement with the recently determined thermodynamic mechanism of allosteric activation through a 'metal relay' that increases the affinity of the enzyme for its natural phosphoenolpyruvate substrate.

Effect of bending strain on the torsion elastic constant of DNA.

The torsion constants of both circular and linear forms of the same 181 bp DNA were investigated by time-resolved fluorescence polarization anisotropy (FPA) of intercalated ethidium. The ratio of intrinsic ethidium binding constants of the circular and linear species was determined from the relative fluorescence intensities of intercalated and non-intercalated dye in each case. Possible changes in secondary structure were also probed by circular dichroism (CD) spectroscopy. Upon circularization, the torsion constant increased by a factor of 1.42, the intrinsic binding constant for ethidium increased by about fourfold, and the CD spectrum underwent a significant change. These effects are attributed to an altered secondary structure induced by the bending strain. Quantitative agreement between torsion constants obtained from the present FPA studies and previous topoisomer distribution measurements on circular DNAs containing 205 to 217 bp removes a long-standing apparent discrepancy between those two methods. After storage at 4 degrees C for eight months, the torsion constant of the circular DNA increased by about 1.25-fold, whereas that of the linear DNA remained unchanged. For these aged circles, both the torsion constant and intrinsic binding constant ratio lie close to the corresponding values obtained previously for a 247 bp DNA by analyzing topoisomer distributions created in the presence of various amounts of ethidium. The available evidence strongly implies that torsion constants measured for small circular DNAs with less than 250 bp are specific to the altered secondary structure(s) therein, and are not applicable to linear and much larger circular DNAs with lower mean bending strains.

Effects of Na+ and Mg2+ on the structures of supercoiled DNAs: comparison of simulations with experiments.

Recent cryo-electron microscopy (cryo-EM) results suggest that sufficient NaCl concentration (> or approximately 0.1 M) and superhelix density (> or approximately-0.05) cause circular DNAs to adopt highly extended, tightly interwound configurations, in which the strands are laterally contiguous along almost their entire length. Millimolar levels of MgCl2 reportedly act synergistically with NaCl to produce similar conformations. However, Monte Carlo simulations with purely repulsive interduplex forces failed to reproduce such structures. In the present work, solution measurements of particular physical properties were performed both to characterize the effects of Na+ and Mg2+ on DNA structure and to provide quantitative tests of Monte Carlo simulations of circular DNAs. Supercoiled p30 delta DNAs in 10 mM Tris plus 0, 0.122, and 0.1 M NaCl, and 0.1 M NaCl plus 4 mM Mg2+ were examined by static and dynamic light scattering (LS and DLS), time-resolved fluorescence polarization anisotropy (FPA) of intercalated ethidium, and circular dichroism (CD) spectroscopy. Upon addition of 0.122 M NaCl, the radius of gyration (Rg) decreased substantially, which indicates that p30 delta adopts a more compact structure. This contradicts the cryo-EM studies, where molecular extension and Rg both increase upon adding 0.1 M NaCl. In 0.1 M NaCl, the torsion constant measured by FPA is practically invariant to superhelix density, and the plateau diffusion coefficient at large scattering vector (Dplat) is likewise nearly the same at both relaxed and native superhelix densities. Such invariance is difficult to reconcile with any transition from relaxed circles to tightly interwound structures with laterally contiguous strands. Metropolis Monte Carlo simulations were performed to generate canonically distributed sets of structures, from which average Do values and scattered intensity ratios, [symbol: see text]I (zero) [symbol: see text]/[symbol: see text] l(k) [symbol: see text], were calculated. Agreement between simulations and experiments in regard to [symbol: see text] I(O) [symbol: see text] /[symbol: see text] I(k) [symbol: see text], D(zero) and the supercoiling free energy, delta Gsc (delta l), is remarkably good for the most extensively studied p30 delta samples. The simulated structures exhibit no sign of very tight interwinding with extensive lateral contacts, but instead exhibit most probable superhelix diameters of 85 to 90 A. When 4 mM Mg2+ was added to native supercoiled p30 delta in 0.1 M NaCl, Rg decreased, D(zero) increased, and the longest internal relaxation rate (1/tau 2(zero)) increased, all of which indicate a further overall contraction of the molecular envelope. The torsion constant exhibited a slight increase that is hardly statistically significant. In this case, agreement between the simulations and experiments was only semi-quantitative for most samples investigated, although the predicted contraction was exhibited by all five samples of p30 delta and one of pBR322 DNA. The simulated structures in 0.1 M NaCl plus 4 mM Mg2+ again showed no sign of extensive lateral contacts. A plausible explanation is proposed for the highly extended, tightly interwound structures seen in cryo-EM, and explicitly tested by Monte Carlo simulations of a 1000 bp circular DNA at +25 and -50 degrees C. Structures identical to those seen in cryo-EM are in fact the equilibrium structures in the simulations at -50 degrees C, and the estimated time for equilibration (2.3 x 10(-6) second) is much smaller than the estimated time for vitrification (1 x 10(-4) second).

Salmonella excretion by terrapins and the associated hazard to human health.

The isolation of Salmonella java (phage type Worksop) from the water of a terrapin tank is described. Two terrapins excreted the organism throughout a nine month observation period. Attention is drawn to recent reports of human illness associated with these animals kept as domestic pets.

Isolation of Actinobacillus seminis from rams in the United Kingdom.

Actinobacillus seminis was isolated from the semen of five rams on four farms. Four of the rams had abnormal semen and three were also infertile. The isolates of A seminis showed similar phenotypic profiles and electrophoretic protein patterns to the type strain of A seminis but were distinct from Histophilus ovis previously isolated from rams with epididymitis in Scotland. The infection appeared to be subclinical but two of the five rams had palpable abnormalities of their testes. Three rams were treated with antibiotics but the infection persisted. No gross lesions were found in the genitalia of two of three rams examined post mortem but one had necrotic abscesses in the testes and epididymis. A seminis was isolated from the seminal vesicles and epididymis of one ram without gross lesions but not from the genitalia of the other two. On one farm the infection in a recently purchased ram led to the detection of another case as a result of the bacteriological screening of 11 stock rams not in contact with the initial case. These five subclinical cases, which included a supposedly healthy stock ram, suggest that A seminis infection may be widespread and should be considered in cases of infertility.

Prophylactic analgesia for daycase termination of pregnancy. A double-blind study with controlled release dihydrocodeine.

The incidence and severity of pain and nausea experienced by 40 primigravid day patients who presented for vaginal termination of pregnancy were examined. Controlled-release dihydrocodeine had no effect upon the incidence or severity of these minor sequelae. The requirements for escape analgesia and antiemetic therapy were less than anticipated and possible explanations are discussed. The low incidence of significant nausea and vomiting recorded in this study confirms that vaginal termination of pregnancy may be safely performed as day cases.

Ovine internal parasitism in the Yemen Arab Republic.

Internal parasitism was studied in five flocks in different regions of the Yemen Arab Republic. Ewes and lambs were faeces sampled from the spring of 1977 to the spring of 1978 and helminth-free tracer lambs were subsequently run with three of the flocks from October 1978 to November 1979. The findings indicated that ovine helminthiasis and fascioliasis may be of importance with the following regional variations: Haemonchosis all the year round in the Tihammah coastal plain. Haemonchosis and trichostrongylosis in the periparturient period in the Southern Uplands and Western Foothills. Fascioliasis and nematodiriasis in the Inter-Montane Plains of the Highlands. Possible control regimes are suggested for further investigation.

Aphosphorosis in North Yemen cattle.

A syndrome of unthriftiness, stiffness, anorexia, recumbency and pica in cattle of the Yemen Arab Republic was investigated for the first time. The condition is virtually confined to cows in the Central and Northern Highlands where they are housed continuously and hand fed fresh lucerne and dried sorghum stalks frequently supplemented with domestic scraps. Affected cows had a low mean blood inorganic phosphorus (0.6 mmol/litre) but normal serum calcium and magnesium. Blood phosphorus and serum calcium levels were negatively correlated and fluctuated seasonally, possibly associated with rainfall. The basic diet of lucerne and sorghum stalks was adequate for maintenance but deficient in phosphorus, energy and protein for milk production. The high calcium content of the lucerne portion resulted in wide calcium: phosphorus ratios of 8.8 to 16.5:1. The similarity of the syndrome to osteomalacia caused by phosphorus deficiency in other parts of the world is discussed.

Modified occlusion tests for the Bain breathing system.

Undetected defects in the inner tube of the Bain coaxial anaesthetic breathing system may result in a greatly increased apparatus deadspace. Several authorities have advocated tests intended to detect inner tube problems; however, the efficacy of these tests has never been validated. In this study none of the tests were able to detect all the induced defects. A modification of an existing test using the backbar pressure-relief valve and a new double occlusion test were sufficiently sensitive to detect all defects.

On the origin of the temperature dependence of the supercoiling free energy.

Monte Carlo simulations using temperature-invariant torsional and bending rigidities fail to predict the rather steep decline of the experimental supercoiling free energy with increasing temperature, and consequently fail to predict the correct sign and magnitude of the supercoiling entropy. To illustrate this problem, values of the twist energy parameter (E(T)), which governs the supercoiling free energy, were simulated using temperature-invariant torsion and bending potentials and compared to experimental data on pBR322 over a range of temperatures. The slope, -dE(T)/dT, of the simulated values is also compared to the slope derived from previous calorimetric data. The possibility that the discrepancies arise from some hitherto undetected temperature dependence of the torsional rigidity was investigated. The torsion elastic constant of an 1876-bp restriction fragment of pBR322 was measured by time-resolved fluorescence polarization anisotropy of intercalated ethidium over the range 278-323 K, and found to decline substantially over that interval. Simulations of a 4349-bp model DNA were performed using these measured temperature-dependent torsional rigidities. The slope, -dE(T)/dT, of the simulated data agrees satisfactorily with the slope derived from previous calorimetric measurements, but still lies substantially below that of Duguet's data. Models that involve an equilibrium between different secondary structure states with different intrinsic twists and torsion constants provide the most likely explanation for the variation of the torsion constant with T and other pertinent observations.

Recovery after day-case anaesthesia. A 24-hour comparison of recovery after thiopentone or propofol anaesthesia.

Sixty patients who presented for day-case dilatation and curettage were allocated randomly to receive either thiopentone or propofol for induction and maintenance of anaesthesia. One anaesthetist administered all the anaesthetics whilst all assessments were made by one other. The results indicate that early recovery of memory function, critical flicker fusion frequency and subjective feelings of tiredness, drowsiness and alertness were superior in the propofol group. There was a significant difference in subjective feelings of tiredness and drowsiness recorded by the two study groups at 24 hours. Memory function assessed by Wechsler logical memory function passages at 24 hours was impaired in the propofol group in comparison to a group of 'reference' subjects.

Latency of brachial plexus block. The effect on onset time of warming local anaesthetic solutions.

A double-blind study was set up to investigate the effect of warming local anaesthetic solutions on the latency of onset of subclavian perivascular brachial plexus blocks. Twenty-four adult patients were randomly allocated into two equal groups. In group A the local anaesthetic was injected at room temperature, while in group B the local anaesthetic solution was prewarmed to 37 degrees C in a thermostatically controlled heating block. All blocks were performed using 0.5 ml/kg of a solution prepared by mixing equal volumes of 0.5% bupivacaine with adrenaline 1:200,000, and 1% prilocaine. The speed of onset of sensory blockade was significantly increased when the temperature of the local anaesthetic solution was increased to 37 degrees C. There were no adverse side effects in either group.