Tetanus Toxin-Induced Effects on Extracellular Amino Acid Levels in Rat Hippocampus: An In Vivo Microdialysis Study

Abstract: Tetanus toxin is a potent neurotoxin that is widely considered to produce its effect through impairment of inhibitory neurotransmission. We report the effect of a single unilateral intrahippocampal injection of tetanus toxin on extracellular levels of neuroactive amino acids in freely moving rats, at times ranging between 1 and 7 days posttreatment. Tetanus toxin treatment did not alter extracellular levels of aspartate, glutamate, and taurine at any time during the study. However, although extracellular GABA levels were unaffected by toxin injection 1, 2, and 3 days after treatment, they were reduced (45 ± 8% of contralateral vehicle-injected level) at day 7. Challenge with a high K⁺ concentration, 7 days after treatment, produced elevations in extracellular levels of taurine and GABA in both vehicle- and toxin-injected hippocampi, with evoked levels of GABA being lower in the toxin-treated side (39 ± 16% of contralateral vehicle-injected level). Aspartate and glutamate levels were not increased by high-K⁺ infusion. These findings are discussed in relation to the possible role that an imbalance in excitatory/inhibitory tone may play in the production of tetanus toxin-induced neurodegeneration.     

Demographic and social characteristics of adults with cystic fibrosis in the United Kingdom.

OBJECTIVE--To obtain information about social and demographic characteristics and lifestyle of adult patients with cystic fibrosis, including those who do not attend major specialist clinics. DESIGN--Confidential self completion postal questionnaire to adult patients with cystic fibrosis, asking about social and demographic characteristics, social class and occupation, employment, education, insurance and social security benefits, symptom severity, and medical care. SETTING--National association for adults with cystic fibrosis. SUBJECTS--1052 adult members of the Association of Cystic Fibrosis Adults UK, accounting for 68% of those with cystic fibrosis in the United Kingdom population over 16 years of age and over 80% of those over 25 in June 1990. RESULTS--The response rate was 82% (397 women, 423 men). Most adults with cystic fibrosis were found to be living fulfilling lives into adulthood. Significantly fewer men were married or cohabiting than women (110 (26%) men, 175 (44%) women). 420 (55%) responders were working, and of these 235 (56%) had less than two weeks'' sick leave a year. Half of those not employed gave ill health as the reason. Revealing that they had cystic fibrosis at job interviews reduced likelihood of being employed for those with mild to moderate disease. People with cystic fibrosis had been less successful than the general population in achieving O level or equivalent qualifications, but more successful in achieving A level or higher qualifications. Achievement of any qualifications enhanced employment prospects irrespective of disease severity. CONCLUSION--Contrary to an image of chronic ill health and disability, a high proportion of adults with cystic fibrosis are living full and productive lives.     

Kinetics and mechanism of action of muscle pyruvate kinase

1. The mechanism of rabbit muscle pyruvate kinase was investigated by measurements of fluxes, isotope trapping, steady-state velocity and binding of the substrates. All measurements were made at pH8.5 in Tris/HCl buffer and at 5mm-free Mg(2+). 2. Methods of preparing [(32)P]phosphoenolpyruvate from [(32)P]P(i) in high yield and determining [(32)P]-phosphoenolpyruvate and [8-(14)C]ADP are described. 3. The ratio Flux of ATP to ADP/Flux of ATP to phosphoenolpyruvate (measured at equilibrium) increased hyperbolically with ADP concentration from unity to about 2.1 at 2mm-ADP, but was unaffected by phosphoenolpyruvate concentration. Since the ratio is greater than unity, one pathway for the addition of substrates must involve phosphoenolpyruvate adding first to the enzyme in a rate-limiting step. However, the substrates must also add in the alternative order, because of the non-linear increase in the ratio with ADP concentration and because the rate of increase is very much less than that predicted from the steady-state velocity data for an ordered addition. The lack of influence of phosphoenolpyruvate on the ratio is consistent with the rapid addition of ADP in the alternative pathway. At low ADP concentrations the alternative pathway contributes less than 33% to the total reaction. 4. Isotope trapping was observed with [(32)P]phosphoenolpyruvate, confirming that when phosphoenolpyruvate adds first to the enzyme it is in a rate-limiting step. The release of phosphoenolpyruvate from the ternary complex must also be a slow step. Trapping was not observed with [8-(14)C]ADP, hence the addition of ADP to the free enzyme must be rapid unless its dissociation constant is very large (>20mm). 5. Binding studies showed that 4mol of [(32)P]phosphoenolpyruvate binds to 1mol of the enzyme, probably unligated to Mg(2+), with a dissociation constant appropriate to the mechanism indicated above. Binding of [8-(14)C]ADP could not be detected, and hence the binding of ADP occurs by a low-affinity step. The latter is also demanded by the steady-state velocity data. 6. The ratio Flux of phosphoenolpyruvate to ATP/Flux of phosphoenolpyruvate to pyruvate (determined from the incorporation of label into phosphoenolpyruvate from [3-(14)C]-pyruvate or [gamma-(32)P]ATP during the forward reaction) did not differ significantly from unity. Steady-state velocity data predicted grossly different flux ratios for ordered dissociations of the products, and the results indicate that the dissociation must be rapid and random. The data also exclude a Ping-Pong mechanism. 7. Permissible rate constants for the above mechanism are calculated. The results indicate a high degree of cooperativity in binding, whatever the order of addition of substrate.     

Incorporation von [D-4,5-3H]-Ieucine und [2-14C]-Acetat in die photosynthetischen Pigmente und Chinone von Chlorella pyrenoidosa

Der Metabolismus der photosynthetischen Pigmente und Chinone von Chlorella wurde mit Hilfe von ¹⁴CO₂, [2-¹⁴C]-Acetat und [D-4,5-³H]-Leucin als Vorstufen der Isoprenoidsynthese untersucht. Auch wurde untersucht, ob Leucin als Vorstufe in der Biosynthese der Terpenoide fungiert. Die Verwendung der Doppelmarkierung sollte bestehende Unterschiede in der Regulation und Biosynthese der Isoprenoide der Chloroplasten, Mitochondrien und des Zytoplasmas aufdecken. Neben der Verwendung von Radioisotopen wurde die Inkorporation von Deuterium in die Carotinoide verwendet, um den turnover der Prenyl-Lipide direkt nachzuweisen. In tracer-kinetischen Untersuchungen mit ¹⁴CO₂ konnte gezeigt werden, daß ¹⁴-C-α-Carotin zu ¹⁴C-Lutein und ¹⁴C-ß-Carotin zu ¹⁴C-Zeaxanthin umgesetzt wird. Eine Vorstufenfunktion läßt sich auch für Plastohydrochinon-9 nachweisen, das in Plastochinon-9 umgesetzt wird. Die Markierungskinetik der einzelnen Prenyl-Lipide deutet auf einen schnellen turnover hin. Anhand der ¹⁴C-Inkorporationskinetik wurden Halbwertszeiten im Bereich von 30 min bis 220 min ermittelt, während sich aus den Dekorporationskinetiken Zeiten von 9 bis 113 h ergeben. Prenyl-Lipide wie die Carotine, Chlorophyll a und Plastochinon-9, die einerseits Vorstufenfunktion besitzen, andererseits aber auch direkt an der Photosynthese beteiligt sind, werden schneller umgesetzt als ihre Folgeprodukte. Somit scheint ein direkter Zusammenhang zwischen der Funktion des Prenyl-Lipides im Chloroplasten und seinem Umsatz zu bestehen. Sowohl Acetat wie auch Leucin werden von Chlorella als Vorstufen in der Terpenoidbiosynthese verwendet. Acetat wird aber wesentlich schneller inkorporiert als Leucin, was vielleicht darauf zurückzuführen ist, daß Leucin im Zytoplasma zu Acetat oder Mevalonsäure metabolisiert wird und dann in den Chloroplasten gelangt. Auch im tracer-kinetischen Experiment mit ¹⁴C-Acetat und ³H-Leucin läßt sich eine Vorstufen-Produkt-Beziehung für die Chlorophylle, Carotinoide und Chinone zeigen. Im Vergleich zu den Experimenten mit ¹⁴CO₂ zeigen die tracer-kinetischen Experimente mit ¹⁴C-Acetat und ³H-Leucin, daß die Chloroplasten-Isoprenoide wesentlich langsamer metabolisiert werden und ihre Umsatzraten mehr jenen entsprechen, die aus den ¹⁴C-Dekorporationskinetiken des ¹⁴CO₂-Experimentes resultieren. Es zeigt sich aber auch hier, daß jene Prenyl-Lipide, die direkt an der Photosynthese beteiligt sind, schneller metabolisiert werden. Daß die Chloroplasten-Isoprenoide in autotroph kultivierten Chlorellen einem ständigen turnover unterliegen, läßt sich auch sehr eindrucksvoll mit Hilfe der Deuterium-Inkorporation für die Carotine zeigen. Die aus der Deuterium-Inkorporation erhaltenen Umsätze der Carotine entsprechen in etwa denen, die sich aus ihrer Dekorporationskinetik im tracer-kinetischen Experiment mit [2-¹⁴C]-Acetat und [D-4,5-³H]-Leucin ergeben, zeigen allerdings, daß a-Carotin wesentlich schneller umgesetzt wird als ß-Carotin. Die Untersuchungen wurden durch ein Stipendium der Deutschen Forschungsgemeinschaft im Austausch mit der British Royal Society an K.H.G. unterstützt, wofür wir uns bedanken.     

Size specific predation by herons and its effect on the sex-ratio of natural populations of the mosquito fish Gambusia affinis baird and girard

Summary Sex-ratios of Gambusia affinis populations in freshwater marshes in the Camargue (Rhône Delta), are highly biased in favour of males, whereas the sex-ratios in ditches are close to unity. Studies of the diet of free living birds and experimental studies on prey size selection in captivity show that the abnormal sex-ratios in marshes can be attributed to differential heron predation. Ditches are relatively free from predation. Mature female Gambusia are larger, and have an energy content 5–25 times greater than that of mature males. Handling times of Grey Heron (Ardea cinerea) and Little Egret (Egretta garzetta) are only slightly longer for female Gambusia than males. Hence, females represent a much more profitable prey.Analysis of nestling regurgitates show that Gambusia makes up a considerable proportion of the diet of four species of Camargue herons, and that the majority of Gambusia taken are females. Under experimental conditions, captive herons consume almost exclusively female fish, even when offered in ratios where they are heavily outnumbered by males.The relevance of these results to optimal diet theory is discussed.     

Oxonol dyes as monitors of membrane potential. Their behavior in photosynthetic bacteria

The responses of oxonol dyes to single and multiple single turnovers of the photosynthetic apparatus of photosynthetic bacteria have been studied, and compared with the responses of the endogenous carotenoid pigments. The absorbance changes of the oxonols can be conveniently measured at 587 nm, because this is an isosbestic point in the ‘light-minus-dark’ difference spectrum of the chromatophores.The oxonols appear to respond to the light-induced ‘energization’ by shifting their absorption maxima. In the presence of K⁺, valinomycin abolished and nigericin enhanced such shifts, suggesting that the dyes respond to the light-induced membrane potential. Since the dyes are anions at neutral pH values, they probably distribute across the membrane in accordance with the potential, which is positive inside the chromatophores. The accumulation of dye, which is indicated by a decrease in the carotenoid bandshift, poises the dye-membrane equilibrium in favor of increased dye binding and this might be the cause of the spectral shift.The dye response has an apparent second-order rate constant of approx. 2 · 10⁶ M^?1 · s^?1 and so is always slower than the carotenoid bandshift. Thus the dyes cannot be used to monitor membrane potential on submillisecond timescales. Nevertheless, on a timescale of seconds the logarithm of the absorbance change at 587 nm is linear with respect to the membrane potential calibrated with the carotenoid bandshift. This suggests that under appropriate conditions the dyes can be used with confidence as indicators of membrane potential in energy-transducing membranes that do not posses intrinsic probes of potential.     

Mechanism of "L"-type pyruvate kinase from rabbit liver. Evidence against phosphoenzyme formation.

The "L"-type pyruvate kinase from rabbit liver does not catalyse exchange between phosphoenol[1-14C]pyruvate and pyruvate at either pH 8.5 or 6.2. Spectrophotometric experiments at pH 8.5 and 6;2 and gel-filtration experiments with [32P]phosphoenolpyruvate at pH 8,5 also fail to demonstrate phosphoenzyme formation. It is concluded that it is very unlikely that the enzyme has a phosphoenzyme mechanism.     

Mechanism of carbamoyl-phosphate synthetase. Binding of ATP by the rat-liver mitochondrial enzyme.

This paper demonstrates, by pulse-chase techniques, the binding to rat liver mitochondrial carbamoyl phosphate synthetase of the ATP molecule (ATPB) which transfers its gamma-phosphoryl group to carbamoyl phosphate. This bound APTB can react with NH3, HCO-3 and ATP (see below) to produce carbamoyl phosphate before it exchanges with free ATP. Mg2+ and N-acetylglutamate, but not NH3 or HCO-3, are required for this binding; the amount bound depends on the concentration of ATP (Kapp = 10--30 microns ATP) and the amount of enzyme. At saturation at least one ATPB molecule binds per enzyme dimer. Binding of ATPB follows a slow exponential time course (t1/2 8--16 s, 22 degrees C), independent of ATP concentration and little affected by NH3, NCO-3 or by incubation of the enzyme with unlabelled ATP prior to the pulse of [gamma-32P]ATP. Formation of carbamoyl phosphate from traces of NH3 and HCO-3 when the enzyme is incubated with ATP follows the kinetics expected if it were generated from the bound ATPB, indicating that the latter is a precursor of carbamoyl phosphate ('Cbm-P precursor') in the normal enzyme reaction. This indicates that the site for ATPB is usually inaccessible to ATP in solution but becomes accessible when the enzyme undergoes a periodical conformational change. Bound ATP becomes Cbm-P precursor when the enzyme reverts to the inaccessible conformation. Pulse-chase experiments in the absence of NH3 and HCO-3 (less than 0.2 mM) also demonstrate binding of ATPA (the molecule which yields Pi in the normal enzyme reaction), as shown by a 'burst' in 32Pi production. Therefore, (in accordance with our previous findings) both ATPA and ATPB can bind simultaneously to the enzyme and react with NH3 and HCO-3 in the chase solution before they can exchange with free ATP. However, at low ATP concentration (18 micron) in the pulse incubation, only ATPB binds since ATP is required in the chase (see above). Despite the presence of two ATP binding sites, the bifunctional inhibitor adenosine(5')pentaphospho(5')adenosine(Ap5A) fails to inhibit the enzyme significantly. A more detailed modification of the scheme previously published [Rubio, V. & Grisolia, S. (1977) Biochemistry, 16, 321--329] is proposed; it is suggested that ATPB gains access to the active centre when the products leave the enzyme and the active centre is in an accessible configuration. The transformation from accessible to inaccessible configuration appears to be part of the normal enzyme reaction and may represent to conformational change postulated by others from steady-state kinetics. The properties of the intermediates also indicate that hydrolysis of ATPA must be largely responsible for the HCO-3-dependent ATPase activity of the enzyme. The lack of inhibition of the enzyme by Ap5A indicates substantial differences between the Escherichia coli and the rat liver synthetase.