The investigation of critical parameters in the glycolytic response of single living cells by rapid microspectrofluorometric analysis

Summary NAD(P)H fluorescence emission spectra are recorded from single living cells, by a recently developed multichannel microspectrofluorometric technique, in correlation with the intracellular microelectrophoretic addition of substrate (i. e., glucose-6-P). These spectra may be used as a reference basis in establishing the critical parameters to be followed when the same studies are extended to a variety of cells, submitted to various drug effects or physical treatments. The sum-spectra corresponding to channel by channel (wavelength by wavelength) summation of spectra obtained from various cells within a series, before and after addition of substrate, and their difference spectrum may be normalized and evaluated comparatively. The NAD(P)H emission maximum prior to addition of substrate seems to present a mixture of dehydrogenase-bound and free coenzyme. There is a suggestion that immediately after substrate, i. e., at 5 sec, an increase in free NADH is first observed. While the overall changes in fluorescence intensity associated with substrate are quite large (50–150% increase), the counts (i. e., an expression of photons) associated with shifts in the emission maximum (free vs. bound NAD(P)H changes) are at times barely above noise. Rapid microspectrofluorometry provides in principle the most direct approach for the identification of coenzyme bound to various dehydrogenases in single living cells, but further improvements in spectral resolution and signal-to-noise ratio are required, for a better definition of the spectral shifts which may be observed.

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Zusammenfassung Mit Hilfe einer kürzlich entwickelten Mehrkanal-Mikrospektrofluorometer-Methode wurden von einzelnen lebenden Zellen nach intrazellulär mikroelektrophoretischer Substratzugabe (z. B. Glucose-6-P) NAD(P)H Fluoreszenz-Emissionsspektren aufgenommen. Diese Spektren können als Vergleichsbasis bei der Festsetzung der entscheidenden Parameter verwendet werden, wenn die gleichen Untersuchungen auf eine Reihe von Zellen ausgedehnt werden sollen, die verschiedenen Medikamenteffekten oder physikalischer Behandlung ausgesetzt werden. Die Summenspektren, die der kanalmäßigen (wellenlängenmäßigen) Summierung der Spektren von verschiedenen Zellen innerhalb einer Serie, vor und nach Substratzugabe entsprechen, sowie ihre Differenzspektren können normalisiert und vergleichsweise ausgewertet werden. Das NAD(P)H-Emissionsmaximum vor der Substratzugabe scheint ein Gemisch von freiem und dehydrogenasegebundenem Coenzym darzustellen. Unmittelbar nach Substratzugabe (d. h. nach 5 sec) ist ein Anstieg an freiem NADH das erste Mal zu beobachten. Während die mit dem Substrat einhergehenden Gesamtveränderungen der Fluoreszenzintensität recht groß sind (50–150% Anstieg), sind die Impulse (als Effekt der Photonen), die mit einer Verschiebung im Emissionsmaximum verbunden sind (Veränderungen von freiem und gebundenem NAD(P)H) zu manchen Zeiten kaum höher als das Rauschen. Die rasche Mikrospektrofluorometrie stellt im Prinzip die direkteste Methode zur Identifizierung von Coenzymen dar, die an verschiedenen Dehydrogenasen in einzelnen lebenden Zellen gebunden sind. Weitere Verbesserungen der Spektralauflösung und der Empfindlichkeit (signal-to-noise ratio) sind notwendig, um die Spektralverschiebungen, die beobachtet werden, besser auswerten zu können.

     

A systematic formula for the asymptotic expansion of singular integrals

Asymptotic theories like the lifting-line, the slender body or the slender ship lead to lineintegrals with singular kernels. Sometimes these integrals are improper, that is to say that they are defined only by their Finite Part. To find asymptotic expansions of these integrals, the Matched Asymptotic Expansion Method is widely used along with other more specific methods depending on the kernel type. The first method is laborious and not systematic, and the other methods are sometimes too much specific to treat general cases. Moreover, all of them are not well adapted to deal with Finite Part integrals.Here, a new method is proposed to avoid the previous difficulties. This method is systematic for homogeneous kernels and gives approximations up to any order, as long as the derivative of the weight function exists at this given order. Moreover the occurrence of logarithmic terms in the expansion is explained and easily predictable. An elliptic integral and the classical lifting-line theory are treated to illustrate the ease of this method.

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Résumé Les théories asymptotiques telles que la ligne portante, le corps élancé ou le navire de grand allongement conduisent à des intégrales curvilignes à noyaux singuliers. Parfois, ces intégrales sont impropres c'est à dire qu'elles sont définies en Parties Finies. Différentes méthodes ont été mises au point pour trouver les développements asymptotiques de ces intégrales. Généralement elles dépendent fortement de la nature du noyau, et c'est finalement la méthode des développements raccordés qui est utilisées quand le noyau est trop compliqué. Cependant, cette méthode est laborieuse et comme les précèdentes non adaptée aux intégrales défines par leur Partie Finie.Une nouvelle méthode est proposée pour surmonter ces difficultés. Cette méthode est systématique pour les noyaux homogènes et donne les approximations à tout ordre pourvu que les dérivées de la fonction poids existent jusqu'à cet ordre. De plus la présence de termes logarithmiques dans le développement est expliquée et aisément prédictible.Une intégrale elliptique, ainsi que la fameuse théorie de la ligne portante sont traités pour illustrer les possibilités de la méthode.

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Nomenclature D domain of integration - f(x) weight function - FP Finite Part - h(x) weight function - I ,I _o bounded intervals - j, J integers - K(x, ) singular kernel - L, L integers - M integer defining the approximation order - P _k (x) Legendre polynomial - R set of real numbers - R(ß) equals 1 if is an integer and 0 if not - R _{f, J} ,R _{K, L} remainders of Taylor developments - S () equals either 1 or the sign function:sgn() - t, u, v, x variable of integration - , real numbers - homogeneity order of the kernel - F () Euler's integral (gamma function) - small parameter - [.] integer part of     

Percolation in strongly correlated systems: The massless Gaussian field

We derive a number of new results for correlated nearest neighbor site percolation onZ ^d. We show in particular that in three dimensions the strongly correlated massless harmonic crystal, i.e., the Gaussian random field with mean zero and covariance –, has a nontrivial percolation behavior: sites on whichS _x h percolate if and only ifh _c . with0 _c < . This provides the first rigorous example of a percolation transition in a system with infinite susceptibility.     

A propos de conjectures de Serre et Sullivan

Summary We first give a new proof of a conjecture of J.-P. Serre on the homotopy of finite complexes, which was recently proved by C. McGibbon and J. Neisendorfer. The emphasis is on a property of the mod. 2 homology of certain spaces: their quasi-boundedness as right modules over the Steenrod algebra. This property is preserved when one goes from a simply connected space to its loop space and also when one takes a covering of anH-space. Then we show that this notion of quasi-boundedness simplifies H. Miller's proof of D. Sullivan's conjecture on the contractibility of the space of pointed maps from the classifying space of the groupe /2 into a finite complex.     

Oriented immobilization of Desulfovibrio gigas hydrogenase onto carbon electrodes by covalent bonds for nonmediated oxidation of H2

The orientation of hydrogenase bound covalently to a pyrolytic graphite edge electrode modified with a 4-aminophenyl monolayer can be modulated via electrostatic interactions during the immobilization step. At low ionic strength and when the amino groups of the electrode surface are mostly protonated, the hydrogenase is immobilized with the negatively charged region that surrounds its 4Fe4S cluster nearer to the protein surface facing the electrode. This allows direct electron transfer between the immobilized hydrogenase and the electrode, which is observed by the strong catalytic currents measured in the presence of the H2 substrate. Therefore, a very stable enzymatic electrode is produced that catalyzes nonmediated H2 oxidation.