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     

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.     

The synthesis of imidazole-2-thione nucleosides

Ribosylation of the trimethylsilyl derivative ( 1b ) of imidazole-2-thione ( 1a ) using either stannic chloride or silver perchlorate as catalyst resulted in the formation of the acylated derivatives of 1-(β-D-ribofuranosyl)imidazole-2-thione ( 3c ) and 1,3-di-(β-D-ribofuranosyl)imidazole-2-thione ( 4c ) with the latter predominating ( 4c:3c , ca. 2:1 ). The diribosylated nucleoside 4c was shown to be the N,N-disubstituted product rather than the N,S-disubstituted product by ¹H nmr and ¹³C nmr spectroscopy. Employment of the iodine-catalyzed fusion procedure reversed the aforementioned product ratios and provided the monoriboside 3c in excellent yield. When the trimethylsilyl derivative ( 5b ) of 2-methylthioimidazole ( 5a ) was reacted with 2,3,5-tri-O-benzoyl-D-ribofuranosyl bromide ( 2d ) in acetonitrile, the major product was 1,3-di-(2,3,5-tri-O-benzoyl-β-D-ribofuranosyl)-imidazole-2-thione ( 4b ). The formation of 4b in this reaction is thought to arise via the Hilbert-Johnson mechanism.     

Synthesis,coordination to Rh(I), and hydroformylation catalysis of new beta-aminophosphines bearing a dangling nitrogen group: an unusual inversion of a Rh-coordinated P center

Variants of the beta-aminophosphine L(1) [Ph(2)PCH(2)CH(Ph)NHPh] containing additional nitrogen donor functions have been prepared. These functions are branched off the C atom adjacent to the P atom, or the P atom itself. Ligand [Ph(2)PCH(o-C(6)H(4)NMe(2))CH(Ph)NHPh] has been obtained as a mixture of two diastereomers L(3A) and L(3B) by lithiation of L(2) [Ph(2)PCH(2)(o-C(6)H(4)NMe(2))] with n-BuLi followed by PhCH=NPh addition and hydrolysis. The diastereomers have been separated by fractional crystallization from ethanol. Ligand Et(2)NCH(2)P(Ph)CH(2)CH(Ph)NHPh has been obtained as a mixture of two diastereomers L(5A) and L(5B)(starting with P-Ph reductive cleavage of L(1) by lithium and subsequent hydrolysis to give PhP(H)CH(2)CH(Ph)NHPh (mixture of two diastereomers L(4A) and L(4B)). The latter reacts with diethylamine and formaldehyde to afford the L(5) diastereomeric mixture. Complexes RhCl(CO)(L) (L = L(3A), 1(A); L(3B), 1(B); L(5A/B), 2(A/B)) were obtained by reaction of [RhCl(CO)(2)](2) and the appropriate ligand or ligand mixture. Complexes 1(A), 1(B), and 2(A) have been isolated in pure form and characterized by classical techniques and by single-crystal X-ray diffraction. All structures exhibit a bidentate kappa-P,kappa-N(NHPh) mode similar to the complex containing L(1). While complexes 1(A) or 1(B) are stable in CDCl(3) solution, complex 2(A) slowly converts to its diastereomer 2(B). This unexpected epimerization appears to take place by inversion at the Rh-coordinated P center, an apparently unprecedented phenomenon. A mechanism based on a reversible P-C bond oxidative addition is proposed. The influence of the pendant nitrogen function of the diaminophosphines L(3A) and L(5A/B) on the rhodium catalytic activity in styrene hydroformylation has been examined and compared to that of the aminophosphines L(1) or L(2). The observed trends are related to the basicity of the dangling amine function and to its proximity to the metal center.     

Solid-phase synthesis using a new polyacrylic resin : Synthesis of the fragment 14–21 of the amino acid sequence of histone H4

The solid-phase synthesis of the octapeptide 1 AcGly-Ala-Lys-Arg-His-Arg-Lys-ValOMe, which represents the fragment 14-21 of the amino acid sequence of the chromosomal histone H4, as well as of the structurally related nonapeptide 2 AcGly-Ala-Lys-Leu-Arg-His-Arg-Lys-ValOMe, is described using a new polyacrylic resin containing a glycolamide ester linkage(resin-NHCO-CH₂-OCO-peptide) acting as a labile anchoring moiety between the resin and the peptide.After elongation of the polypeptide chain using classical protecting groups, i.e. t-butyloxycarbonyl for the α-NH₂ function, benzyloxycarbonyl, nitro and 2,4-dinitrophenyl groups for the side-chains of Lys, Arg and His respectively, both peptides 1 and 2 were obtained in good yields and with a high purity as shown by high-pressure liquid chromatography, by amino-acid analysis and by high-field proton NMR spectroscopy.This work demonstrates the ability of the newly introduced polyacrylic resin to act as a convenient support for solid-phase peptide synthesis.     

Ab initio calculation of the heptamer (NH3)7 as a reasonable starting point for a description of the ammonia crystal

Quasi-Wannier functions for the ammonia crystal have been determined from optimized ab initio calculations of the heptamer (NH₃)₇, thus accounting for all the first neighbor interactions. The calculated geometry and binding energy are in good agreement with their experimental counterparts.