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.

     

Mass spectrometry of onium salts—I. Studies on anilinium oxides

The mass spectra of the three isomeric trimethylanilinium oxides and their methyl-d₃ analogues show that the m- and p- isomers undergo intermolecular trans-O-alkylation before evaporation. In the o-isomer, only 10% transalkylated product is observed and there is strong evidence that most of this isomer evaporates without undergoing structural changes. By indirect introduction, however, the o-isomer showed only transalkylated product. The most important fragmentation patterns on electron-impact are α-cleavage on the N-methyl carbon or expulsion of the O-substituent with formation of a quinoid structure. The latter dominates for the o- and p-methyl ethers while the former is the most important pathway for the m-isomer and for the corresponding phenols. Lower fragments are of modest intensity.     

Sheaf cohomology and free resolutions over exterior algebras

We derive an explicit version of the Bernstein-Gel'fand-Gel'fand (BGG) correspondence between bounded complexes of coherent sheaves on projective space and minimal doubly infinite free resolutions over its ``Koszul dual' exterior algebra. Among the facts about the BGG correspondence that we derive is that taking homology of a complex of sheaves corresponds to taking the ``linear part' of a resolution over the exterior algebra.

We explore the structure of free resolutions over an exterior algebra. For example, we show that such resolutions are eventually dominated by their ``linear parts" in the sense that erasing all terms of degree 1$"> in the complex yields a new complex which is eventually exact.

As applications we give a construction of the Beilinson monad which expresses a sheaf on projective space in terms of its cohomology by using sheaves of differential forms. The explicitness of our version allows us to prove two conjectures about the morphisms in the monad, and we get an efficient method for machine computation of the cohomology of sheaves. We also construct all the monads for a sheaf that can be built from sums of line bundles, and show that they are often characterized by numerical data.

     

Sheet metal welding using a pulsed Nd: YAG laser-robot

This paper presents a pulsed Nd: YAG laser-robot system for spot and seam welding of mild steel sheets. The study evaluates the laser beams behaviour for welding, and then investigates pulsed Nd: YAG laser spot and seam welding processes. High pulse power intensity is needed to initiate the key-hole welding process and a threshold pulse energy to reach full penetration. In seam welding, a weld consists of successive overlapping spots. Both high pulse energy and high average power are needed to keep the key-hole welding going. A 70% overlap is used to define overlapping spot welding as seam welding and to optimize process parameters because a high tensile strength joint compatible with the strength of the base material can be obtained when the overlap is ≥70%; at the same time a smooth seam with full penetration is obtained. In these cases, the joints in pulsed Nd: YAG laser welding are comparable in strength to those obtained with CO₂ laser welding. Robot positioning and motion accuracies can meet the demands of Nd: YAG laser sheet metal welding, but its cornering accuracy affects the welding processes. The purpose of the study is to evaluate the YAG laser-robot system for production in the automotive industry.     

Imaging and quantifying transverse flow velocity with the Doppler bandwidth in a phase-resolved functional optical coherence tomography

The Doppler bandwidth extracted from the standard deviation of the frequency shift in phase-resolved functional optical coherence tomography (F-OCT) was used to image the velocity component that is transverse to the optical probing beam. It was found that above a certain threshold level the Doppler bandwidth is a linear function of flow velocity and that the effective numerical aperture of the optical objective in the sample arm determines the slope of this dependence. The Doppler bandwidth permits accurate measurement of flow velocity without the need for precise determination of flow direction when the Doppler flow angle is within +/-15 degrees perpendicular to the probing beam. Such an approach extends the dynamic range of flow velocity measurements obtained with the phase-resolved F-OCT.     

Serial MR imaging of intracranial metastases after radiosurgery

Purpose: To evaluate the spatiotemporal evolution of radiosurgical induced changes both in metastases and in normal brain tissue adjacent to the lesions by serial magnetic resonance (MR) imaging. Methods and Materials: Thirty-five intracranial metastases of different primaries were treated in 25 patients by single high-dose radiosurgery. MR images acquired before radiosurgery were available in all patients. Sixty-three follow-up MR studies were performed in these patients including T₂- and contrast-enhanced T₁-weighted MR images. The average follow-up time was 9 ± 5 months (mean ± standard deviation [SD]). Based on contrast-enhanced T₁-weighted MR images, tumor response was radiologically classified in the following four groups: stable disease was assumed if the average tumor diameter after treatment did not show a tumor shrinkage of more than 50% and an increase of more than 25%, partial remission as a shrinkage of tumor size of more than 50%, a disappearance of contrast-enhancing tumor as a complete remission, and an increase of tumor diameter of more than 25% as tumor progress. Moreover, we analysed signal changes on T₂-weighted images in brain parenchyma adjacent to the enhancing metastases. Results: The overall mean survival time was 10.5 ± 7 months, with a 1-year actuarial survival rate of 40%. Stable disease, partial or complete remission of the metastatic tumor was observed in 22 patients (88%). Central or homogeneous loss of contrast enhancement appeared to be a good prognostic sign for stable disease or partial remission. This association was statistically significant (p < 0.05). Three patients (12%) suffered from tumor progression. In eight patients (32%) with stable disease or partial remission, signal changes on T₂-weighted images were observed in tissue adjacent to the contrast enhancing lesions. A progression of the high signal on T₂-weighted images was seen in seven of the eight patients between 3 and 6 months after therapy, followed by a signal regression 6–18 months after irradiation. Conclusion: MR imaging is a sensitive imaging tool to evaluate tumor response as well as the presence or absence of adjacent parenchymal changes following radiosurgery. Loss of homogeneous or central contrast enhancement on Gd-enhanced MR images appeared to be a good prognostic sign for tumor response. Tumor shrinkage seems not to be dependent on time. In addition, most cases of radiation induced changes in normal brain parenchyma observed on T₂-weighted images seem to be self limited.     

Winding Clusters in Percolation on the Torus and the Möbius Strip

Using a simulation technique introduced recently, we study winding clusters in percolation on the torus and the Möbius strip for different aspect ratios. The asynchronous parallelization of the simulation makes very large system and sample sizes possible. Our high accuracy results are fully consistent with predictions from conformal field theory. The numerical results for the Möbius strip and the number distribution of winding clusters on the torus await theoretical explanation. To our knowledge, this study is the first of its kind.