Bo Thorell

MICROSPECTROFLUOROMETRIC APPROACH TO THE STUDY OF FREE/BOUND NAD(P)H RATIO AS METABOLIC INDICATOR IN VARIOUS CELL TYPES

Under excitation at 365 nm, the cell fluorescence is mainly due to bound and free NAD(P)H, plus a small contribution from flavins. Resolution is first attempted in the simplest case. i.e. the increase spectrum (δIf) due to microinjection of glucose‐6‐phosphate (G6P) into EL2 ascites cells. Above 510 nm, δIF is identical to the spectrum of free NADH. Below 510 nm. the presence of a second component is suggested, i.e. the intensity of the free NADH spectrum is lower than the measured δIF level. The difference between δIf and the free NADH spectrum (maximum at 475 nm) yields a spectrum suggestive of bound NADH with maximum at 450 nm. Thus, with free and bound NADH, the entire δIF can be reconstructed, with some assumptions as to the relative quantum yields of the two components. This seems to leave no place for a flavin component.

Cyclic variations in the peptidase and catheptic activities of yeast cultures synchronized with respect to cell multiplication

The total “over-all” dipeptidase and proteinase A activities of normal and X-irradiated cultures of Saccharomyces cerevisiae were assayed during various physiological states. Yeast cultures first starved for some hours in succinate buffer were then made to undergo two or three somewhat synchronous cell divisions in a yeast extract growth medium. The cytological characteristics were followed as well as the content of UV-absorbing cytoplasmic materials. The enzymic activities were expressed on a per-cell basis and also per protein content. During the starvation phase the protein content per cell remained high but a marked decrease of dipeptidase activity per cell was noted, while the proteinase A activity showed a minor decrease. During the subsequent growth and cell division period marked cyclic variations occurred in the enzyme activities, more marked for the peptidase and less for the proteinase. The highest enzymic activities always coincide with stages preparatory to cell division, and the lowest levels were reached during each subsequent budding period. After 15,000 r of X-irradiation the first division occurred normally in timing as in protein and enzyme content. The second division was delayed and many of the cells inhibited from budding. After this division the nitrogen content per cell increased to about 150 per cent of the normal, and the enzyme content remained high. The cells were left in a state preparatory to cell division. No appreciable enzyme activity was detected in the culture medium during the various phases of normal yeast cell growth or in the post-irradiation period. The results indicate that cycles of increased proteolysis form essential steps during certain stages of cell growth preparatory to cell division. The idea is advanced that the proteinases and peptidases may be operative in the intracellular reactions aiming at the replenishment of the metabolic amino acid pool necessary for protein synthesis.

DNA, RNA and hemoglobin during erythroblast maturation. A cytophotometric study.

Abstract Cytophotometric determination of the DNA, RNA, dry mass, and hemoglobin content was made in individual erythropoietic bone marrow cells from 4 normal subjects. The Hb content and concentration increased, and RNA content and concentration decreased with increasing maturation of erythropoietic cells. Hemoglobin was accumulated mainly during G1 and early S phases of DNA synthesis in proliferating cells. In middle and late S and G2 phases of DNA synthesis the mean Hb content of each maturation stage was mainly unchanged and the Hb concentration decreased, due to dilution by newly synthesized non-heme-proteins. It is postulated that DNA is repressed in G1 phase of DNA synthesis through interaction of hemoglobin at a certain Hb concentration, in this work, at MCHC of about 20%.

Ribonuclease and desoxyribonuclease activities in normal and regenerating bone marrow homogenates.

Abstract 1. 1. Phenylhydrazine was used to induce regeneration of chicken bone marrow. 2. 2. The mitotic frequencies were determined and showed up to a 10-fold increase in the regenerating tissues. 3. 3. The pentose and desoxypentose nucleic acids were determined in the two types of tissue. A consistent increase in the PNAP/DNAP quotients was noted in the regenerating tissues. 4. 4. The ribonuclease activity in the bone marrow homogenates has its pH optimum near 7.5. A correlation between the enzymic activity and the state of growth was found. 5. 5. The desoxyribonuclease activity in the bone marrow homogenates shows a pH optimum near 5.7. The influence of magnesium at different pH values was studied. A good correlation between the desoxyribonuclease activity and the state of growth of the tissue was found. 6. 6. The relationship between the metabolism of the pentose and desoxypentose nucleic acids and the corresponding enzymic activities is discussed.

An electron microscopic study of the nuclear abnormalities in erythroblasts in beta-thalassaemia major.

Summary. Erythroblasts in the peripheral blood of 20 splenectomized patients with β‐thalassaemia were examined by electron microscopy. A number of alterations in the structure and integrity of the nucleus and nuclear membrane was noted and these included apparent widening of the nuclear pores, partial absence and areas of reduplication of the nuclear membrane. Intranuclear inclusions were found, some identical to the electron‐dense inclusions (Heinz bodies) present in the cytoplasm, while others contained aggregates of finer particles, probably iron micelles. Some erythroblast nuclei appeared to contain portions of cytoplasm with similar inclusions. Nuclear alterations were most prominent when inclusion bodies were abundant in the cytoplasm and when they were in close approximation to or in contact with nuclear material. These nuclear abnormalities may be related to the recognized disturbance in proliferation of thalassaemic erythroblasts and the ineffective erythro‐poiesis encountered in this disease.

32‐msec scan of the NAD(P)H fluorescence spectrum in single living cells

A multichannel microspectrofluorometer based on an electron bombardment silicon camera tube, a multiscaling computer, and a miniature Amici prism for spectrometer, allows the scanning of the NAD(P)H fluorescence emission spectrum from a 30 μ region in a single living EL2 ascites cancer cell, within 32 msec. Observations of changes in the relative maximum emission intensities of two fluorochromes within a same cell are quite possible (for emission maxima ∼ 10–20 nm apart). Thus, in the fluorescence emission spectrum of cells incubated with benzpyrene or dibenzanthracene, the 423 nm (polycyclic hydrocarbon)/443 nm [NAD(P)H] ratio is 1.25 or over, while it decreases to 1.0 or less upon microelectrophoretic addition of glucose‐6‐phosphate [NAD(P) reduction].

Rapid multichannel microfluorometry for metabolic and spectral studies in single living cells

Abstract A multichannel microspectrofluorometer has been developed for operation on two modes, a ‘morphological’ mode for the assay of intracellular fluorochromes (e.g. NAD(P)H) in correlation with topography, and a ‘spectral’ mode for wavelength analysis of natural cell fluorescence. This instrument is based on an electron bombardment silicon camera tube (EBS) operated in conjunction with a multiscaling computer. The total NAD(P)H emission from 2 × 30 μm cell strips can be analysed in real time (32.8 msec frame scan) with a signal-to-noise ratio over 100:1. The metabolic changes in cytoplasmic regions are compared with those in regions comprising cytoplasm + nucleus, where the major contribution may be nuclear (cf earlier studies). The observation of a ‘multilocalized’ and asynchronous metabolic response is facilitated with substrates such as glucose-1-phosphate, associated with a longer lag period before the initiation of fluorescence changes. The latter largely occur in the 440–480 nm region. Fluorescence spectra recorded from intracellular regions are nearly super-posable to the spectrum obtained from NAD(P)H crystals.

Enzyme-substrate kinetics associated with transient state conditions in single living cells

Abstract 1. 1. The transient changes in NAD+ reduction (fluorescence pulse) which result from the microelectrophoretic addition of substrate in stepwise increasing amounts can be followed in single living EL2 cells by microfluorimetry. With glucose 6-phosphate ( Glc -6-P ) as substrate, each fluorescence pulse represents the summated contributions of glyceraldehyde phosphate dehydrogenase (NAD+-reducing) and lactic dehydrogenase (NADH-reoxidizing). 2. 2. The plot of Glc -6-P concentration against the area of the fluorescence pulse reveals sigmoidal kinetics, which are altered by adenine nucleotides, lactic dehydrogenase inhibitor and substrates, etc. In all these cases the kinetics of NAD+ reduction in vivo favour a higher-order relationship with regard to substrate in the optimum concentration range. 3. 3. When Glc -6-P is increased gradually metabolic block sets in more easily than if optimal doses are added at once or suboptimal doses by continuous slow drip. 4. 4. While the substrate levels required for half-maximal and maximal NAD+ reduction are somewhat lowered in presence of ADP and P1 as compared to Glc -6-P alone, there is considerable parallelism between the rates of substrate utilization under all these conditions. The rate is severely reduced in presence of ATP and maximal NAD+ reduction occurs at a much lower concentration of Glc -6-P . 5. 5. At suboptimal or optimal levels of substrate, lactic dehydrogenase is quite efficient in reoxidizing NADH, but the latter can accumulate considerably when the cell is overloaded with substrate. In presence of lactate and oxamate the rates of Glc -6-P utilization are considerably lowered, but the NAD+ reduction proceeds quite adequately and occurs at substrate concentrations much lower than with Glc -6-P alone. 6. 6. The EL2 cells seem self-sufficient in terms of endogenous activators of the glycolytic chain, so that they depend mainly on the supply of substrate. However, in the living cell the interplay between mitochondrial and extramitochondrial compartments is suggested by succinate and ATP experiments.

Pathways of glucose metabolism in irradiated and non-irradiated tissue culture ascites cells☆☆☆

Abstract The microfluorimetric-microelectrophoretic technique which allows the fluorescence assay of reduced pyridine nucleotides in localized cellular portions during microelectrophoretic treatment with metabolites, has been used for comparative experiments between irradiated giant tissue culture cells (EL2G) and their non-irradiated counterparts (EL2SG). There are preliminary indications that the extramitochondrial fluorescence responses to glucose or microelectrophoretic mixtures containing glucose-6-phosphate, fructose-1,6-diphosphate and 6-phosphogluconate are more intense in the irradiated cell, while there is a higher response to uridine-diphosphoglucose in the non-irradiated cell. The responses to a multipotential component such as fructose-6-phosphate are similar in both cell types. The non-irradiated cells are more sensitive to Rotenone (an inhibitor of DPNH-cytochrome b reductase) which enhances the extramitochondrial fluorescence response to substrates of glucose catabolism. The higher glycolytic activity and lower sensitivity to Rotenone seen in the irradiated cell may be due to radiation damage of their respiratory membrane structures, e.g. the mitochondrial lipoprotein membranes.

A topographic analysis of metabolic pathways in single living cells by multisite microfluorometry

Abstract Multichannel (multisite) microfluorometry in conjunction with microinjection of metabolic intermediates (e.g. glycolytic phosphate esters) was used for in situ topographic analysis of metabolic transients (e.g. NAD(P)NAD(P)H) in correlation to structure and compartmentalization of single living EL2 and L cells. In cells submitted to repeated microinjections with different doses of substrate (glucose-6-P or -1-P, 6-phosphogluconate, allosteric factors) rate laws were derived by a power or exponential approximation. On this basis intracellular metabolic rates were evaluated topographically and the multisite-multicomponent control of enzyme pathways in integrated biochemical systems was assessed. From different simulation trials it appeared that the transients observed are best simulated by a difference of exponentials, accounting for NAD(P) reducing and reoxidizing pathways. The determination of intracellular metabolic rate laws, their multisite-multicomponent control and the extent to which topographic discrimination of compartmentalization is possible provide the basis for application to specific problems in cell physiology, specialized cell function or the understanding of multicellular steady states.