Fluorometric determination of DNA in fixed tissue using ethidium bromide

The measurement of DNA in tissue samples fixed in ethanol/acetic acid is described. Small, fixed tissue samples are digested by warm alkaline treatment followed by neutralization with HCl, and DNA is determined by complex formation with the dye ethidium bromide (EB). When standard DNA from calf thymus was treated similarly, a hyperchromicity of 8–12% and a reduction in fluorescence intensity of the EB-DNA complex to 55% was observed. The NaOH concentration (0.5–2.0 mol/liter) or the temperature (50–60°C) used for the digestion of tissue, as well as subsequent ribonuclease or protease treatment had no effect on the observed tissue DNA concentrations.     

Properties of detergent-solubilized and membranous (Ca2+ + Mg2+)-activated ATPase from sarcoplasmic reticulum as studied by sulfhydryl reactivity and ESR spectroscopy. Effect of protein-protein interactions

(1) Sulfhydryl reactivity and electron spin resonance spectra of nitroxide maleimide spin labels, covalently attached to sarcoplasmic reticulum ATPase, were examined on both detergent-solubilized and membranous material. Monomeric and oligomeric ATPases were prepared by the use of dodecyloctaethylene glycol monoether as a solubilizing detergent. (2) Immediately after solubilization, the reaction curve of nonomeric ATPase with 5,5'-dithiobis(2-nitrobenzoate) was characterized by positive cooperativity (S-shaped as a function of time). In contrast, the SH reactivity of both oligomeric and membranous ATPases obeyed usual first-order kinetics and could be analyzed in terms of three classes of reactive site. All enzymatically active ATPase preparations responded to addition of ADP with a decrease in SH reactivity. During enzymatic inactivation of monomeric ATPase, the SH-modification rate was dramatically enhanced with loss of cooperative features. Ca2+ removal from the high-affinity sites stimulated SH reactivity before inactivation had taken place. (3) ESR spectroscopy indicated less motional constraints on monomeric than on oligomeric and membranous ATPases. Arrhenius plots of ESR spectral parameters suggest a conformational transition in both membranous and solubilized ATPases at about 22 degrees C. The transition was also present in EGTA-, but not in heat-inactivated ATPase. Although SH reactivity of monomeric ATPase was dramatically enhanced by EGTA inactivation, the results of ESR, circular dichroism and analytical ultracentrifugation experiments indicate limited conformational changes induced by EGTA treatment. (4) The data indicate marked differences in the properties of monomeric ATPase on the one hand and oligomeric and membranous enzymes on the other hand. They are consistent with previous functional evidence for the presence of ATPase in an associated state in the membrane (M?ller, J.V., Lind, K.E. and Andersen, J.P. (1980) J. Biol. Chem. 255, 1912-1920).     

Absolute rates of cholesterol synthesis in extrahepatic tissues measured with 3H-labeled water and 14C-labeled substrates.

This study was undertaken to develop techniques for measuring absolute rates of sterol synthesis in extrahepatic tissues in vitro and to estimate the magnitude of the errors inherent in the use of various 14C-labeled substrates for such measurements. Initial studies showed that significant errors were introduced when rates of synthesis were estimated using [3H]water since about 20 nmol of water were bound to each mg of tissue cholesterol isolated as the digitonide. This source of error could be eliminated by subtracting apparent incorporation rates obtained at 0 degrees C from those obtained at 37 degrees C or by regenerating and drying the free sterol. In a second set of experiments, the H/C incorporation ratio in cholesterol was determined in the liver by measuring the absolute rates of hydrogen and acetyl CoA flux into sterols. The ratio of 0.69 +/- 0.03 was found to be independent of the rate of hepatic cholesterol synthesis, the rate of hepatic acetyl CoA generation, or the source of the acetyl CoA. In a third set of studies, rates of incorporation of [3H]water or 14C-labeled acetate, octanoate, and glucose into digitonin-precipitable sterols were simultaneously measured in nine different extrahepatic tissues. Assuming that the H/C ratio measured in the liver also applied to these tissues, the [3H]water incorporation rates were multipled by the reciprocal of the H/C ratio to give the absolute rates of sterol synthesis in each tissue. When these were compared to the incorporation rates determined with the 14C-labeled substrates the magnitude of the errors in the rates of sterol synthesis obtained with these substrates in each tissue could be assessed. Only [14C]octanoate gave synthesis rates approaching 100% of those obtained with [3H]water and this occurred only in the intestine and kidney; in the other extrahepatic tissues this substrate gave rates of 6--66+ of the absolute rates. Rates of [14C]acetate incorporation in sterols varied from 4 to 62% of the [3H]water incorporation rates while those obtained with [14C]glucose were only 2--88% of the true rates. These studies document the large and highly variable errors inherent in estimating rates of sterol synthesis in extrahepatic tissues using 14C-labeled substrates under in vitro conditions.     

Effect of penicillin on the morphology of a Neisseria meningitidis strain liberating free endotoxin. An electron microscope study

An endotoxin-liberating strain of Neisseria meningitidis plasmolysed extensively after 2 h of exposure to 100 times MIC values of benzypenicillin. The peptidoglycan layer could be demonstrated after 2 h of treatment in places where the cytoplasm still was close to the cell wall. After 20 h, however, this layer was complete undetectable. In untreated cells the peptidoglycan layer could more easily be found in older cultures than in very young cultures. An increased adhesiveness and aggregation to other bacterial cells and to cell wall material could be observed after 2 h of penicillin treatment, and more pronouncedly after 20 h. A high yield of free cell wall material could be demonstrated after 2 h of penicillin treatment. This corresponded well to an increased content of free endotoxin in the filtrates from the cell cultures treated with penicillin, compared to untreated controls. After 20 h of treatment, free cell wall material had formed large aggregates or was adherent to the cell walls of ghost cells. The corresponding endotoxin analysis showed a reduced content of filtrable endotoxin. Possible implications of the structural changes in relation to penicillin treatment of patients are discussed.     

Sesquiterpenes of thalloid liverworts of the genera Conocephalum,Lunularia, Metzgeria and Riccardia

Thalloid liverworts of orders Metzgeriales and Marchantiales elaborate essential oils distinguishable from those of the Jungermanniales by the absence of β-barbatene and anastreptene. Riccardia sinuata elaborates a novel tricyclic exomethylene sesquiterpene of as yet undetermined structure. Conocephalum conicum elaborates cadinene-type sesquiterpenes. β-Cadinene from the latter species is clearly enantiomeric to the same product from vascular plants.     

The pyridine nucleotide and non-pyridine nucleotide dependence of L-glutamate dehydrogenase in the histochemical system

Summary Under histochemical conditions (fresh frozen sections from liver, kidney and cerebellum of the rat) it was shown that the oxidation of L-glutamic acid was carried out by the NAD-dependent L-glutamate dehydrogenase (E.C. 1.4.1.2) and/or the NAD- or NADP-dependent L-glutamate dehydrogenase (E.C. 1.4.1.3) as well as by an enzyme system which is not dependent on externally added NAD, NADP, FAD, FMN or CoQ₁₀ for activity.This non-pyridine dependent activity was related to the L-glutamate dehydrogenases proper, owing to the following: a) the localization of activity noticed corresponds to that obtained with the NAD- or NADP-containing media, b) the incubation time needed for initial formation of red and blue formazans is similar to that with coenzyme-containing media, c) pre-extraction experiments reveal similarity in enzyme diffusion rates, d) the named activity is influenced by the same agents and to the same extent as the activity obtained by the inclusion of NAD or NADP (e.g. dissociation of the dehydrogenase molecule into subunits due to urea, inhibition of activity due to N-ethyl maleimide and 1.10-phenanthroline, activation due to the allosteric effect of ADP and to high substrate concentration, allosteric inhibition caused by GTP and inhibition caused by -ketoglutaric acid, no inhibitory effect of KCN), and e) the named activity was not affected by added PMS (excluding activity due to L-aminoacid oxidase).In the in situ localization of enzyme activity it was found that L-glutamate dehydrogenases E.C. 1.4.1.2 and E.C. 1.4.1.3 co-exist in the cells of kidney and cerebellum, while the L-glutamate dehydrogenase E.C. 1.4.1.3 only was present in liver cells.Finally, it was stated that incubation time should be kept as short as possible in order to avoid Nothing dehydrogenase reaction as well as inhibition due to accumulation of -ketoglutaric acid. Only gel incubation media should be applied.Recipient of a research grant from the Danish Ministry of Education     

Does abscisic acid influence proline accumulation in stressed leaves?

Root treatments of barley (Hordeum distichum L.) plants with 10^-7 to 10^-4 M abscisic acid (ABA) caused an increase in proline content, especially at higher concentrations, within 2–3 h. Even 3 h after the removal of ABA from the medium the plants continued to accumulate proline. The higher the concentration of the ABA, the higher was the proline level at 6 h. When the highest ABA concentration, 10^-4 M, was tested with polyethylene glycol (PEG) (-5.0 bars) in the medium, the ABA treatment resulted in a higher proline content than in control plants. The treatments PEG alone and PEG + ABA resulted in heavy accumulation of proline, especially, 3 h after releasing the plants from the stress. The proline content in PEG+ABA-treated plants was always higher than plants treated with PGE or ABA alone. In peas (Pisum sativum L. cv Alaska) the same trend occurred although to a lesser degree. These findings indicate an influence of ABA on proline accumulation in water-stressed plants.Abbreviations ABA abscisic acid - PEG polyethylene glycol - RWC relative water content     

Synthesis of diastereomeric bis-unsaturated prostaglandins

With the report given herein all diastereomers of PGF₂, PGE₂, and PGD₂ which bear the naturally recognized 15-S hydroxylated center, whether in the natural or $\text{ent}$-prostanoic acid skeleton, have been prepared by a route involving initial introduction of the carboxyl (α) chain (1). A major advantage of the initial α-ylation⁺ route is the facile reduction of the 13,14-en-15-one system with methanolic NaBH₄ which proceeds without competing 1,4-reduction. The products are thus free of 13,14-dihydro-PG₂ contaminants (2). The initial pharmaco logical evaluation of these diastereomers will be submitted for publication in this journal (3).