Pyridine nucleotide metabolism in mammalian cells in culture

The biosynthesis of pyridine nucleotides has been examined in a number of mammalian cell lines in culture. In all lines examined, nicotinamide is incorporated by a biochemical pathway distinct from the Preiss-Handler pathway for nicotinic acid. In at least the human cell line D98/AH2, there is no detectable endogenous synthesis of the pyridine ring from tryptophan. Although most cell lines examined (hamster BHK 21/13, mouse L929 and human D98/AH2) use either nicotinic acid or nicotinamide as a precursor for DPN and TPN, two mouse cell lines, 3T3-4E and LM CIID, are unable to utilize nicotinic acid as a source of the pyridine ring. If nicotinic acid is present in the medium, substantial amounts of intracellular desamido DPN accumulate suggesting that the last step (desamido DPN→DPN) is limiting in the Preiss-Handler pathway. With nicotinamide, the only compound which accumulates in substantial amounts apart from DPN and TPN is nicotinamide ribose; there is no detectable NMN. The results of pulse-labeling experiments suggest that nicotinamide ribose may be an intermediate in the nicotinamide pathway. Following growth of D98/AH2 cells in high concentrations of niacin, biosynthesis of DPN from nicotinamide was completely inhibited for at least six hours. The converse experiment revealed no inhibition of niacin incorporation. This observation suggests that a niacin pathway intermediate, which present evidence indicates is desamido-DPN. can inhibit nicotinamide utilization. Newly synthesized DPN turns over with a half-life of two hours in azaserine-treated D98/AH2 cells. In the absence of azaserine, the nicotinamide moiety of newly synthesized DPN is lost from D98/AH2 cells to the medium with a half-life of eight hours. About 80% of the nicotinamide is lost to medium as nicotinamide ribose.     

Bacterial mesosomes: Real structures of artifacts?

The ultrastructural study of membrane organization in gram-positive bacteria related to the OsO₄ fixation conditions revealed that large, complex mesosomes are observed only when the bacteria are subjected to an initial fixation with 0.1% OsO₄ in the culture broth, as in the prefixation step of the Ryter-Kellenberger procedure. Evidence was obtained suggesting that the large mesosomes are produced by this prefixation. The kinetic study of the membrane morphological alterations occurring during the prefixation of Bacillus cereus with 0.1% OsO₄ in the culture broth showed that the amount of mesosome material increases linearly from zero to a maximum observed at 1.7 min of prefixation and that at about this time a maximum is reached for the number of mesosomes per unity of cell area and for the average individual mesosome area. The large mesosomes observed in gram-positives fixed by the complete Ryter-Kellenberger procedure would be the result of the membrane-damaging action of 0.1% OsO₄. Such damaging action was deduced from the observation that 0.1% OsO₄ quickly lyses protoplasts and induces a quick and extensive leakage of intracellular K⁺ from B. cereus and Streptococcus faeculis. In support of that interpretation is the observation that in bacteria subjected to several membrane-damaging treatments, mesosome-like structures are seen after three different fixation procedures. In bacteria initially fixed with 1% OsO₄, 4% OsO₄ or 2.5% glutaraldehyde, no large, complex mesosomes are observed, small and simple invaginations of the cytoplasmic membrane being present. The size of these minute mesosomes is inversely proportional that causes of fixation. Uranyl acetate was found among the studied fixatives the one to the rate the least damage to bacterial membranes. This fixative satisfactorily preserves protoplasts. In bacteria initially fixed with uranyl acetate no mesosomes were found. The results of the present work throw serious doubts on the existence of mesosomes, both large and small, as real structures of bacterial cells. It is proposed that a continuous cytoplasmic membrane without infoldings (mesosomes) would be the real pattern of membrane organization in gram-positives.     

Identification of Isn1 and Sdt1 as Glucose- and Vitamin-regulated Nicotinamide Mononucleotide and Nicotinic Acid Mononucleotide 5��-Nucleotidases Responsible for Production of Nicotinamide Riboside and Nicotinic Acid Riboside

Recently, we discovered that nicotinamide riboside and nicotinic acid riboside are biosynthetic precursors of NAD⁺, which are utilized through two pathways consisting of distinct enzymes. In addition, we have shown that exogenously supplied nicotinamide riboside is imported into yeast cells by a dedicated transporter, and it extends replicative lifespan on high glucose medium. Here, we show that nicotinamide riboside and nicotinic acid riboside are authentic intracellular metabolites in yeast. Secreted nicotinamide riboside was detected with a biological assay, and intracellular levels of nicotinamide riboside, nicotinic acid riboside, and other NAD⁺ metabolites were determined by a liquid chromatography-mass spectrometry method. A biochemical genomic screen indicated that three yeast enzymes possess nicotinamide mononucleotide 5′-nucleotidase activity in vitro. Metabolic profiling of knock-out mutants established that Isn1 and Sdt1 are responsible for production of nicotinamide riboside and nicotinic acid riboside in cells. Isn1, initially classified as an IMP-specific 5′-nucleotidase, and Sdt1, initially classified as a pyrimidine 5′-nucleotidase, are additionally responsible for dephosphorylation of pyridine mononucleotides. Sdt1 overexpression is growth-inhibitory to cells in a manner that depends on its active site and correlates with reduced cellular NAD⁺. Expression of Isn1 protein is positively regulated by the availability of nicotinic acid and glucose. These results reveal unanticipated and highly regulated steps in NAD⁺ metabolism.     

Fine Structure of Bacillus subtilis : I. Fixation

The fine structure of Bacillus subtilis has been studied by observing sections fixed in KMnO₄, OsO₄, or a combination of both. The majority of examinations were made in samples fixed in 2.0 per cent KMnO₄ in tap water. Samples were embedded in butyl methacrylate for sectioning. In general, KMnO₄ fixation appeared to provide much better definition of the boundaries of various structures than did OsO₄. With either type of fixation, however, the surface structure of the cell appeared to consist of two components: cell wall and cytoplasmic membrane. Each of these, in turn, was observed to have a double aspect. The cell wall appeared to be composed of an outer part, broad and light, and an inner part, thin and dense. The cytoplasmic membrane appeared (at times, under KMnO₄ fixation) as two thin lines. In cells fixed first with OsO₄ solution, and then refixed with a mixture of KMnO₄ and OsO₄ solutions, the features revealed were more or less a mixture of those revealed by each fixation alone. A homogeneous, smooth structure, lacking a vacuole-like space, was identified as the nuclear structure in a form relatively free of artifacts. Two unidentified structures were observed in the cytoplasm when B. subtilis was fixed with KMnO₄. One a tortuous, fine filamentous element associated with a narrow light space, was often found near the ends of cells, or attached to one end of the pre-spore. The other showed a special inner structure somewhat similar to cristae mitochondriales.     

STUDIES ON THE FIXATION OF ARTIFICIAL AND BACTERIAL DNA PLASMS FOR THE ELECTRON MICROSCOPY OF THIN SECTIONS

The process of fixation of DNA-containing plasms is investigated by macroscopical and electron microscopical observations on solutions of DNA, nucleohistones, as well as on bacterial nuclei. The following treatments were found to produce a gelation of a solution of DNA or nucleohistones: (a) OsO₄ fixation at pH 6 in the presence of amino acids (tryptone) and Ca⁺⁺. (b) Exposure to aqueous solutions of uranyl acetate. (c) Exposure to aqueous solutions of indium chloride. Observed in the electron microscope, these gels show a fine fibrillar material. From experiments in which solutions of DNA or nucleohistones are mixed with bacteria and treated together, it is concluded that the behavior of the bacterial nucleoplasm is similar to that of the DNA solutions. The appearance of birefringence indicates that uranyl acetate and indium chloride produce an orientation of the molecules of a DNA solution during gelation. Bacterial chromosomes fixed by these agents also show a certain order, while those fixed by the OsO₄-amino acid-Ca⁺⁺ formula do not. Whether or not the order can be considered to be artificial is discussed, and a tentative conclusion is presented: (a) Uranyl acetate may induce artificial order. (b) Fixatives which do not gel DNA probably result in the grossest artifacts. (c) OsO₄ fixation at pH 6 in the presence of amino acids (tryptone) and Ca⁺⁺ may give the most accurate preservation of the in vivo disposition of DNA (RK⁺ fixation).     

Quantitative assessment of cellular changes provoked by microwave enhanced fixation of parathyroids

Summary Rat parathyroids fixed by microwave enhancement, i.e. microwave irradiation in the presence of glutaraldehyde for 8 s and postfixation with OsO₄ after a delay of 5 min, were compared with parathyroids fixed by perfusion with glutaraldehyde followed by immersion in glutaraldehyde and finally in OsO₄. Morphometric analysis revealed that microwave enhanced fixation led to larger mean cell volume, to larger cell surface area, and to larger suface area in membranes of RER and secretory granules. Though it is not known by which method parathyroid cells are conserved closer to the living state it is obvious that microwave enhanced fixation retains more membranes but provokes centrifugal dislocation of membranes mimiking exocytosis.     

Turnover at nicotinamide adenine dinucleotide in cultures of human cells.

The rate of turnover of nicotinamide adenine dinucleotide (NAD) in the human cell line, D98/AH2, has been estimated by measuring the rates of entry into and exit from NAD molecules of 14C-adenine. In one set of experiments, cells were labeled by growth in medium containing 14C-adenine for six hours and then shifted to medium without labeled adenine. The loss of 14C-adenine from the adenine nucleotide and pyridine nucleotide pools was measured, and the data were analyzed using an analytical treatment which corrects for the relatively slow turnover of precursor pools. The loss of 14C-adenine from the NAD pool and from the precursor ATP pool could be related to the absolute rate of NAD breakdown. Under the experimental conditions used, the rate of NAD turnover ranged from 83,000 to 126,000 molecules per second per cell. In a complementary experiment cells were grown in the presence of unlabeled adenine, then shifted into medium containing 14C-adenine and the rate of entry of 14C-adenine into adenine and pyridine nucleotides was measured. The data were treated using a similar analysis to relate the rate of entry of 14C-adenine into NAD and the precursor ATP pools to the absolute turnover rate of NAD. This analysis gave a value for NAD turnover of 78,000 molecules per second per cell in excellent agreement with results from the pulse-chase experiments. The results from both types of experiment indicate that within D98/AH2 cells the half-life of an intact NAD molecule is 60 +/- 18 minutes. Thus, in a human D98/AH2 cell growing with a generation time of 24 hours, NAD is turning over at twice the rate found in Escherichia coli with a generation time of half an hour.     

Profiles of the biosynthesis and metabolism of pyridine nucleotides in potatoes (<Emphasis Type="Italic">Solanum tuberosum</Emphasis> L.)

As part of a research program on nucleotide metabolism in potato tubers (Solanum tuberosum L.), profiles of pyridine (nicotinamide) metabolism were examined based on the in situ metabolic fate of radio-labelled precursors and the in vitro activities of enzymes. In potato tubers, [³H]quinolinic acid, which is an intermediate of de novo pyridine nucleotide synthesis, and [¹⁴C]nicotinamide, a catabolite of NAD, were utilised for pyridine nucleotide synthesis. The in situ tracer experiments and in vitro enzyme assays suggest the operation of multiple pyridine nucleotide cycles. In addition to the previously proposed cycle consisting of seven metabolites, we found a new cycle that includes newly discovered nicotinamide riboside deaminase which is also functional in potato tubers. This cycle bypasses nicotinamide and nicotinic acid; it is NAD → nicotinamide mononucleotide → nicotinamide riboside → nicotinic acid riboside → nicotinic acid mononucleotide → nicotinic acid adenine dinucleotide → NAD. Degradation of the pyridine ring was extremely low in potato tubers. Nicotinic acid glucoside is formed from nicotinic acid in potato tubers. Comparative studies of [carboxyl-¹⁴C]nicotinic acid metabolism indicate that nicotinic acid is converted to nicotinic acid glucoside in all organs of potato plants. Trigonelline synthesis from [carboxyl-¹⁴C]nicotinic acid was also found. Conversion was greater in green parts of plants, such as leaves and stem, than in underground parts of potato plants. Nicotinic acid utilised for the biosynthesis of these conjugates seems to be derived not only from the pyridine nucleotide cycle, but also from the de novo synthesis of nicotinic acid mononucleotide.     

INFLUENCE OF GLUTARALDEHYDE AND/OR OSMIUM TETROXIDE ON CELL VOLUME, ION CONTENT, MECHANICAL STABILITY, AND MEMBRANE PERMEABILITY OF EHRLICH ASCITES TUMOR CELLS

Effects of fixation with glutaraldehyde (GA), glutaraldehyde-osmium tetroxide (GA-OsO₄), and osmium tetroxide (OsO₄) on ion and ATP content, cell volume, vital dye staining, and stability to mechanical and thermal stress were studied in Ehrlich ascites tumor cells (EATC). Among variables investigated were fixation time, fixative concentration, temperature, osmolality of the fixative agent and buffer, total osmolality of the fixative solution, osmolality of the postfixation buffer, and time of postfixation treatment in buffer (Sutherland, R. M., et al. 1967. J. Cell Physiol. 69:185.). Rapid loss of potassium, exchangeable magnesium, and ATP, and increase of vital dye uptake and electrical conductivity occurred with all fixatives studied. These changes were virtually immediate with GA-OsO₄ or OsO₄ but slower with GA (in the latter case they were dependent on fixative temperature and concentration) (Foot, N. C. 1950. In McClung's Handbook of Microscopical Technique. 3rd edition. 564.). Total fixative osmolality had a marked effect on cell volume with OsO₄ but little or no effect with GA or GA-OsO₄. Osmolality of the buffer had a marked effect on cell volume with OsO₄, whereas with GA or GA-OsO₄ it was only significant at very hypotonic buffer osmolalities. Concentration of GA had no effect on cell volume. Osmolality of the postfixation buffer had little effect on cell volume, and duration of fixation or postfixation treatment had no effect with all fixatives. Freezing and thawing or centrifugal stress (up to 100,000 g) had little or no effect on cell volume after all fixatives studied. Mechanical stress obtained by sonication showed that OsO₄ alone produced poor stabilization and that GA fixation alone produced the greatest stabilization. The results indicate that rapid membrane permeability changes of EATC follow fixative action. The results are consistent with known greater stabilizing effects of GA on model protein systems since cells were also rendered relatively stable to osmotic stress during fixation, an effect not noted with OsO₄. After fixation with GA and/or OsO₄ cells were stable to osmotic, thermal, or mechanical stress; this is inconsistent with several earlier reports that GA-fixed cells retain their osmotic properties.     

Amplification of pyridine nucleotide pools in mitogen-stimulated human lymphocytes

NAD⁺ levels in resting human lymphocytes obtained from 20 donors were found to be 69.9 ± 21.7 pmols/10⁶ cells. After 3 days of phytohemagglutinin (PHA) stimulation the NAD⁺ levels rose to 452 ± 198 pmols/10⁶ cells. NADH, NADP⁺ and NADPH also increased in mitogen-stimulated lymphocytes, but the major portion of the increase in total pyridine nucleotide pools was accounted for by the increase in NAD⁺. When PHA-stimulated lymphocytes were incubated in nicotinamide-deficient growth medium, there was no significant increase in their total pyridine nucleotide pools; however, the ratios of oxidized to reduced pyridine nucleotides changed in a similar fashion to cells grown in medium containing nicotinamide. When lymphocytes in nicotinamide-deficient medium were stimulated with PHA they increased their levels of DNA synthesis and cell replication in a similar fashion to cells growing in nicotinamide-supplemented media. Human lymphocytes were able to synthesize pyridine nucleotides from nicotinamide or nicotinic acid; however, in the absence of a preformed pyridine ring they did not efficiently use tryptophan for the synthesis of NAD. Uptake of [carbonyl-¹⁴C]nicotinamide and conversion to NAD was markedly increased in PHA-stimulated lymphocytes; these cells also showed a marked increase in activity of the enzyme adenosine-triphosphate-nicotinamide mononucleotide (ATP-NMN) adenylyl transferase.     

Relative speed of fixation of glutaraldehyde and osmic acid in plant cells measured by grana appearance in chloroplasts

Summary Brief fixation in a mixture of glutaraldehyde and OsO₄ caused stacked chloroplast grana membranes in leaf cells of wheat, barley, tobacco, maize, cowpea, pigweed or bean plants to distend and vesiculate. Fixation with glutaraldehyde followed by OsO₄ prevented this fixation artifact. In a fixative mixture, OsO₄ apparently reacted with cell contents before glutaraldehyde.     

Electron microscopic characterization of calcium-binding physodes in the green alga Mougeotia scalaris

Summary Effect of the covalently cross-linking agents glutardialdehyde and osmium tetroxide, and of adsorption of the vital dye, neutral red, to the matrix of the calcium-binding vesicles from the green alga Mougeotia scalaris has been analysed in situ, both in terms of structural preservation and of the calcium-binding capacity of the vesicles. Upon cell fixation in glutardialdehyde without OsO₄, the vesicles appear to dissolve, but upon simultaneous fixation in glutardialdehyde with OsO₄ (1% w/v), the vesicles retain a globular form, are evenly stained by osmium and appear to be surrounded by a membrane-like structure. This structure was also observed around the vesicles in cells preincubated for 10 min in 0.1 mM neutral red and then fixed in glutardialdehyde/OsO₄ for 1 h. More detailed information of the matrix structure is obtained when simultaneous fixation of the Mougeotia cells was shortened to 15 min: a membrane-like structure was no longer observed around the vesicles. After cell treatment in the presence of neutral red, no calcium at all was found inside the vesicles. A small amount of calcium remained, when cells were fixed simultaneously and extensively in the absence of neutral red. However, calcium was found, to a considerable extent, inside the vesicles after short simultaneous fixation of the cells in the absence of neutral red. Based on the ultrastructural and elemental features presented here, the calcium-binding vesicles in Mougeotia appear to represent a member of the large family of (calcium-binding) physodes in lower plants (CaBP).     

Redox involvement in acid secretion in the amphibian gastric mucosa

Summary Gastric fundic metabolism was studied by spectroscopic observation in frog mucosa during transitions of secretory status in vitro and by direct measurement of pyridine nucleotides and associated metabolites in biopsies of dog fundic mucosa also during secretory oxidation of the redox components from flavin adenine dinucleotide (FAD) to cytochromea ₃. Addition of histamine resulted in reduction of these components with onset of secretion by about 50%. In contrast, the effect of apparently, burimamide and subsequently histamine on the ratio of nicotinamide adenine dinucleotide to nicotinamide adenine dinucleotide, reduced (NAD⁺/NADH) was relatively slight. Further, the presence of burimamide substantially reduces the effect of amytal on the pyridine nucleotide spectrum and abolishes the effect of amytal on FAD and the cytochromes. Measurements of lactate, pyruvate, -ketoglutarate, NH₃ and glutamate in the dog showed that whereas the calculated NAD⁺/NADH ratio in the cytoplasm declined with onset of secretion, the calculated mitochondrial ratio rose. No change was noted in the nicotinamide adenine dinucleotide phosphate/nicotinamide adenine dinucleotide phosphate, reduced (NADP⁺/NADPH) ratio. It is concluded that (1) H₂ antagonists act by blocking substrate flow into the mitochondrial respiratory chain, (2) conversely, histamine stimulation acts at the level of substrate mobilization, and (3) there may be a cross-over in the mitochondrial chain between NAD⁺ and FAD.     

Verluste radioaktiv markierter Substanzen aus Pisum-Wurzeln nach Verfütterung von D-Glucose-14C im Verlauf unterschiedlicher Präparationsmethoden für die Elektronenmikroskopie

Zusammenfassung Die Verluste an radioaktiv markierten, aus d-Glucose-¹⁴C in Pisum-Wurzeln entstandenen Substanzen, die im Verlauf unterschiedlicher Präparationsmethoden für die Elektronenmikroskopie auftreten, werden mit einem Flüssigszintillationszähler bestimmt. Die Identifizierung der Metabolite erfolgt an Hand von Dünnschichtchromatogrammen und Autoradiographien. Nach Flüssigfixierung mit Glutaraldehyd (Glut.) und OsO₄ wird weniger äthanollösliche Radioaktivität, die zu ca. 76% in Glucose, sonst aber in Aminosäuren und N-freien Säuren vorliegt, extrahiert, als nach Einzelfixierung mit einem der genannten Fixierungsmittel.Durch stufenlose Acetonentwässerung, anstelle von Entwässerung in Aceton-bzw. Äthanol-Stufen und Propylenoxyd, ferner durch Zusetzen von Saccharose zu den Fixierungs- und Entwässerungs-Medien, kann die Extraktion weiter reduziert werden. Nach OsO₄-Räucherung und stufenloser Entwässerung bleibt die größte Menge an Radioaktivität im Gewebe. Der Aufenthalt in den hohen Aceton- und Äthanolstufen bringt hier keine hohen Verluste mit sich, dasselbe gilt für Propylenoxyd und Epon. Glut. und Acrolein unterscheiden sich hinsichtlich der Retention an Radioaktivität nicht wesentlich.In allen Präparationen besteht der größte Teil der verbleibenden Radioaktivität aus Glucose. Es wird vermutet, daß diese hauptsächlich auf Grund von Membranveränderungen zurückbleibt, die Aminosäuren zusätzlich durch ihre Reaktion mit den Fixantien.

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The effect of different preparation schedules for electron microscopy on the loss of labelled substances in roots of Pisum after feeding with ¹⁴C-glucose

Summary The losses of marked metabolites of ¹⁴C-glucose in Pisum roots were determined by means of liquid scintillation counting during different electron microscopic preparation schedules. ¹⁴C-glucose was applied to the roots. Metabolites were identified in thinlayer chromatographs and radioautographs. Fixation with glutaraldehyde (glut.) followed by OsO₄ rendered the radioactivity in the tissue less susceptible to ethanolic extraction than treatment with either glut. or OsO₄. The ethanolic extracts showed 76 per cent of their radioactivity in glucose, the remainder in amino acids and nitrogen-free acids. Continuous dehydration by acetone instead of stepwise dehydration by acetone, ethanol, and propylene oxide further reduces extraction. Even better results are achieved by adding sucrose to the fixation and dehydration media. Finally, after fixation with OsO₄ fumes followed by continuous dehydration, most of the incorporated radioactivity is retained in the tissue. The passage through high acetone and ethanol concentrations as well as through propylene oxide and epon does not cause significant losses. Glut. and acroleine do not differ significantly with respect to the retention of radioactivity. In all preparations the larger part of the retained radioactivity is found in glucose. It is suggested that glucose is predominantly retained because of membrane changes during fixation, whereas the amino acid retention is enhanced by reactions with the fixing agents.

     

Parathyroid cell variants may be provoked during immersion fixation

Summary Parathyroid glands of cattle, dogs, cats, mice and rats were immersed in glutaraldehyde or mixtures consisting of glutaraldehyde, formaldehyde and acrolein in either Na-phosphate, Na/K-phosphate or Na-cacodylate buffer, and postfixed with OsO₄ in the same buffers or, alternatively, in s-collidine.Excellent preservation of bovine, feline and murine parathyroid glands was achieved with fixation mixtures containing 1% glutaraldehyde, 1.5–2% formaldehyde and 2.5–5% acrolein in 0.1 M Na-cacodylate with or without Ca²⁺ and Mg²⁺, Na-phosphate or Na/K-phosphate at 4°C followed by postfixation with 1% OsO₄ in the same buffers or in s-collidine containing sucrose, Ca²⁺ and Mg²⁺. This procedure largely abolished the occurence of parathyroid cell variants. Bovine parathyroid glands were also satisfactorily preserved with 1% glutaraldehyde and 2% formaldehyde whereas 1% glutaraldehyde and 2.5 or 5% acrolein, lower or higher buffer osmolarity, or immersion at room temperature led to vacuolization of RER and to breakdown of membranes. In contrast, all fixation protocols led to the formation of dark and light cell variants and to multinucleated syncytial cells in dog and rat parathyroids. The results thus show that parathyroid cell variants arise during immersion fixation and that aldehydes, buffers and temperature are important factors for provoking parathyroid cell variants.     

ELECTRON MICROSCOPE OBSERVATIONS ON FROZEN-DRIED CELLS

To explore the problem of artefacts which may be produced during usual fixation, dehydration, and embedding, the authors have examined pancreas, liver, and bone marrow frozen at about -180°C., dried, at -55 to -60°C., embedded in methacrylate, sectioned, and floated on a formol-alcohol mixture. By these treatments the labile structure of living cells can be fixed promptly and embedded in methacrylate avoiding possible artefacts caused by direct exposure to chemical fixatives. Cell structures are ultimately exposed to a fixative when the sections are floated on formol-alcohol, but at this stage artefacts due to chemical fixation are expected to be minimized, as the fixatives act on structures tightly packed in methacrylate polymer. In the central zone of tissue blocks so treated, the cells are severely damaged by ice crystallization but at the periphery of the blocks the cell structure is well preserved. In such peripherally located cells, elements of the endoplasmic reticulum (ER), Palade's granules, homogeneously dense mitochondria, and nuclear envelopes and pores, can be demonstrated without poststaining with OsO₄. The structural organization in the nucleus is distorted by vacuolization. The mitochondrial membranes and cristae, cellular membrane, and the Golgi apparatus, however, are detected only with difficulty. The Golgi region generally appears as a light zone, in which some ambiguous structures are encountered. After staining the sections with OsO₄ or Giemsa solution, an inner mitochondrial structure which resembles the cristae seen in conventional OsO₄-fixed specimens appears, but the limiting membrane is absent. Treatment with OsO₄ or Giemsa solution also renders more distinct the membrane of the ER and Palade's granules but not the Golgi apparatus and cellular membrane. Treatment with ribonuclease results in the disappearance of Palade's granules. On the strength of these observations the authors conclude that OsO₄ fixation gives a satisfactory preservation of such cell structures as the nuclear envelope, endoplasmic reticulum, and Palade's granules, though it may induce slight swelling of these cell components.     

Heterotrophic Fixation of CO2 in Soil

The occurrence of heterotrophic CO₂ fixation by soil microorganisms was tested in several mineral soils differing in pH and two artificial soils (a mixture of silica sand, alfalfa powder, and nutrient medium inoculated with a soil suspension). Soils were incubated at ambient (∼0.05 vol%) and elevated (∼5 vol%) CO₂ concentrations under aerobic conditions for up to 21 days. CO₂ fixation was detected using either a technique for determining the natural abundance of ¹³C or by measuring the distribution of labeled ¹⁴C-CO₂ in soil and bacteria. The effects of elevated CO₂ on microbial biomass (direct counts, chloroform fumigation extraction method), composition of microbial community (phospholipid fatty acids), microbial activity (respiration, dehydrogenase activity), and turnover rate were also measured. Heterotrophic CO₂ fixation was proven in all soils under study, being higher in neutral soils. The main portion of the fixed CO₂ (98–99%) was found in extracellular metabolites while only ∼1% CO₂ was incorporated into microbial cells. High CO₂ concentration always induced an increase in microbial activity, changes in the composition of the microbial community, and a decrease in microbial turnover. The results suggest that heterotrophic CO₂ fixation could be a widespread process in soils.     

Silicotungstic acid for cytochemical localization of water soluble substance(s) of cholinergic motor nerve terminal

Summary Silicotungstic acid (STA), an electron dense substance and a powerful precipitating agent of quaternary ammonium salts such as choline and acetylcholine, was employed on the frog motor end-plate in order to prove that STA reacts with diffusible substance(s) in nerve terminals. Thus, STA treatment and osmium tetroxide (OsO₄) fixation were performed in three different ways. No reaction was detectable when STA treatment followed osmification, while simultaneous treatment with STA and OsO₄ darkened both presynaptic and synaptic vesicle membranes. When STA was employed directly on fresh tissues which were subsequently fixed by OsO₄, small black precipitates were observed in the synaptic vesicles and none on other synaptic structures. The possible reaction of STA with acetylcholine is discussed.     

Direct visualization of intracellular calcium in rat osteoblasts by energy-filtering transmission electron microscopy

Osteoblasts are the highly specialized bone cells responsible for matrix mineralization. Mineralization is a complex, incompletely understood, process involving intracellular calcium homeostasis. Rapid changes in ionized calcium concentration ([Ca²⁺]_i) occur in these cells, but the intracellular distribution of total calcium, which may be involved in matrix mineralization, remains unknown. We have therefore investigated the distribution of total calcium in osteoblasts either ex vivo from rapidly mineralizing neonatal rat bones or in the same cells cultured to confluence before they had entered the mineralization phase, and without stimulation for mineralized matrix formation. All cells were examined bone-untreated (controls) or following the addition of the ionophore ionomycin that induced a large and sustained increase in [Ca²⁺]_i. Cryomethods, quick-freezing and freeze-drying, and OsO₄ vapor fixation were employed to preserve the original calcium distribution, and the preservation was verified by secondary ion mass spectrometry (SIMS). Intracellular calcium distribution was identified by energy-filtering transmission electron microscopy (EELS). Scarce calcium signals were recorded from all osteoblasts maintained in buffer (controls). Ionomycin addition resulted in the accumulation of calcium in mitochondria, and more calcium was stored in the mitochondria of osteoblasts involved in mineralization than in those of osteoblasts before mineralization. Moreover, in the former, strong calcium signals were recorded around the junctions between mitochondria and the endoplasmic reticulum. Thus EELS allowed to obtain high-resolution total calcium maps in defined intracellular structures, but only at elevated calcium levels.     

Tissue fixation and osmium black formation with nonvolatile octavalent osmium compounds

Summary Several compounds of osmium^VIII, including potassium osmiamate and coordination complexes of OsO₄ with ammonia and various heterocyclic nitrogen compounds, have been synthesized and characterized. They have also been evaluated as substitutes for OsO₄ in postfixation of biological specimens and in light and electron microscopic cytochemical methods resulting in osmium black formation.The most useful of these osmic compounds, a molecular addition complex of hexamethylenetetramine (methenamine) with OsO₄, has a negligible vapor pressure of OsO₄. It has the molecular formula C₆H₁₂N₄.2OsO₄ and has been designated osmeth. Although it has only limited solubility, aqueous solutions of the compound (or of OsO₄) can be rapidly prepared by dissolution in a minimal amount of dimethylformamide and subsequent dilution with distilled water or buffer. Although stable in the solid state, the complex in solution undergoes partial dissociation releasing OsO₄, and the odor of OsO₄ becomes apparent.Such solutions of osmeth are (0.25%) considerably less concentrated with respect to OsO₄ than solutions (1–2%) ordinarily employed for ultrastructural preservation or in cytochemical studies. Osmeth has limited value for postosmication after glutaraldehyde fixation because the generation (release) of OsO₄ appears to be slow. Adequate osmication of tissue blocks exists only at the surface, but effective osmication can be achieved throughout tissue sections. In cytochemical reactions resulting in the formation of osmium blacks, the osmeth solutions are as effective as OsO₄ solutions of equivalent concentrations. Our findings indicate that OsO₄ solutions of less than 1% may be satisfactorily utilized in many cytochemical studies.Osmeth is safer and more convenient to handle than OsO₄ because small amounts may be solubilized as needed. It should be considered as a substitute for OsO₄ in ultrastructural cytochemistry.This investigation was supported by NIH research grant number DE 02668 from the National Institute of Dental Research and by NIH grant number RR 05333 from the Division of Research Facilities and ResourcesVisiting Professor, Dental Research Center, University of North Carolina at Chapel Hill, Jan.-May, 1975. Supported in part by USPHS Grant HD 09209