R.b.e. and o.e.r. of extended-Bragg-peak helium ions: survival and development of rat embryos.

Rats were exposed under aerobic or hypoxic conditions to 200-1200 rads of 60Co gamma-rays or extended-Bragg-peak helium ions on the eighth day of gestation. Uterine contents were examined on the twentieth day of gestation. At the 50 per cent embryonic survival level, helium ion r.b.e. was 1(.0) (aerobic) and 1(.2) (hypoxic). Maximum attainable gamma-ray and helium-ion o.e.r.s. were 2(.2) and 1(.7) respectively, indicating an oxygen-effect gain (o.e.g.) of 1(.2). At the 10 per cent survival level helium ion r.b.e. was 1(.1) (aerobic) and 1(.4) (hypoxic). Gamma-ray and helium-ion 0.e.r.s. were 2(.0) and 1(.5) respectively, indicating a helium ion o.e.g. of 1(.3). These data demonstrate that the small fraction of high-LET radiation present in this helium ion beam has a neglible effect on the aerobic r.b.e., but lowers the effective o.e.r. of the beam approximately 25 per cent relative to that of gamma-rays. Helium ions were significantly more effective than gamma-rays in killing embryos under hypoxic conditions, in producing congenital abnormalities under aerobic conditions, and in stunting foetal growth under both conditions.     

Oxygen effect onγ-irradiated DNA

Summary DNA from bacteriophage T1 or calf thymus DNA was-irradiated in dilute solution at 200 µg/ml in absence and in presence of oxygen. A variety of physico-chemical and biological properties was measured and the respective oxygen enhancement ratios (o.e.r.) were calculated. For the occurrence of single and double strand breaks, formation of terminal phosphate end groups and for the base damage o.e.r.-values between 2 to 4 are measured. Whereas the formation of actual breaks results to an o.e.r. of 1.8, the induction of alkalilabile lesions in the nucleotide strand seems to occur rather exclusively in presence of oxygen. In contrast to physico-chemical alterations, the inactivation of biological functions does not show any oxygen effect as determined by the priming activity of thymus DNA with respect to enzymatic RNA synthesis or by the infectivity of T1-DNA in a spheroplast system withE. coli hcr⁺ (K12) or hcr^– (K12 A16). The discrepancy between the o.e.r. for biological inactivation and that for the structural damage in DNA is discussed.     

Adding two components of radiosensitization by oxygen

It has been shown, or inferred, in various contexts that radiosensitization of cells by oxygen is the sum of two (or more) components. If the component sensitivities conform with the Alper and Howard-Flanders equation their sum cannot also conform, but, in practice, even the most meticulous experimental techniques will fail to reveal lack of conformity unless one of the component K values is at least nine times the other. Thus, despite the many results that have demonstrated conformity with the equation, the existence of at least two components may well be a general phenomenon. The killing of cells by radiation is attributable to a summation of lesions in different structures; different K values for the contributing components are therefore to be expected, since neither oxygen nor its competitors are likely to be present in uniform concentration in all elements of the cell nucleus. Provided the components have intrinsic values of o.e.r. greater than one, their addition results in sensitivity that increases monotonically with PO2, approaching asymptotically to the overall o.e.r. which is a weighted average of the component o.e.r.s. In a curve plotted with PO2 on a linear scale a point of inflection can occur only if one component o.e.r. has a value less than one (i.e. oxygen is protective for that component), and then only if relationships between the other parameters satisfy certain conditions. In cases in which points of inflection in the sensitivity curve has been observed these are unlikely to be accounted for by the addition of two components. The analysis of the consequences of adding two components of oxygen sensitization could apply also to chemical sensitization of hypoxic cells.     

Bacterial respiration-linked proton translocation and its relationship to respiratory-chain composition.

1. The relationship between chain composition and the efficiency of respiration-linked proton translocation was studied in nine bacterial species of widely differing taxonomic and ecological status. 2. All the bacteria investigated contained respiratory chain dehydrogenases, ubiquinone and/or menaquinone, cytochrome b and cytochrome oxidase aa3 and/or o. In addition, some of these organisms also contained pyridine nucleotide transhydrogenase and/or cytochrome c. 3. leads to H+/O ratios of whole cell suspensions oxidising endogenous substrates were in the approximate range 4-8 mol H+ translocated per g-atom oxygen consumed. It was concluded from the observed leads to H+/O ratios of cells loaded with specific substrates that proton-translocating loops 1 and 2 were present in all of the organisms investigated, but that loops 0 and 3 were dependent upon the presence of pyridine nucleotide transhydrogenase and cytochrome c respectively. 4. The wide range in energy conservation efficiency which was observed in these organisms is discussed in relation to their respiratory chain composition and natural habitat.     

Xylitol formation by Candida boidinii in oxygen limited chemostat culture

Summary Production of xylitol by Candida boidinii NRRL Y-17213 occurs under conditions of an oxygen limitation. The extent to which substrate is converted to xylitol and its coproducts (ethanol, other polyols, acetic acid), and the relative flow rates of substrate to energetic and biosynthetic pathways is controlled by the degree of oxygen limitation.With decrease in oxygen concentration in the inlet gas, for a constant dilution rate of 0.05 1/h. the specific oxygen uptake rate decreased from 1.30 to 0.36 mmol/gh Xylitol was not produced at specific oxygen uptake rates above 0.91 mmol/gh. Upon shift to lower oxygen rates, specific xylitol production rate increased more rapidly than specific ethanol production rate:Nomenclature D dilution rate (1/h) - DOT dissolved oxygen tension (%) - m_o2 maintenance coefficient (mmol O₂/g cell mass h) - q_o2 specific oxygen uptake rate (mmol O₂/g cell mass h) - q_s specific xylose uptake rate (g xylose/g cell mass h) or (mmol xylose/g cell mass h) - q_x specific xylitol production rate (g xylitol/ g cell mass h) or (mmol xylitol/ g cell mass h) - q_e specific ethanol production rate (g ethanol/ g cell mass h) or (mmol ethanol/ g cell mass h) - q_CO2 specific carbon dioxide production rate (mmol CO₂/g cell mass h) - S xylose concentration (g/1) - Y_cm/s cell mass yield coefficient, (g cell mass/mmol xylose) or (g cell mass/ g xylose consumed) - Y_cm/O2 cell mass yield coefficient, (g cell mass/mmol O₂) - Y_X/S xylitol yield coefficient (g xylitol/g xylose consumed) - Y_x/O2 xylitol yield coefficient (g xylitol/mmol O₂) - Y_e/s ethanol yield coefficient (g ethanol/g xylose consumed) - OUR oxygen uptake rate (mmol O₂/1h) - specific growth rate (1/h)     

The influence of oxygen on the survival and yield of DNA double-strand breaks in irradiated yeast cells.

Survival and induction of DNA double-strand breaks were studied in cells of Saccharomyces cerevisiae irradiated under oxic or anoxic conditions with 30 MeV electrons. A linear relationship between DNA double-strand breakage and dose was found in both cases. The o.e.r.-value for colony forming ability was found to be 1.9 +/- 0.2, whereas the o.e.r.-value for DNA double-strand breakage was 3.0 +/- 0.1. These results are not inconsistent with the idea that DNA double-strand breaks are involved in killing of yeast cells. The frequency of induction of DNA double-strand breaks was found to be 0.74 x 10(-11) double-strand breaks per g/mol per Gy when cells were irradiated under oxygen and 0.24 x 10(-11) double-strand breaks per g/mol per Gy under nitrogen.     

Pure exogenous singlet oxygen: nonmutagenicity in bacteria

Singlet oxygen (1 delta gO2) is the lowest energy-excited state of molecular oxygen, and more reactive than the triplet ground-state molecule. Although singlet oxygen has been implicated in a variety of biological effects, including reactions with DNA or some of its components, evidence for mutagenesis by singlet oxygen has remained unclear. We have previously described a system for bacterial exposure to pure exogenous singlet oxygen that eliminates ambiguity regarding the identity of the reactive species responsible for observed results. Despite the potent toxicity of pure singlet oxygen for several different strains of bacteria, we have found no evidence for mutagenicity of singlet oxygen in 26 Salmonella typhimurium histidine-auxotrophic strains killed to 35% survival. These strains included a variety of base-pair substitution or frameshift target sequences for reversion, including targets responsive to oxidative damage and targets rich in GC base pairs. Some strains combined histidine mutations with one or more mutations affecting DNA-repair capacity. 4 strains possessing the hisG46 mutation also were not mutated when exposed to dose ranges killing less than 28% and up to 99% of the bacteria. The relative frequency of small inphase deletions was assayed in hisG428 bacteria exposed to single oxygen and found to be the same as the spontaneous level. In addition to lack of induction of mutation in these strains, the 8-azaguanine forward mutation assay yielded no evidence of mutagenesis by singlet oxygen in strains killed to 15% survival. No induction of genetic changes by singlet oxygen was seen in an assay for duplication of approximately 1/3 of the bacterial chromosome. Tests for the ability of singlet oxygen to induce lambda prophage in E. coli K12 also proved negative. These studies collectively indicate that pure singlet oxygen generated outside the bacterial cell does not react significantly with the bacterial chromosome in ways leading to base-pair substitutions, frameshift mutations, small or large deletions, large duplications, or damage that interferes with DNA replication and induces the SOS system.     

Plastid Class I and Cytosol Class II Aldolase of Euglena gracilis (Purification and Characterization)

The plastidic class I and cytosolic class II aldolases of Euglena gracilis have been purified to apparent homogeneity. In autotrophically grown cells, up to 81% of the total activity is due to class I activity, whereas in heterotrophically grown cells, it is only 7%. The class I aldolase has been purified to a specific activity of 20 units/mg protein by anion-exchange chromatography, affinity chromatography, and gel filtration. The native enzyme (molecular mass 160 kD) consisted of four identical subunits of 40 kD. The class II aldolase was purified to a specific activity of 21 units/mg by (NH4)2SO4 fractionation, anion-exchange chromatography, chromatography on hydroxylapatite, and gel filtration. The native enzyme (molecular mass 80 kD) consisted of two identical subunits of 38 kD. The Km (fructose-1,6-bisphosphate) values were 12 [mu]M for the class I enzyme and 175 [mu]M for the class II enzyme. The class II aldolase was inhibited by 1 mM ethylenediaminetetraacetate (EDTA), 0.8 mM cysteine, 0.5 mM Zn2+, or 0.5 mM Cu2+. Na+, K+, Rb+, and NH4+ (but not Li+ or Cs+) enhanced the activity up to 7-fold. After inactivation by EDTA, the activity could be partially restored by Mn2+, Cu2+, or Co2+. A subclassification of class II aldolases is proposed based on (a) activation/inhibition by Cys and (b) activation or not by divalent ions.     

Catalytic properties of phenol carboxylase. In vitro study of CO2: 4-hydroxybenzoate isotope exchange reaction

Phenol is metabolized in a denitrifying bacterium in the absence of molecular oxygen via para-carboxylation to 4-hydroxybenzoate (biological Kolbe-Schmitt synthesis). The enzyme system catalyzing the presumptive carboxylation of phenol, tentatively named 'phenol carboxylase', catalyzes an isotope exchange between 14CO2 and the carboxyl group of 4-hydroxybenzoate (specific activity 0.1 mumol 14CO2 incorporated into 4-hydroxybenzoate x min-1 x mg-1 cell protein) which is considered a partial reaction of the overall enzyme catalysis; 14C from [14C]phenol was not exchanged into 4-hydroxybenzoate ring positions to a significant extent. The 14CO2 isotope exchange reaction was studied in vitro. The reaction was dependent on the substrates CO2 and 4-hydroxybenzoate and required K+ and Mn2+. The actual substrate was CO2 rather than HCO3-. The apparent Km values were 1 mM dissolved CO2, 0.2 mM 4-hydroxybenzoate, 2 mM K+, and 0.1 mM Mn2+. The cationic cocatalysts could be substituted by ions of similar ionic radius: K+ could be replaced to some extent by Rb+, but not by Li+, Na+, Cs+, or NH4+; Mn2+ could be replaced to some extent by Fe2+ greater than Mg2+, Co2+, but not by Ni2+, Zn2+, Ca2+, or Cu2+. The exchange reaction was not strictly specific for 4-hydroxybenzoate, however it required a p-hydroxyl group; derivatives of 4-hydroxybenzoate with OH, CH3 or Cl substituents in m-position did react, whereas those with substitutions in the o-position were inactive or were inhibitory. The enzyme was induced when cells were grown on phenol, but not on 4-hydroxybenzoate. Comparison of SDS/PAGE protein patterns of cells grown on phenol or 4-hydroxybenzoate revealed several additional protein bands in phenol-grown cells. The possible role of similar enzymes in the anaerobic metabolism of phenolic compounds is discussed.     

Delayed ultraviolet light-induced cessation of respiration by inadequate aeration of Escherichia coli.

Inadequately aerated Escherichia coli B/r cultures did not shut their respiration off 60 min after ultraviolet light (52 M/m2 at 254 nm) as they did when well supplied with oxygen. Since cessation of respiaration is associated with cell death, the result suggested that oxygen toxicity by superoxide radicals generated by cell metabolism might be responsible for cell death. The specific activity of superoxide dismutase, which scavenges O2- radicals, increased twofold after 90 min of adequate aeration, but the specific activity of catalase remained constant. Respiration and viability of irradiated cells were affected not at all by the presence of superoxide dismutase and only slightly by the presence of catalase. Metal ions such as Mn2+ and Fe2+ inducers of superoxide dismutase, had no effect on respiration and viability. When irradiated cells were incubated under N2 for 90 min, the respiration, growth, and viability time-course responses were the same as for the cells not exposed to anareobiosis. We conclude that superoxide anions generated at the time of irradiation play no part in cessation delays the ultraviolet light-induced synthesis of proteins responsible for the irreversible cessation of respiration.     

Factors controlling the intracellular concentration of calcium and the spontaneous activity of the ureter

The role of the electrogenic Na(+)-Ca(2+)-exchange mechanism in regulating the spike activity of the ureter was studied. The ureter cells were shown to be capable of generating action potentials (AP) in sodium-free Krebs solution. The time during which the spikes are generated is in exponential dependence on the concentration of calcium ions in the medium, [Ca2+]o within 2.5 to 15 mmol/l. Simultaneously with the generation of the spikes, accumulation of calcium in the muscles is observed, proportional to the increase of [Ca2+]o. The addition of as little as 20 mmol/l Na+ or Li+ ions into the solution restores the prolonged electrical activity of the ureter. Under these conditions, the decrease of intracellular Ca2+ within 5 min was more than two times larger as compared with that in sodium-free medium. Upon substituting Ba2+ ions for Ca2+ ions in Krebs solution AP are generated within an interval which was the longer the higher the Ba2+ concentration in the medium. Li+ ions can replace Na+ ions in maintaining AP and in extruding calcium from the cell. It is supposed that the generation of the stable spike activity of the ureter depends on the functioning of Na(+)-Ca(2+)-exchange mechanism.     

Osmolytes responsible for volume reduction under isosmotic or hypoosmotic conditions in Barnacle muscle cells.

In numerous animal cells, experimental manipulations that increase the intracellular free Ca2+ concentration induce cell volume reduction. This may occur under isosmotic conditions, e.g. when external Ca2+ (Ca(o)) is replaced by Mg2+ (42) or during exposure to hypoosmotic conditions (i.e. regulatory volume decrease, RVD) in the presence of Ca(o). We determined the osmolytes responsible for volume reduction under isosmotic and hypoosmotic conditions in barnacle muscle cells. Organic osmolytes (i.e. free amino acids and methylamines) and inorganic ions accounted for approximately 78% and 22% of the intracellular isosmotic activity, respectively. Isosmotic Ca(o) removal induced a net loss of KCI (with a ratio of 1K:1Cl) and free amino acids (FAA, mainly glycine and taurine). During RVD. the same ions (but in a proportion of 2K:1Cl) and FAA were lost. Since RVD was accompanied by extracellular alkalinization, the 2K:1Cl loss may be explained by the presence of a K+/H+ exchanger (or K+-OH- co-transporter) or Cl-/OH- exchanger. The lack of RVD in the absence of Ca(o) cannot be attributed to the loss of intracellular osmolytes during isosmotic Ca(o) removal because addition of Ca(o) during cell swelling promoted RVD.     

Clinical aspects of continuous neonatal oxygen monitoring.

Twenty newborn infants (gestational age 30--40 weeks, weight 980--3400 g) were studied in two groups to compare two commercially available systems for continuous in vivo oxygen monitoring: the So2 catheter and the transcutaneous Po2 (TcPo2) electrode, and their respective electronic systems. Measurements from these systems were correlated with determinations made from samples intermittently drawn and measured by conventional So2 and Pao2 in vitro methods , respectively. Information about these two in vivo oxygen monitoring systems was then related to our previous experience with the bare-wire earlobe O2a electrode. Measurements from the two in vivo monitoring techniques studied showed good correlations with their respective in vitro oxygen measurements: So2 catheter, y = x - 3.08, r = 0.98 (range studied 74% to 100%) and transcutaneous electrode, y = 0.98x + 0.57, r = 0.89 (range studied 34 to 92 mm Hg). It was concluded that all three systems give a good reflection of central arterial oxygen (either Sao2 or Pao2)). The system to be used in specific clinical situations should depend on condition of the baby and stage of treatment, need for an umbilical line to measure other variables, equipment available, and training of personnel.     

The relationship between o.e.r., r.b.e. and number of fractions for X-ray- or neutron-induced skin damage.

The skin reactions in aerated and hypoxic mouse tails after single or fractionated doses of 250 kV X-rays or fast neutrons (6 MeV deuterons on beryllium) have been measured. The o.e.r. for one to sixteen fractions of X-rays remains constant, while that for one to ten fractions of neutrons decreases with increasing neutron fractionation and decreasing neutron dose/fraction. The o.e.r. for X-rays was 1.7, for single-neutron doses 1.4, and for ten fractions of neutrons 1.25. It was anticipated that the o.e.r. for neutron-induced damage would decrease further as neutron fractionation is increased because the contribution to damage from the highest LET components of dose, the alpha and heavy recoil particles, would increase relative to the lowe LET components. The r.b.e. values obtained for skin damage were higher at all neutron doses/fraction examined in this study on tails than all those previously obtained in studies on skin at other sites on four species. This may be due to the influence of hypoxia on the r.b.e. measurements in the mouse tail.     

Negative air ions as a source of superoxide

The physico-chemical characteristics and possible formation mechanisms of negative air ions are considered. It was found that the products of oxygen and nitrogen negative ionization reduce ferricytochromec and nitroblue tetrazolium, and that these reactions were inhibited by superoxide dismutase. The interaction of negatively ionized oxygen with water led to hydrogen peroxide accumulation, which was inhibited by tetranitromethane or catalase. Nitrogen ionization under these conditions caused the formation of the hydrated electron e _aq ^— and the superoxide anion O ₂ ^— . The data obtained indicate that the biological activity of negative air ions may be dependent on superoxide. The generation of reactive oxygen ions in the gas phase and also at a gas/water interface is described. A scheme for superoxide production under oxygen and nitrogen ionization is proposed.     

Effects of bicarbonate and oxygen concentration on photoinhibition of thylakoid membranes

The effects of bicarbonate and oxygen on photoinhibition of thylakoid membranes were investigated by varying their concentrations independently of each other. A pretreatment of the thylakoid suspension which lowered the bicarbonate concentration of the medium without affecting its oxygen content, increased the degree of photoinhibition upon illumination. This showed that the normal bicarbonate content of a thylakoid suspension, originating from dissolved carbondioxide from the air, protects against photoinhibition. The resistance against photoinhibition was further increased by addition of extra NaHCO₃ up to about 5 mM. The normal oxygen content can be decreased profoundly without affecting the degree of photoinhibition; in contrast, even small changes from the normal bicarbonate content affected photoinhibition.At oxygen concentrations approximately below 25 M, added NaHCO₃ not only did not protect, but caused a more severe PS 2 inactivation. This was due to a blockage by added NaHCO₃ of the recovery from a reversible photoinhibited state.Furthermore, it is shown that if the bicarbonate ions bound to high-affinity sites in PS 2 were replaced by formate ions, the thylakoid membranes became less susceptible to photoinhibition under normal oxygen tension.Abbreviations chl chlorophyll - HEPES (N-[2-Hydroxyethyl]piperazine-N-[2-ethanesulfonic acid]) - PpBQ phenyl-p-benzoquinone - PS 2 Photosystem 2 - Q_A and Q_B primary and secondary quinone acceptors of Photosystem 2     

Superoxide dismutase enhances the formation of hydroxyl radicals in the reaction of 3-hydroxyanthranilic acid with molecular oxygen.

Superoxide dismutase (SOD) enhanced the formation of hydroxyl radicals, which were detected by using the e.s.r. spin-trapping technique, in a reaction mixture containing 3-hydroxyanthranilic acid (or p-aminophenol), Fe3+ ions, EDTA and potassium phosphate buffer, pH 7.4. The hydroxyl-radical formation enhanced by SOD was inhibited by catalase and desferrioxamine, and stimulated by EDTA and diethylenetriaminepenta-acetic acid, suggesting that both hydrogen peroxide and iron ions participate in the reaction. The hydroxyl-radical formation enhanced by SOD may be considered to proceed via the following steps. First, 3-hydroxyanthranilic acid is spontaneously auto-oxidized in a process that requires molecular oxygen and yields superoxide anions and anthranilyl radicals. This reaction seems to be reversible. Secondly, the superoxide anions formed in the first step are dismuted by SOD to generate hydrogen peroxide and molecular oxygen, and hence the equilibrium in the first step is displaced in favour of the formation of superoxide anions. Thirdly, hydroxyl radicals are generated from hydrogen peroxide through the Fenton reaction. In this Fenton reaction Fe2+ ions are available since Fe3+ ions are readily reduced by 3-hydroxyanthranilic acid. The superoxide anions do not seem to participate in the reduction of Fe3+ ions, since superoxide anions are rapidly dismuted by SOD present in the reaction mixture.     

The pyruvate-dehydrogenase complex from Azotobacter vinelandii. 3. Stoichiometry and function of the individual components.

Labelling studies with N-ETHYLMALEIMIDE SHOW THAT EITHER IN THE PRESENCE OF Mg2+, thiamine pyrophosphate (TPP) and pyruvate or in the presence of NADH the overall activity of the pyruvate dehydrogenase complex from Azotobacter vinelandii is inhibited without much inhibition of the partial reactions. The complex undergoes a conformational change upon incubation with NADH. The inhibition by bromopyruvate is less specific. Specific incorporation of a fluorescent maleimide derivative was observed on the two transacetylase isoenzymes. Binding studies with a similar spin label analogue show that 3 molecules/FAD are incorporated by incubation of pyruvate, Mg2+ and TPP, whereas 2 molecules/FAD are incorporated via incubation with NADH. The spin label spectra support the idea that in the complex the active centres of the component enzymes are connected by rapid rotation of the lipoyl moiety. Three acetyl groups are incorporated in the complex by incubation with [2-14C]pyruvate. Time-dependent incorporation supports the view that the two transacetylase isoenzymes react in non-identical ways with the pyruvate dehydrogenase components of the complex. The results show that the complex contains 2 low-molecular-weight transacetylase molecules and 4 molecules of the high-molecular-weight isoenzyme. Mn2+-binding studies show that the complex binds 10 ions, with different affinities. 2 Mn2+ ions are bound with a 20-fold higher affinity than the remaining 8 Mn2+ ions. The latter 8 ions bind with equal affinities and are thought to reflect binding to the pyruvate dehydrogenase components of the complex. It is concluded that the complex contains 8 pyruvate dehydrogenase molecules, 4 high-molecular-weight transacetylase molecules, 2 low-molecular-weight transacetylase molecules and 1 dimeric (2-FAD-containing) symmetric molecule of lipoamide dehydrogenase. Evidence comes from pyruvate-dependent inactivation and labelling studies that the pyruvate dehydrogenase components contain either an - SH group or an S-S bridge which participates in the hydroxyethyl transfer to the transacetylase components.     

Silver enhancement of quantum dots resulting from (1) metabolism of toxic metals in animals and humans, (2) in vivo, in vitro and immersion created zinc-sulphur/zinc-selenium nanocrystals, (3) metal ions liberated from metal implants and particles

Autometallographic (AMG) silver enhancement is a potent histochemical tool for tracing a variety of metal containing nanocrystals, e.g. pure gold and silver nanoclusters and quantum dots of silver, mercury, bismuth or zinc, with sulphur and/or selenium. These nanocrystals can be created in many different ways, e.g. (1) by manufacturing colloidal gold or silver particles, (2) by treating an organism in vivo with sulphide or selenide ions, (3) as the result of a metabolic decomposition of bismuth-, mercury- or silver-containing macromolecules in cell organelles, or (4) as the end product of histochemical processing of tissue sections. Such nano-sized AMG nanocrystals can then be silver-amplified several times of magnitude by being exposed to an AMG developer, i.e. a normal photographic developer enriched with silver ions. The present monograph attempts to provide a review of the autometallographic silver amplification techniques known today and their use in biology. After achieving a stronghold in histochemistry by Timm's introduction of the "silver-sulphide staining" in 1958, the AMG technique has evolved and expanded into several different areas of research, including immunocytochemistry, tracing of enzymes at LM and EM levels, blot staining, retrograde axonal tracing of zinc-enriched (ZEN) neurons, counterstaining of semithin sections, enhancement of histochemical reaction products, marking of phagocytotic cells, staining of myelin, tracing of gold ions released from gold implants, and visualization of capillaries. General technical comments, protocols for the current AMG methods and a summary of the most significant scientific results obtained by this wide variety of AMG histochemical approaches are included in the present article.     

Role of the testa epidermis in the leakage of intracellular substances from imbibing soybean seeds and its implications for seedling survival

The generation of ethylene from 1-aminocyclopropane-1-carboxylic acid (ACC) added to a cell-free preparation from etiolated pea ( Pisum sativum L. cv. Alaska) epicotyls was found not to be due to a specific ACC oxidase or to oxygen radicals. Rather, endogenously produced H₂O and manganese ions are coupled in a reaction sequence which produces ethylene from ACC. In a model system, H₂O and Mn²⁺ converted ACC to ethylene under conditions similar to those in the pea preparation. Ultrafiltration of the pea preparation inhibited ethylene production, but it could be reconstituted either by adding an H₂O₂-generating system to the ultrafiltrate or Mn²⁺ to the retentate. H₂O₂-generating systems could reconstitute ethylene formation in a heat-inactivated cell-free sample while the loss of ability to produce ethylene upon dialysis of the pea preparation correlated with the loss of Mn²⁺ from the sample. Studies using cell-free preparations to investigate ethylene synthesis should take care to exclude the possible involvement of H₂O₂ and Mn^2+.