Effects of alkylating agents on lymphocytes from controls and from patients with Fanconi's anemia

Summary The frequency of sister chromatid exchanges (SCE) and chromosome aberrations and the dynamics of cell division in peripheral blood lymphocytes of four patients with Fanconi's anemia were studied after in vitro exposure to alkylating agents TEPA and mitomycin.SCE frequency was significantly increased even after very low doses of mutagens, while chromosome aberrations were significantly increased only after high doses (0.160 g/ml mitomycin and 10^-5 M TEPA). The responses of Fanconi's anemia cells and control cells did not differ significantly. The increased frequency of both SCE and chromosome aberrations was accompanied by gradual delay of cell division, which was most conspicuous in cells from patients with Fanconi's anemia.     

Modulation of brain mitochondrial membrane permeability and synaptosomal Ca2+ transport by dopamine oxidation

Effects of dopamine on the membrane permeability transition, thioredoxin reductase activity, production of free radicals and oxidation of sulfhydryl groups in brain mitochondria and the Ca²⁺ uptake by Na⁺-Ca²⁺ exchange and sulfhydryl oxidation in brain synaptosomes were examined. The brain mitochondrial swelling and the fall of transmembrane potential were altered by pretreatment of dopamine in a dose dependent manner. Depressive effect of dopamine on mitochondrial swelling was reversed by 10 g/ml catalase, and 10 mM DMSO. The activities of thioredoxin reductase in intact or disrupted mitochondria were decreased by dopamine (1-100 M), 25 M Zn²⁺ and 50 M Mn²⁺. Dopamine-inhibited enzyme activity was reversed by 10 g/ml SOD and 10 g/ml catalase. Pretreatment of dopamine decreased Ca²⁺ transport in synaptosomes, which was restored by 10 g/ml SOD and 10 mM DMSO. Dopamine (1-100 M) in the medium containing mitochondria produced superoxide anion and hydrogen peroxide, while its effect on nitrite production was very weak. The oxidation of sulfhydryl groups in mitochondria and synaptosomes were enhanced by dopamine with increasing incubation times. Results suggest that dopamine could modulate membrane permeability in mitochondria and calcium transport at nerve terminals, which may be ascribed to the action of free radicals and the loss of reduced sulfhydryl groups.     

Biotransformation of cholesterol by Micrococcus species mediated by plasmid DNA

A steroid-biotransforming strain RJ6 was identified as Micrococcus roseus. This bacterium has a 10kb plasmid pMQV10. Curing mediated through cultivation of the culture with a low concentration (200ng/ml) of mitomycin C is described. Loss of cholesterol degradation (chol⁺) and streptomycin resistance (Sm^r) phenotypes as a consequence of the loss of plasmid indicate the extrachromosomal location of these two genes in this strain. An electroporation procedure was developed for transformation of cured strain of Micrococcus (RJC6) by plasmids. Frequency of greater than 10⁵ transformants/g DNA was achieved, which is 100-fold higher than the standard transformation procedure that yielded 5.3×10³ transformants/g DNA in the same strain.     

Effect of aldosterone on incorporation of amino acids into renal medullary proteins

Summary Studies on the effects of pretreatment with aldosterone on the incorporation of³H leucine or³H methionine into proteins in renal slices were carried out in Joklik-modified minimal essential medium. Administration of aldosterone (2 g/100 g body wt) to adrenalectomized rats increased³H leucine incorporation into trichloroacetic acid insoluble fractions of crude homogenates of cortical slices by 15.5±0.4% and of medullary slices by 53.5±1.3%. No increase in isotope incorporation was observed in slices of renal papilla or spleen prepared from the same rats. Aldosterone had no effect on the³H-leucine content of the trichloroacetic acid-soluble fractions of all three renal zones and the spleen. The dose of aldosterone that elicited a half-maximal increase in³H-methionine incorporation into proteins of renal medullary slices (0.45 g of aldosterone/100 g body wt) was indistinguishable from that needed to elicit a halfmaximal increase in the urinary K⁺/Na⁺ ratio (0.35 g of aldosterone/100 g body wt). Dexamethasone, a potent glucocorticoid, at a dose of 0.8 g/100 g body wt did not augment³H-leucine incorporation into renal medullary proteins but was effective at 8 g/100 g body wt. Spirolactone (SC-26304), a potent anti-mineralocorticoid, abolished the effect of aldosterone on amino acid incorporation into medullary proteins when administered at a 100-fold higher dosage [i.e., 80 gvs. 0.8 g (per 100 g body wt)]. These results imply that the action of aldosterone on amino acid incorporation is mediated by the mineralocorticoid rather than the glucocorticoid pathway, presumably the mineralocorticoid receptors. Moreover, pretreatment of the rats with actinomycin D (70–80 g/100 g body wt) erased the effect of aldosterone (0.8 g/100 g body wt) on amino acid incorporation into medullary proteins.In paired experiments with³H and³⁵S methionine, aldosterone (0.8 g/100 g body wt) increased methionine incorporation into trichloroacetic acid precipitable proteins of subcellular fractions of the renal medulla. The effect of aldosterone on incorporation of methionine into medullary cytosol proteins was analyzed further by polyacrylamide gel electrophoresis at pH 8.3 in tris-glycine buffer. The gel profiles indicate that aldosterone significantly increased methionine incorporation into at least one protein (independent of the isotope) with a molecular weight of 31,000. This increase was inhibited by either pretreatment of the rat with actinomycin D (70–80 g/100 g body wt or SC-26304 (80 g/100 g body wt). Dexamethasone (0.8 g/100 g body wt) did not increase incorporation of methionine into the medullary cytosol proteins resolved by polyacrylamide gel electrophoresis.     

Estimation ofEscherichia coli mortality in seawater by the decrease in3H-label and electron transport system activity

The mortality ofEscherichia coli in seawater was assessed by viable counts, electron transport system activity, and cellular³H-labelling. Filtration was used to assess the grazing mortality. Cellular radiolabelling and electron transport system activity were useful methods for assessingE. coli survival in seawater. The decrease in the³H-label as a method to assess bacterial mortality was validated by using viable counts and metabolic activity assays. The particulate fraction that passed 2 m but was retained on 0.2 m pore-size filters was the primary reason forE. coli mortality in seawater.     

Assignment of the genes for human lysosomal acid lipases A and B to chromosomes 10 and 16

Summary This paper gives the results of studies on the effects of malathion on human lymphocytes stimulated by PHA, including cell survival, chromosomal aberration and nucleic acid content. Increasing malathion doses (10–70 g/ml) were introduced into cultures of human lymphocytes at different times relative to the time of PHA addition.At a concentration of 10 g/ml, malathion induced an increase in the RNA and DNA fractions in lymphocyte cultures. Higher malathion concentrations (50 and 70 g/ml), especially the dose of 70 g/ml, induced both a significant decrease in the content of RNA and DNA and reduced survival of cultured cells. The insecticide damaged the chromosome structure, but no correlation was revealed between the dose and the level of chromosome aberrations.     

Use of mitochondrial inhibitors to differentiate kinetic properties of the ATP-dependent Ca2+ uptake system in synaptic membranes

Synaptosomal membranes accumulate 3–6 times more Ca²⁺ in the presence of ATP (50–1000 M) than basal Ca²⁺ accumulation (-ATP). The location of this Ca²⁺ accumulation appears to reside on the cytosolic face of the synaptosome since lysed synaptosomes accumulate 4-times more Ca²⁺ than intact synaptosomes. The inclusion of mitochondrial inhibitors, oligomycin (0.7 g/ml), sodium azide (100 M) and dinitrophenol (100 M) differentiate mitochondrial from nonmitochondrial Ca²⁺ accumulation under conditions that are [Ca²⁺]- and ATP-dependent. In the presence of low concentrations of ATP (<150 M) and Ca _free ²⁺ (2.5 or 6.8 M), Ca²⁺ accumulation occurs as one process in both lysed synaptosomal membranes and purified synaptic plasma membranes in the presence and/or absence of MI. When ATP levels are increased (>200 M), the Ca²⁺ accumulation process remains independent of the presence of mitochondrial inhibitors when Ca _free ²⁺ =2.5 M. When Ca _free ²⁺ is increased to 6.8 M, mitochondrial inhibitors differentiate mitochondrial from nonmitochondrial accumulation. These studies suggest that optimal conditions for the measurement of Ca²⁺ accumulating mechanisms in synaptosomal membranes depend on both [Ca²⁺] and ATP. Use of these assay conditions provide evidence that ATP-dependent Ca²⁺ uptake may be a viable mechanism for the regulation of synaptosomal Ca²⁺ levels.     

Effect of the insecticides methylpyrimifos and chlorpyrifos on soil microflora in an agricultural loam

A study of the effects of two selected organophosphorus insecticides methylpyrimifos and chlorpyrifos on soil microflora in an agricultural loam was made. The insecticides had concentrations of 10 to 300 g g^–1. The presence of methylpyrimifos at concentrations of 100 to 300 g g^–1 or chlorpyrifos at concentrations from 10 to 300 g g^–1 significantly decreased aerobic dinitrogen fixing bacteria and dinitrogen fixation. Nitrifying bacteria decreased at concentrations of 200 and 300 g g^–1 of methylpyrimifos. The presence of 10 to 300 g g^–1 of chlorpyrifos decreased the total number of bacteria. However, fungal populations and denitrifying bacteria were not affected as a consequence of the addition of the organophosphorus insecticides to the agricultural soil, showing that these microorganisms can tolerate high amounts of those insecticides.     

Mitochondria in fine afferent nerve fibres of the knee joint in the cat: a quantitative electron-microscopical examination

The distribution of mitochondria, their content and concentration (expressed as the ratio of the mean volume of mitochondria and the surface of the sensory axon) were determined in group-III and-IV nerve fibres innervating the knee joint capsule in the cat. Mitochondria mainly accumulated in axonal swellings (beads) and end bulbs of the terminal branches. Between single nerve fibres, marked differences in the content and the concentration of mitochondria were obtained in proximal portions (inside of the perineurium) and in distal portions (unmyelinated sensory endings). In group-III nerve fibres, the mitochondrial concentration ranged from 0.005 to 0.030 m³/m² (proximal portion) and from 0.016 to 0.080 m³/m² (distal portion). In unmyelinated group-IV nerve fibres, the values also showed a broad variation ranging from 0.001 to 0.011 m³/m² (proximal portion) and from 0.003 to 0.019 m³/m² (distal portion). The wide range of mitochondrial concentrations may reflect different energy consumption during receptive processes: nerve fibres with a low mechanical threshold and a high probability of excitatory events may be rich in mitochondria, whereas fibres with a high mechanical threshold and a low probability of excitatory events may be poor in mitochondria.     

The use of antibiotics in the culture of non-sterile plant protoplasts

Summary The use of antibiotics to control infections in cultures of protoplasts of leaf mesophyll cells has been examined. The antifungal agents nystatin and amphotericin B were non-toxic to protoplasts at concentrations that controlled fungal growth (25 units and 2.5 g/ml respectively). Of the antibacterial agents examined, only carbenicillin and, to a lesser extent, gentamicin were active against the bacteria usually encountered whilst still permitting normal protoplast metabolism and regeneration. The most satisfactory control of contaminating microorganisms was obtained with a combination of nystatin (25 units/ml) or amphotericin B (2.5 g/ml) and carbenicillin (250 g/ml).     

Endogenous phosphorylation of rat brain synaptosomal plasma membranes in vitro: Some methodological aspects

The time course of endogenous phosphorylation in vitro of total or separted synaptic plasma membrane proteins (SPM) has been correlated with that of hydrolysis of the phosphate donor (ATP) in the incubation medium. The ATP/SPM ratio in the medium was varied. In a low-ratio medium (7.5 M ATP; 2.2 g SPM/l) a complete hydrolysis of ATP occurred almost instantaneously as was measured by the release of free phosphate in and the disappearance of ATP from the medium. As a consequence, only a very short peak of phosphorylation, followed by dephosphorylation was observed. However, when higher ATP/SPM ratios were used (200 M ATP; 0.4 g SPM/l and 500 M ATP; 0.4 g SPM/l), the incorporation of phosphate into SPM proteins was linear for 20 sec, and the maximum level of phosphate incorporation was increased. Similar results were obtained after separation of³²P-labeled phosphoproteins by slab gel electrophoresis. However, analysis of the autoradiographs obtained fromone SPM preparation under different ATP/SPM ratios revealed dependence of phosphorylation of individual protein bands on the conditions used.     

Ultrastructure of Sporothrix schenckii treated with iodine-potassium iodide solution

The ultrastructural changes produced by iodine-potassium iodide solution on yeast cells of Sporothrix schenckii were investigated by transmission electron microscopy in order to clarify the mechanism of oral potassium iodide therapy for sporotrichosis. Yeast cells were dipped with solutions containing various concentrations of iodine. The rate of germination decreased markedly between the range of iodine concentrations from 0.63 g/ml to 5.0 g/ml. No significant ultrastructural changes were seen at the concentration of the iodine of 1.25 g/ml (80% germination) or less. In the concentration of 2.5 g/ml (50% germination), normal cells and degenerated cells coexisted. When the cells were treated with 5.0 g of iodine per ml (0% germination) or more, their interior structures were completely destroyed. It is assumed that iodine treatment of the organism causes rapid destruction in the whole cell.     

Regulation of antibiotic production by iron and oxygen during defined medium fermentations of Streptomyces clavuligerus

Summary When grown in a chemically defined medium, Streptomyces clavuligerus excreted cephamycin C, in addition to other components, throughout most of the growth phase. Ferrous iron and oxygen are required for the biosynthesis of this antibiotic and the concentration of these cofactors was manipulated to maximize cephamycin C production. The iron content of the chemically defined medium was shown to be sub-optimal for antibiotic production and the addition of 130 g/ml ferrous iron almost doubled the cephamycin C levels to 200 g/ml. When dissolved oxygen was maintained at saturation levels, only 60–80 g/ml cephamycin C was produced, and the intermediate penicillin N accumulated to high levels (50 g/ml). This suggests that the high concentration of dissolved oxygen had a greater effect on the enzymes catalysing the conversion of penicillin N to cephamycin C, than on those involved in the earlier steps of the pathway leading to the formation of penicillin N.     

3H-thymidine incorporation at the end of the S phase in cultured human lymphocytes

Summary Cultured human lymphocytes of female origin were exposed to 3 Ci/ml ³H-thymidine for 2, 21/4, 21/2, 23/4, and 3 hrs, respectively; a proportion of 0, 1.95, 6.4 and 16.7% of labeled metaphases were found among a total of over 3000 metaphases examined. 25 metaphases with little overall radioactivity were analyzed in detail by grain count and grain distribution. Earlier studies were confirmed that the grain distribution at the extreme end of the S phase is non-random in several regions of the chromosomal complement, but that a distinct interval (Z interval) probably does not exist.

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Zusammenfassung In der vorliegenden Arbeit wird das Ende der DNA-Synthese mittels autoradiographischer Methoden an normalen menschlichen Lymphocyten untersucht. Das Reduplikationsverhalten der Chromosomen am Ende der DNA-Synthese wird näher beschrieben und diskutiert.Das Reduplikationsmuster der letzten 30 min der S-Phase als auch das Verhalten des späten X-Chromosoms sowie der Vergleich zum Markierungsverhalten der Autosomen sprechen gegen die Existenz eines sogenannten Z-Intervalls am Ende der Synthese-Phase.

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This investigation was supported in part by grants from the Deutsche Forschungsgemeinschaft and is part of a thesis by D. B. in fulfillment of requirements for the M. D., at the University of Hamburg.     

Inhibition of somatic embryo maturation in Sitka spruce [Picea sitchensis (Bong.) Carr.] by butylated hydroxytoluene,a volatile antioxidant released by parafilm

Head-space volatiles above embryogenicPicea sitchensis (Bong.) Carr. (Sitka spruce) tissues cultured in glass Petri dishes sealed with Parafilm M or cling-film, were captured on Tenax adsorption traps and analysed by gas chromatography / mass spectrometry. Each sealing system released a single major compound into the head-space; butylated hydroxytoluene from Parafilm M and 2-ethyl-1-hexanol from cling-film. After two weeks sealed under Parafilm M butylated hydroxytoluene accumulated to 1.1 g g^–1 FW in tissues and subsequent somatic embryo maturation was prevented. When butylated hydroxytoluene was supplied via the head-space (100 g/250 ml flask) 0.5 g g^–1 FW accumulated in tissues after two weeks and no somatic embryo maturation occurred. Potentially phytotoxic metabolites of butylated hydroxytoluene included a substituted stilbenequinone, butylated hydroxytoluene quinone methide and butylated hydroxytoluene dimer.     

Effects of monensin on ATP levels and cell functions in rat liver and lung in vitro

Summary Effects of the proton-alkali cation-exchanging ionophore, monensin, on aspects of cellular metabolism and ionic exchanges have been studied in rat tissues in vitro. Incubation of liver slices at 38°C with 0.1 m monensin induced timedependent vesiculation, initially in the Golgi region, reduction of ATP content and of protein synthesis. At 1 m, monensin also reduced net, active movements of K⁺, Na⁺, Cl^– and water in liver slices and inhibited state 3 respiration in isolated mitochondria. The respiratory inhibitor, amytal, similarly reduced ATP content and protein synthesis at concentrations lower than those inhibiting ion transport in slices. Low concentrations of monensin (0.1–1.0 m) had similar effects on ATP and ion transport in slices of adult lung. By contrast, late-fetal liver and lung were much less sensitive to monensin; in these tissues, glycolysis sustained substantial levels of ATP. Monensin also induced vesiculation of the Golgi apparatus in fetal lung cells. It is concluded that by lowering ATP levels, monensin can markedly alter various metabolic activities in those cells which depend primarily on oxidative phosphorylation for their metabolic energy.     

Ability of bisbenzylputrescine and propanediamine analogs to modulate the intracellular polyamine pool of P388D1 cells

The effects of a series of bisbenzyldiamine analogs have been tested on P388D1 cell line in vitro. Their effects on cell growth, polyamine oxidase (PAO) activity and intracellular polyamine content were determined. The cytotoxicity tests were performed in culture medium supplemented with 100 mol/L aminoguanidine (I), 100 mol/L aminoguanidine and 100 mol/L N,N-bis(2,3-butadienyl)-1,4-butanediamine (MDL 72,527) (II), and finally 100 mol/L aminoguanidine and 200 mol/L D,L-difluoromethylornithine (DFMO) (III). The IC50 values under conditions I and III were similar, suggesting that inhibition of ornithine decarboxylase by DFMO did not affect the biological effect of our derivatives. Spermine and spermidine remained nontoxic in conditions I and III. However in the condition II, the toxicity of all tested compounds (excepted spermidine) was increased, suggesting that the inhibition of cellular PAO increased their toxicity.The enzymatic test of PAO showed that at high doses inhibition of this enzyme by putrescine analogs occurred, while the N-methylated propanediamine derivative increased the enzyme activity; however, these results do not correlate with cytotoxicity tests. When these derivatives were incubated for 48 h with the cells, all of them increased the cell content in putrescine (160%) and spermine (145%) and decreased the spermidine content (75%) without any modification of the total amount of polyamine.The correlation between the cytotoxic results and the intracellular polyamine determination shows that the increase in spermine content along with the inhibition of retroconverting PAO enzyme increases the toxic effect of tested compounds (including spermine), suggesting that spermine toxicity is more important in the absence of intracellular oxidation processes.     

Copper but not iron limitation increases astaxanthin production by Phaffia rhodozyma in a chemically defined medium

The influence of copper (0–32 M) and iron (0–108 M) on growth and astaxanthin production by Phaffia rhodozyma was studied. Copper below 3.2 M increased the astaxanthin content of the cells (from 220 to 287 g g^–1) but at the expense of a slightly decreased growth (from 11.3 to 10.2 mg ml^–1). In contrast, iron below 1 M decreased both the growth and astaxanthin content of the cells. Using copper limitation instead of toxic respiratory inhibitors to improve astaxanthin production has obvious advantages from the product quality, environmental and process operation points of view.     

Protamine reversibly decreases paracellular cation permeability inNecturus gallbladder

Summary Protamine, a naturally occurring arginine-rich polycationic protein (pI 9.7 to 12), was tested inNecturus gallbladder using a transepithelial AC-impedance technique. Protamine sulfate or hydrochloride (100 g/ml=20 m), dissolved in the mucosal bath, increased transepithelial resistance by 89% without affecting the resistance of subepithelial layers. At the same time, transepithelial voltage ( ^ms ) turned from slightly mucosapositive values to mucosa-negative values of approximately +1 to –5 mV. The effect of protamine on transepithelial resistance was minimal at concentrations below 5 g/ml but a maximum response was achieved between 10 and 20 g/ml. Resistance started to increase within 1 min and was maximal after 10 min. These effects were not inhibited by serosal ouabain (5×10^–4 m) but could be readily reversed by mucosal heparin. The sequence of protamine effect and heparin reversal could be repeated several times in the same gallbladder. Mucosal heparin, a strong negatively charged mucopolysaccharide, or serosal protamine were without effect. Mucosal protamine reversibly decreased the partial ionic conductance of K and Na by a factor of 3, but did not affect Cl conductance. Net water transport from mucosa to serosa was reversibly increased by 60% by protamine. We conclude that protamine reversibly decreases the conductance of the cation-selective pathway through the tight junction. Although this effect is similar to that reported for 2,4,6-triamino-pyrimidinium (TAP), the mechanism of action may differ. We propose that protamine binds to the apical cell membrane and induces a series of intracellular events which leads to a conformational alteration of the tight junction structure resulting in decreased cationic permeability.     

A pressure-retaining deep ocean sampler and transfer system for measurement of microbial activity in the deep sea

A deep ocean sampler (DOS) has been developed for microbiological sampling and is capable of aseptically collecting 400-ml water samples from any depth in the world oceans. The instrument maintains samples under in situ pressure and temperature. A hyperbaric transfer system has also been developed, enabling transfer of sample volumes up to 150 ml, without decompression or dilution, to pressurized incubation chambers. Utilization of¹⁴C-glutamate (21 to 96g/l) and¹⁴C-acetate (4.6g/l) by microbial populations in undecompressed water samples from the N.W. Atlantic and the Cape and Angola Basins was recorded over incubation periods of 2 to 18 weeks. Rates of substrate utilization ranged from 1 to 38×10^–2 g/l/day.