The effect of 5-azacytidine on the growth and thymidine kinase activity of coleoptile sections ofTriticum

The growth of coleoptile sections ofTriticum and its stimulation by indole-3-acetic acid (IAA) are inhibited by 5-azacytidine added into the cultivation medium. 50 per cent depression of the elongation was observed at 2×10^?3M 5-azacytidine concentration. Thymidine kinase activity in cell-free extracts prepared from coleoptile sections treated with 5-azacytidine, and caleulated per 10 mg of their wet weight, is increased while IAA administration resulted in its depression. The observed changes in thymidine kinase activity can be explained assuming the different uptake of water due to 5-azacytidine and IAA treatment.     

The effects of biogenic phosphates on proteinase-induced protein cleavage and functioning of plasminogen activator

We showed, using the method of lysis of fibrin plates and five substrate proteins in a thin layer of agar gel, that inorganic orthophosphate (0.001–0.06 M) enhances by 50–250% the activatory functions of streptokinase, urokinase, and tissue plasminogen activator and, in general, by 1.2–12.0 times enhances protein lysis by trypsin, α-chymotrypsin, subtilisin, papain, bacterial metalloprotease, and even pepsin at a concentration < 4 mM. At higher concentrations, phosphate sharply inhibited pepsin activity and inhibited by 40–50% gelatin lysis by papain and gelatin (at a peak concentration) and casein lysis by metalloprotease. Inorganic pyrophosphate ions at concentrations of 10^?8–10^?1 M enhanced the cleavage of a number of proteins by serine proteinases and, at concentrations of 10^?5–10^?3 M, the activities of pepsin, plasminogen tissue activator, and streptokinase by 100 and 40%, respectively. The pyrophosphate concentrations of >10^?3 and >10^?4 M inhibited pepsinand metalloproteinase-catalyzed lysis of vritually all proteins. ATP increased casein lysis by serine proteinases, metalloproteinase, and pepsin by 20–60% at concentration of >10^?3 M and by 30–260% at 10^?2 M concentration. At concentrations of 10^?2 M, it inhibited the cleavage of some proteins by trypsin, chymotrypsin, papain, and metalloproteinase by 20–100%, and, at concentrations of 10^?3 M, lysis of albumin by pepsin and other proteins (except for fibrinogen) by metalloproteinase. A GTP concentration of 10^?7–10^?2 M increased protein degradation by serine proteinases, papain, and gelatin lysis by pepsin by 20–90%, whereas albumin lysis was inhibited by 40–70%. The presence of 10^?6–10^?5 M GTP led to a slightly increased degradation of hemoglobin and casein by bacterial metalloproteinase, while ≥10^?3 M GTP induced a drop in the activity of the metalloproteinase by 20–50%. ADP enhanced gelatin lysis by trypsin, casein lysis by pepsin and papain, and inhibited metalloproteinase activity by 20–100% (at ≥10^?3 M). Peculiarities of the effects of AMP and GD(M)P on gelatin lysis were found.     

The effect of 2-chloro, 6-(trichloromethyl) pyridine on the chemoautotrophic metabolism of nitrifying bacteria

Studies were conducted to elucidate the mechanism of action of 2-chloro-6-(trichloromethyl)pyridine or Technical N-SERVE on the nitrification process brought about byNitrosomonas europaea. The growth ofNitrosomonas was completely inhibited in the presence of 0.2 ppm N-SERVE while 1.0 ppm of the chemical was effective in the complete inhibition of ammonia oxidation by fresh cell suspensions. Cells stored at 4 C for a period of three days required somewhat higher concentrations (1.5 ppm) of N-SERVE for the complete inhibition of their ammonia oxidizing ability while the cytochrome oxidase of these cells was inhibited to the extent of 65 to 70 percent in the presence of a corresponding amount of N-SERVE. A 45 – 70 percent reversal of the inhibition of ammonia oxidation caused by N-SERVE was obtained by the addition of 6×10^–4 M Cu⁺⁺. An equivalent concentration of Cu⁺⁺ was also effective for the complete reversal of the inhibition of cytochrome oxidase present in whole cells.Hydroxylamine oxidation by intactNitrosomonas cells was not affected by levels of N-SERVE ranging from 1 – 3 ppm. The cytochrome oxidase effective in hydroxylamine oxidation and present in cell-free extracts was not inhibited by even 100 ppm N-SERVE. Likewise, the hydroxylamine activating enzyme hydroxylamine cytochromec reductase was also not inhibited by such levels of the chemical. Raising the concentration to 170 ppm N-SERVE, however, caused a 90 percent inhibition of the enzyme.Although a 5×10^–6 M concentration of allylthiourea completely inhibited ammonia oxidation byNitrosomonas cells, concentrations up to 10^–3 M of this compound did not affect the cytochrome oxidase activity of whole cells or cell-free extracts. The inhibition of ammonia oxidation caused by 5×10^–6 M allythiourea, unlike the inhibition by N-SERVE, could not be reversed by the addition of 6×10^–4 M Cu⁺⁺.Evidence is presented that the action of N-SERVE is on that component of cytochrome oxidase which is involved in ammonia oxidation.     

Inhibition of Iron-oxidizing Activity by Bisulfite Ion in Thiobacillus ferrooxidans

Growth of Thiobacillus ferrooxidans on iron- and sulfur-salts media and iron oxidizing activity of this bacterium were strongly inhibited by bisulfite ion. The mechanism of inhibition by bisulfite ion of iron-oxidizing activity was studied with the plasma membrane of T. ferrooxidans AP19-3. The c-type cytochrome in the plasma membrane was reduced by ferrous ion and the cytochrome reduced by Fe²⁺ was oxidized by cytochrome c oxidase in the plasma membrane. In contrast, c-type cytochrome was reduced by bisulfite ion, but it was not oxidized by cytochrome c oxidase in the membrane. Cytochrome c-oxidizing activity was also inhibited by the ion when mammalian cytochrome c was used as an electron donor, suggesting that cytochrome c oxidase, one of the component of iron oxidase, is the site of inhibition by bisulfite ion.     

GLYOXYLATE CYCLE ENZYME ACTIVITIES IN THE CYANOBACTERIUM ANACYSTIS NIDULANS1

The enzymes of the glyoxylate cycle, isocitrate lyase (EC.4.1.3.1) and malate synthase (EC.4.1.3.2), were measured in cell-free extracts from the cyanobacterium Anacystis nidulans Drouet during photoautotrophic growth in medium aerated with ordinary air (0.03% CO₂). Isocitrate lyase had an average specific activity of 112 nmoles·min^?1·mg protein^?1 whereas malate synthase had an average specific activity of 12.5 nmoles·min^?1·mg protein^?1. Unpurified isocitrate lyase showed classical Michaelis kinetics with a K_m of 8 mM. Isocitrate lyase activity was strongly inhibited by numerous cellular metabolites at 10 mM concentration. The previously reported low specific activity for isocitrate lyase may be due to metabolite inhibition caused by growth in high CO₂ concentrations. The activities reported for isocitrate lyase and malate synthase suggest the operation of the glyoxylate cycle in Anacystis nidulans under CO₂-limiting growth conditions.     

Isolation and Characterization of Phenol-Degrading Soil Bacterium Xanthobacter flavus

A soil bacterium isolated from a contaminated site degraded phenol when provided as the sole carbon and energy source in the medium. The bacterium was identified as Xanthobacter flavus MTCC 9130. This microbial strain was able to tolerate phenol up to 1000 mg L^?1 concentration. The lag phase increased with the increase in phenol concentration. The optimum growth temperature was 37°C. The organism efficiently utilized phenol and could degrade it completely within 120 h when initial concentration was less than 600 mg L^?1. Degradation of phenol was through ortho pathway, enzyme assay through cell-free extract exhibited the presence of catechol 1,2-dioxygenase. The specific activity was 0.146 μ mol min^?1 mg^?1 protein. However, higher concentrations of phenol in the medium had a negative effect on the growth of the bacterium. Hence this ability of Xanthobacter flavus can be effectively used for bioremediation studies of phenol-contaminated sites.     

Ricerche sulla fisiologia dell' acido ascorbico. XXVI. Sull' azione inibente dell' acido βindolacetico nei confronti dell' inverdimento e sulla capacità dell' acido ascorbico di impedirne l' azione

Abstract

INIBITION OF GREENING BY INDOLACETIC ACID AND ITS PREVENTION BY ASCORBIC ACID. — Stem apex portions from etiolated pea plants (Pisum sativum var. Alaska) were grown in a dark room thermoregulated at 25°C until the development of the third internode and after excission kept in light for 20 hours. Greening on these isolated portions is sharply inhibited by indolacetic acid at concentrations varying from 10^?3M to 10^?6M. The highest inhibition, that is about 40%, correspònds to the highest concentrations (10^?3M). A scarcely significant stimulus is registered at the 10^?6M concentration of indolacetic acid.

Using much younger material (plants 4 days instead of 8 days old) the inhibition caused by treatments with indolacetic acid results greater (the maximum inhibition, always at 10^?3M, reaches about 60%), perhaps as a consequence of the greatest concentration of endogenous auxin.

Treatments with ascorbic acid, both in the reduced and oxydized form, at concentrations ranging from 10^?2M to 10^?1M do not cause any variation in respect of controls.

Ascorbic acid supplied with indolacetic acid greatly reduces the inhibiting effect on greening: some 40% of the inhibition by 5 × 10^?4M indolacetic acid being suppressed by 10^?3M ascorbic acid. Also for the greening process an antagonism between the action of ascorbic acid and that of auxin is thus demonstrated; which was previously demonstrated for various physiological processes (distension growth, water retention, cell multiplication, abscission, etc.) by several studies carried on in this Institute.     

Target of inhibition by the anti-lipogenic antibiotic cerulenin of sterol synthesis in yeast

The antibiotic cerulenin inhibited the incorporation of ¹⁴C-acetyl-CoA by 67% at a concentration of 9 × 10^?6 M but not that of ¹⁴C-HMG-CoA into the non-saponifiable fraction in a cell-free extract of Saccharomyces cerevisiae. Cerulenin markedly inhibited the activity of partially purified HMG-CoA synthase. No inhibition of acetoacetyl-CoA thiolase activity was observed in the same preparation of HMG-CoA synthase. Therefore, cerulenin inhibition of overall sterol synthesis may be accounted for by the specific inhibition of HMG-CoA synthase activity.     

Activity of some Nile River aquatic macrophyte extracts against the cyanobacterium Microcystis aeruginosa

The anti-algal activity of five macrophyte extracts on the cyanobacterium Microcystis aeruginosa in Egypt was investigated in 2013. Extract activity varied according to plant type, extracting solvent and its concentration. The highest inhibitory activity was achieved with ethanol extract at a concentration of 80 mg l^?1, followed by chloroformic extracts, at 60 mg l^?1. Methanolic extracts of Eichhornia crassipes and Polygonum tomentosum inhibited growth of Microcystis aeruginosa at all concentrations. Acetonic extracts inhibited algal growth at 60 mg l^?1, except for the extract of Ceratophyllum subdemersum, which showed stimulation of M. aeruginosa growth. Eichhornia crassipes ethanolic extract exerted the most powerful inhibition by more than five-fold, 570.17%, followed by those of P. tomentosum, Saccharum spontaneum, Ceratophyllum demersum and C. subdemersum, 559.48, 553.99, 544.11 and 366.51%, respectively. Phytochemical screening for the tested plant extracts revealed the presence of biologically active substances of different concentrations, with P. tomentosum having the highest polyphenols, 1.95% of dry weight.     

Formation of Glycerol Dehydrogenase by Microorganisms

The distribution of glycerol dehydrogenase activity was studied with cell-free extracts of bacteria, yeasts, molds and actinomycetes. High activity was found in 4 strains of bacteria and in 3 strains of molds. The enzymes of bacteria were dependent on NAD⁺ and those of molds were dependent on NADP⁺. An isolated gram-positive bacterium, which showed the high activity, was identified as Cellulomonas sp. NT3060. The total and specific activities were associated with growth of this strain and reached the maximum at the early stationary phase. Significant high level activity was detected in cell-free extracts from glycerol and glucose media.     

Inhibition of gibberellin biosynthesis by paclobutrazol in cell-free homogenates ofCucurbita maxima endosperm andMalus pumila embryos

The plant growth retardant paclobutrazol, (PP333) (2RS, 3RS)-1-(4-chlorophenyl)-4,4-dimethyl-2-(1,2,4-triazol-1-yl)pentan-3-ol, inhibits specifically the three steps in the oxidation of the gibberellin-precursorent-kaurene toent-kaurenoic acid in a cell-free system fromCucurbita maxima endosperm. The KI₅₀ for this inhibition is 2×10^?8 M. The KI₅₀ values for the separated2S, 3S, and2R, 3R enantiomers of paclobutrazol in this system are 2×10^?8 M and 7×10^?7 M, respectively. A cell-free preparation from immatureMalus pumila embryos convertsent-kaurene to gibberellin A₉, whereas no conversion occurs in a similar preparation fromMalus endosperm. The conversion ofent-kaurene by the embryo preparation is inhibited by paclobutrazol with KI₅₀ values for the2S,3S and2R,3R enantiomers of 2×10^?8 M and 6×10^?8 M, respectively.     

Growth and Germination Inhibitors in Hog-weed

Properties of a glutathione transport system in T. ferrooxidans strain AP-44 were investigated using a reduced form of ³⁵S-glutathione (³⁵S-GSH). About 71.2% of the total radioactivity taken up into the cells was distributed in the cytosol fraction. The amount of GSH taken up into the cells was in proportion to the amount of ferrous iron oxidized. However, a high concentration of silver ions (50 mm), which completely inhibited an iron-oxidizing activity, did not inhibit the GSH transport. The results suggest that GSH was transported by using a proton electrochemical gradient formed across the cytoplasmic membrane. Since growth inhibition by silver nitrate was decreased by the addition of GSH to both silver ion sensitive-cells and resistant-cells, the GSH transport system may play some role in the silver ion resistance mechanism of the bacterium.     

Ribonucleic acid synthesis in streptomyces antibioticus: Stable ribonucleic acid species synthesized by young and old cells

In studies of RNA synthesis by intact cells and cell-free extracts of $\text{Streptomyces antibioticus}$, it has been found that 48 hr cells (producing actinomycin) and cell-free extracts are less efficient than 12 hr cells (not producing actinomycin) and extracts in the synthesis of RNA. Analysis of the products of “in vivo” and “in vitro” RNA synthesis by sucrose gradient centrifugation reveals that both 12 and 48 hr cultures and cell-free extracts synthesize ribosomal RNA as well as RNA species of higher and lower molecular weights. However, 50–60% of the ³H-uridine labelled RNA synthesized by intact cells sediments as rRNA as compared with only 5–10% of the cell-free product. The addition of 2 × 10^?5 M actinomycin D to incubation mixtures for cell-free RNA synthesis does not significantly alter the relative amounts of the various RNA species synthesized by 12 or 48 hr extracts.     

Effect of trace elements and vitamins on the growth of Methanosarcina barkeri

Growth of Methanosarcina barkeri on methanol as energy source was found to be dependent on cobalt and molybdenum. In the presence of 10^?6 M Co and 5 × 10^?7M Mo optimal growth occurred. Furthermore it could be demonstrated that nickel and selenium each in a concentration of 10^?7 M stimulated the growth of this methanogenic bacterium while the following elements tested in the range of 10^?7 M to 10^?3 had no influence: B, Cr, Cu, Mn, Pb. The requirement of Co and Ni for optimal growth are in accordance with the results that the cells contain the Co containing corrinoid Factor III (0.1 – 0.2 mg 5-hydroxylbenzimidazolylcyanocobamide per g wet cells) and Factor F₄₃₀, a nickel component. Studies on the vitamin dependency of M. barkeri showed that this strain needs only the vitamin riboflavin for the growth in a defined medium. Under these conditions a cell density of 2.6 g dry cells/l could be obtained in a fed batch culture.     

Mannitol Biosynthesis in Pyrenochaeta terrestris II. D-Mannitol-l-phosphatase

Cell-free extracts of mycelial mats of Pyrenochaeta terrestris contained an enzyme which hydrolyzed mannitol-l-phosphate to mannitol and inorganic phosphate. Greatest mannitol-1-phosphatase activity occurred early in the growth period when the mannitol content of the mats was at a maximum. The enzyme was active over a broad pH range with optimum activity between pH 6.5–7.0 in 0.05 M Tris-maleate buffer. Maiinitnl-1-phosphatase was inhibited by reagents known to inhibit enzymes containing -SH groups. A 10-fold purification was attained by a combination of (NII₄)₂ SO₄ fractionation and gel filtration on Sephadex G-100. The partially purified enzyme required Mg^?2 for activity and did not hydrolyze a number of sugar phosphates. Km values for mannitol-l-phosphate and Mg^?2 with the partially purified extract were 3 × 10^?3 M and 1 × 10^?4 M respectively.     

Some kinetic studies on ribonucleosides degradation by extracts of penicillium citrinum

Cell-free extracts of 3–4 days old mats of nitrate-grown Penicillium citrinum catalyze the hydrolytic cleavage of the N-glycosidic bonds of inosine, guanosine and adenosine optimally at pH 4, 0.1 M citrate buffer. The same extracts catalyze the hydrolytic deamination of cytidine at a maximum rate in 0.08 M Tris-acetate buffer pH 6.5, 40°C and 50°C were the most suitable degrees for purine nucleoside hydrolysis and cytidine deamination, respectively. The incubation of the extracts at 60°C, in the absence of cytidine caused a loss in the deaminating activity, while freezing and thawing had no effect on both activities. The deaminating activity seems to be cytidine specific as neither cytosine, adenine, adenosine nor guanosine could be deaminated. Uridine competively inhibited this activity, while ammonia had no effect. The apparent K_m value of this enzyme for cytidine was 1.57×10^?3M and its K_i value for uridine was 7.8×10^?3M. The apparent K_m values of the N-glycosidic bond cleaving enzyme for inosine, guanosine and adenosine were 13.3, 14.2 and 20×10^?3 M, respectively.     

Purification and characterization of an adenosine cyclic 3':5' monophosphate-dependent protein kinase from human erythrocyte membrane.

A cAMP dependent protein kinase was extracted from human erythrocyte membrane with hydrosoluble fraction and partially purified by ammonium sulfate-precipitation and DEAE-cellulose chromatography. The pH of optimal activity is 6.5; the enzyme has an absolute requirement of Mg²⁺ ions at the concentration of 10 mM and is strongly inhibited by Ca²⁺. It uses ATP as phosphate donor with a Km of 3.7 × 10^?6 M. Cyclic AMP stimulates the activity with an apparent Ka of 5 × 10^?8 M; cIMP and cGMP also acts as activators. Enzyme activity is thermolabile and not protected by Mg ATP complex. The enzyme purified from erythrocyte membrane is a type I protein-kinase as proven by DEAE cellulose chromatography and dissociation of the subunits in presence of NaCl 0.5 M and histone.     

Effects of PTH and Ca2+ on renal adenyl cyclase

The effects of calcium ion on the adenylate cyclase system was studied in isolated, renal basal-lateral plasma membranes of the rat. Bovine parathyroid hormone (bPTH) and a guanyl triphosphate analogue, Gpp(NH)p were used to stimulate cyclase activity. Under conditions of maximal stimulation, calcium ions inhibited cyclic adenosine monophosphate (cAMP) formation, the formation rate falling exponentially with the calcium concentration. Fifty percent inhibition of either bPTH- or Gpp(NH)p-stimulated activity was given by approximately 50 μM Ca⁺⁺. Also the Hill coefficient for the inhibition was close to unity in both cases. The concentration of bPTH giving half-maximal stimulation of cAMP formation (1.8 × 10^?8 M) was unchanged by the presence of calcium. These data suggest that calcium acts at some point other than the initial hormone-receptor interaction, presumably decreasing the catalytic efficiency of the enzymic moiety of the membrane complex.     

Cytochrome c-linked reactions in Rhodopseudomonas palustris grown photosynthetically on thiosulfate

The nonsulfur purple bacterium Rps. palustris was adapted to grow photoautotrophically with thiosulfate as substrate. An isolated cell-free fraction catalyzed the enzymatic transfer of electrons from thiosulfate to endogenous and/or added mammalian cytochrome c. Antimycin A, NOQNO, rotenone, amytal and atebrin did not inhibit the thiosulfate-cytochrome c reductase. The products of thiosulfate oxidation were primarily tetrathionate, trithionate, and sulfate, suggesting oxidation via the polythionate pathway. Succinate, formate and NADH were also effective electron donors in this system showing Michaelis constants of 40, 30 and 0.025 mm, respectively for cytochrome c reduction. The NADH-cytochrome c reductase was not inhibited by flavoprotein inhibitors and by Antimycin A or NOQNO. The cell-free extracts also contained an active cytochrome c-O₂ oxidoreductase which was inhibited by cyanide, azide and EDTA, and these inhibitions were overcome by the addition of Cu²⁺. The oxidase activity was stimulated by the addition of uncoupling agents such as CCCP and DNP, as well as by Antimycin A and NOQNO. Reduced + CO minus reduced difference absorption spectra revealed the presence of cytochrome components of the a and o types which may function as the terminal oxidase(s).     

Enzymes Involved in Anaerobic Polyethylene Glycol Degradation by Pelobacter venetianus and Bacteroides Strain PG1

In extracts of polyethylene glycol (PEG)-grown cells of the strictly anaerobically fermenting bacterium Pelobacter venetianus, two different enzyme activities were detected, a diol dehydratase and a PEG-degrading enzyme which was characterized as a PEG acetaldehyde lyase. Both enzymes were oxygen sensitive and depended on a reductant, such as titanium citrate or sulfhydryl compounds, for optimal activity. The diol dehydratase was inhibited by various corrinoids (adenosylcobalamin, cyanocobalamin, hydroxocobalamin, and methylcobalamin) by up to 37% at a concentration of 100 μM. Changes in ionic strength and the K⁺ ion concentration had only limited effects on this enzyme activity; glycerol inhibited the enzyme by 95%. The PEG-degrading enzyme activity was stimulated by the same corrinoids by up to 80%, exhibited optimal activity in 0.75 M potassium phosphate buffer or in the presence of 4 M KCI, and was only slightly affected by glycerol. Both enzymes were located in the cytoplasmic space. Also, another PEG-degrading bacterium, Bacteroides strain PG1, contained a PEG acetaldehyde lyase activity analogous to the corresponding enzyme of P. venetianus but no diol dehydratase. Our results confirm that corrinoid-influenced PEG degradation analogous to a diol dehydratase reaction is a common strategy among several different strictly anaerobic PEG-degrading bacteria.