Synthesis and cytotoxicity studies of bifunctional hybrids of nitrogen mustards with potential enzymes inhibitors based on melamine framework

The new class of hybrid anticancer drugs were obtained by selective functionalization of the triazine scaffold. These were prepared by rearrangement of mono-, bis- and/or tris-(1,3,5-triazin-2-yl)-1,4-diazabicyclo[2.2.2]octanium chlorides leading to formation of 2-chloroethylamino fragments attached to 1,3,5-triazine via one, two or three piperazine rings respectively. Their inhibitory effect was found strongly dependent on the structure of substituents in triazine ring. The anti-proliferative activity of the hybrids evaluated in vitro by using mammalian tumour cells estimated as IC₅₀ was in the range 0.62–139,78 µM. Both cytotoxicity and alkylating activity depended on the substituents of triazine ring, however, also the mono-functional analogues of nitrogen mustards, which are unable to form liaisons between two DNA strands, induced apoptosis and necrosis in the tested cells.     

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) degradation by <Emphasis Type="Italic">Acetobacterium paludosum</Emphasis>

Substrates and nutrients are often added to contaminated soil or groundwater to enhance bioremediation. Nevertheless, this practice may be counterproductive in some cases where nutrient addition might relieve selective pressure for pollutant biodegradation. Batch experiments with a homoacetogenic pure culture of Acetobacterium paludosum showed that anaerobic RDX degradation is the fastest when auxiliary growth substrates (yeast extract plus fructose) and nitrogen sources (ammonium) are not added. This bacterium degraded RDX faster under autotrophic (H₂-fed) than under heterotrophic conditions, even though heterotrophic growth was faster. The inhibitory effect of ammonium is postulated to be due to the repression of enzymes that initiate RDX degradation by reducing its nitro groups, based on the known fact that ammonia represses nitrate and nitrite reductases. This observation suggests that the absence of easily assimilated nitrogen sources, such as ammonium, enhances RDX degradation. Although specific end products of RDX degradation were not determined, the production of nitrous oxide (N₂O) suggests that A. paludosum cleaved the triazine ring.     

Interactions between aldehyde derivatives and the aldehyde binding site of bacterial luciferase

The interaction of trizine aldehydes with the aldehyde binding site of bacterial luciferases was investigated using a series of triazine aldehydes with different aldehyde chain length, and substituents on the s-triazine ring. Substrate activity was determined using luciferase from Photobacterium fischeri and Vibrio harveyi in a dithionite-based luciferases assay. The chain length optimum was determined for two triazine aldehyde classes to be C-10 and C-11, respectively. Only the substrate activity of 10-(4-chloro-6-methyithio-s-triazine-2-yl)aminodecanal (5) was as high as n-decanal, the reference aldehyde. All other triazine derivatives reduced light emission, probably by hindered binding of the substrates. The degree of activity reduction correlated with the volume of the triazine ring moiety. The triazine moiety volume of compound 5 was estimated to be 200 × 10^?30 m³. Triazine aldehydes which showed reduced light emission had an estimated volume of 228 × 10^?30 m³ or greater. All triazine aldehydes showed approximately 10-fold lower activities for Vibrio harveyi than for Photobacterium fischeri luciferase. Substrate specificity was the same for both luciferases. A schematic superposition of quinone aldehydes and triazine aldehydes which showed substrate activities equivalent to n-decanal, indicated potential interaction sites of aldehyde substrates with the aldehyde binding site of bacterial luciferases. The in vivo relevance of the results is discussed.     

Nitrogen nutrition inAtriplex hastata L.

Summary The investigation was carried out in order to ascertain whyAtriplex hastata, previously recorded as a nitrophilous plant, can colonise fly ash which has a low nitrogen content.Sinapis arvensis, a weed which does not grow on fly ash, was selected as a control plant. Solution-culture experiments showed that the two species differed in their responses to high and low levels of nitrogen, the growth rate ofS. arvensis being much more severely affected by low levels of 0 to 50 ppm.A. hastata was found to make very efficient use of its nitrogen supply. Both species grew better with nitrate than with ammonium nitrogen. InA. hastata, uptake of nitrogen was not influenced by pH when only nitrate was present. Where both nitrate and ammonium were available, nitrate was absorbed preferentially at low pH and ammonium at high pH. Some properties of fly ash which render it a suitable medium for growth ofA. hastata are discussed.     

Effect of pH and nitrogen source on pigment production by Monascus purpureus

The effect of pH and nitrogen source on pigment production by Monascus purpureus 192F using glucose as the carbon and energy source, was studied in pH-controlled, batch fermentor cultures using HPLC analysis to determine individual pigment concentrations. A maximum of four pigments were detected in fungal extracts. These were the yellow pigments monascin and ankaflavin, the orange rubropunctatin and the red pigment monascorubramine. Monascorubramine was present as the major product in all instances. Fungal growth and ankaflavin synthesis were favoured at low pH (pH 4.0), whereas production of the other pigments was relatively independent of pH. The nature of the nitrogen source affected fungal growth and pigment production, independent of pH. Ammonium and peptone as nitrogen sources gave superior growth and pigment concentrations compared to nitrate. Ankaflavin was not detected in nitrate cultures. The highest red pigment production was obtained using a glucose-peptone medium at pH 6.5, due to the secretion of red pigments into the medium under these conditions. Correspondence to: M. R. Johns     

Biodegradation of p-nitrophenol by methyl parathion-degrading Ochrobactrum sp. B2

Ochrobactrum sp. B2, a methyl parathion-degrading bacterium, was proved to be capable of using p-nitrophenol (PNP) as carbon and energy source. The effect of factors, such as temperature, pH value, and nutrition, on the growth of Ochrobactrum sp. B2 and its ability to degrade p-nitrophenol (PNP) at a higher concentration (100 mg l⁻¹) was investigated in this study.The greatest growth of B2 was observed at a temperature of 30 °C and alkaline pH (pH 9–10). pH condition was proved to be a crucial factor affecting PNP degradation. Enhanced growth of B2 or PNP degradation was consistent with the increase of pH in the minimal medium, and acidic pH (6.0) did not support PNP degradation. Addition of glucose (0.05%, 0.1%) decreased the rate of PNP degradation even if increased cell growth occurred. Addition of supplemental inorganic nitrogen (ammonium chloride or ammonium sulphate) inhibited PNP degradation, whereas organic nitrogen (peptone, yeast extract, urea) accelerated degradation.     

Oats tempeh

Oats was used as a substrate in tempeh fermentation. The time needed to obtain sufficient mold growth was at least 30 hours at 31°C (Rhizopus oligosporus NRRL 2710). pH was decreasing during the first 32 hours of incubation reaching pH = 5.30. Fermentation of oats led to an increase in water soluble nitrogen, but it did not change protein nitrogen content. R. oligosporus proteinases of optimum pH = 5.50 are postulated to play an important role in oats tempeh fermentation. When a mixture of oats and soybean (1:1) was used, mold growth was faster and the cake tougher. Mixing cereals with legumes to produce good tempeh is recommended.     

Cyanuric acid--a s-triazine derivative as a nitrogen source for some soil microorganisms

Cyanuric acid was not toxic for soil microorganisms examined and was even observed to stimulate the growth of Azotobacter in chernozem. Some isolated fungi were capable of cleaving the ring of cyanuric acid. With the use of 15N-labeled cyanuric acid it was found that the nitrogen taken from this compound by Aspergillus minutus and Pseudogymnoascus sp. was incorporated into their proteins. About 70-90% of 15N derived from cyanuric acid was detected in the biomass of the examined fungi. The ability of soil microorganisms to cleave the triazine ring is of importance in the detoxication of soils treated with triazine herbicides.     

Heterotrophic growth and nutritional aspects of the diatom Cyclotella cryptica (Bacillariophyceae): effect of nitrogen source and concentration

To investigate the nutritional value of the diatom Cyclotella cryptica Reimann, Lewin, and Guillard as an alternative feed for the use in the aquaculture industry, the heterotrophic growth characteristics, total fatty acids, and the resultant fatty acid profile of the microalga were studied when cultivated with sodium nitrate, ammonium chloride, or urea. All three nitrogen sources supported growth under heterotrophic conditions, and their uptake affected the pH of the cultivation medium, even when buffered. The use of sodium nitrate or urea resulted in a significant increase in the pH of the cultivation media, whereas the use of ammonium chloride caused a minor decrease in the pH of the cultivation media. The maximum specific growth rate was highest when urea and ammonium chloride were supplied at a low concentration; however, the total fatty acid content was not significantly affected (P = 0.101) by the nitrogen source when supplied at 10.7 mM nitrogen. The total fatty acid content and fatty acid profile of C. cryptica was more affected by the growth phase (predominately influenced by the initial nitrogen concentration) than by the source of nitrogen.     

The Synthesis of Cyclotene and Its Related Compounds by the Acyloin Condensation

A mutant which required glutamate for growth as the sole nitrogen source was derived from alkalophilic Bacillus No. 8–1 by UV irradiation. The relationship was examined between cell growth and glutamate transport into cells.

Cell growth and glutamate transport into cells were dependent on extracellular pH in the presence of Na⁺, and both were maximum between pH 9 and 10. The quantitative relation between specific growth rate and glutamate uptake rate indicated that the amount of glutamate required for growth at pH 7 and 9 was consistent with that of glutamate transported at pH 7 and 9, respectively. But the amount of glutamate transported at pH 7 was not sufficient to support growth at pH 9. The glutamate transport system of this mutant strain evidently had an effect on growth.     

Competition between soil bacteria and fusarium

Summary The phenomenon of competition has been characterized in liquid medium and sterile soil systems using a variety of soil bacteria andFusarium oxysporum f.cubense as test organisms. For most of the bacteria, suppression of the fungus was the result of a biologically induced nitrogen deficiency, this effect being reversed by the addition of excess inorganic nitrogen. High populations of competitors were found in two soils of neutral pH, but no isolates competed in the acid San Alejo loam.Agrobacterium radiobacter was able to compete when San Alejo loam was limed to about pH 6.6. Inhibition of the fungus by a number of gram-positive, spore-forming rods could not be accounted for in terms of competition for nutrients or by antibiotic production in artificial media.The competitive ability ofA. radiobacter when tested in twelve Central American soils was found to be related to pH in acid and neutral environments but was correlated with texture, organic-matter content and total nitrogen in soils of intermediate pH. In all soils where inhibition occurred, the competitive effect was overcome by additions of inorganic nitrogen. Excluding the group ofBacillus spp., the competitive ability of soil bacteria was related to the ability to develop in the absence of amino acids and growth factors but could not be correlated with growth rates of the bacteria in soil or liquid medium.It is suggested that competition for nutrients is a significant means of ecological control among members of the soil microflora and, together with competitive interactions for space and oxygen, may be the major factors governing the biological control of soil-borne fungi.The investigation was supported in part by a grant from the United Fruit Company. Agronomy Paper No. 471.     

Effect of nutrient medium pH on symbiotic nitrogen fixation byRhizobium leguminosarum andPisum sativum

Summary Experiments were performed to measure the pH-sensitive steps in nodulation and symbiotic fixation byPisum sativum and isolate RP-212-1 ofRhizobium leguminosarum. An aeroponic system with rigorous pH control was used to obtain numerous effective nodules. After exposure to various pH levels, the following responses were measured: (1) legume root growth and development, (2) survival and growth rate of a single effective bacterial isolate, (3) degree of nodulation, (4) rate of nitrogen fixation, (5) plant biomass, and (6) nitrogen content of plants. Both bacterial growth and root development were adequate at all pH levels from 4.4 to 6.6, but efficient nodulation and nitrogen fixation did not occur at pH 4.8 and below. The processes required for symbiosis were about 10 times as sensitive to acidity as either bacterial growth or root growth alone. Nodulation was the most acid-sensitive step.     

Nutritional studies on Colletotrichum gloeosporioides Penz

Summary Colletotrichum gloeosporioides isolated from the diseased leaves ofPolyscias balfuriana could grow and sporulate on a wide range of pH (viz. from 3.0 to 9.0). Maximum growth was recorded at pH 5.5. Mannitol was the best carbon source for growth. Good growth as well as good or excellent sporulation was also recorded on glucose, fructose, maltose and starch. Organic acids (malic and tartaric) supported poor growth.Present organism could utilize a number of nitrogen sources. Nitrates in general were comparatively better sources than ammonium compounds. Aspartic acid was found to be the best nitrogen source for growth. Nitrites were toxic at lower pH values though they supported growth at alkaline medium. Best growth of the organism was obtained on MgSO₄, 7H₂O. The urea supported poor growth. ZnSO₄ inhibited the growth completely. The present organism was incapable of growing in media lacking carbon, nitrogen or sulphur.     

Cryptic dehalogenase and chloroamidase genes in Pseudomonas putida and the influence of environmental conditions on their expression

Mutants of two strains of Pseudomonas putida expressed two cryptic chloroamidases (C-amidase and Hamidase) and one cryptic dehalogenase (DehII). The mutants were selected on either 2-chloropropionamide (2CPA) or 2-monochloropropionate (2MCPA), developing as papillae in parental colonies growing on a metabolisable support substrate. Mutants expressing C-amidase were selected if 2CPA was utilised as either a carbon or a nitrogen source. H-amidase mutants were selected only if 2CPA was used as a nitrogen source. Growth temperature and pH affected the frequency of papillae production, although different temperatures and pHs did not affect the overall growth characteristics of the parental colonies. Decreasing growth temperature increased the frequency of 2cpa⁺ papillae formation, but decreased the frequency of 2mcpa⁺ papillae formation. Low pH (6.0) prevented the formation of 2mcpa⁺ and 2cpa⁺ papillae. However, in the case of the 2cpa⁺ papillae, decreasing the growth temperature also allowed papillae formation at pH 6.0.Abbreviations CAA Chloroacetamide - 2CPA 2-Chloropropionamide - DCA Dichloroacetic acid - HAA Halogenated alkanoic acid - 2MCPA 2-Monochloropropionic acid     

Biosynthesis of Inulinases by Bacillus Bacteria

Biosynthesis of extracellular inulinase by bacteria Bacillus polymyxa 29,B. polymyxa 722, and B. subtilis 68 was studied. The optimal parameters for the producer growth were as follows: pH 7.0, 33–35°C, growth duration 72 h. The presence of reduced mineral nitrogen or organic nitrogen was necessary for the enzyme biosynthesis. The inulinase biosynthesis was sharply activated in the presence of carbohydrates. B. polymyxa 722 and B. polymyxa 29 displayed the maximum activities on a starch-containing culture medium; the maximum activity of B. subtilis 68 was in the presence of sucrose. Inulin did not induce inulinase biosynthesis by the strains studied. The time course of bacteria growth and enzyme biosynthesis was studied.     

On the Origins of Cyanuric Acid Hydrolase: Purification, Substrates, and Prevalence of AtzD from Pseudomonas sp. Strain ADP

Cyanuric acid hydrolase (AtzD) from Pseudomonas sp. strain ADP was purified to homogeneity. Of 22 cyclic amides and triazine compounds tested, only cyanuric acid and N-methylisocyanuric acid were substrates. Other cyclic amidases were found not to hydrolyze cyanuric acid. Ten bacteria that use cyanuric acid as a sole nitrogen source for growth were found to contain either atzD or trzD, but not both genes.     

Gum Arabic coated magnetic nanoparticles with affinity ligands specific for antibodies

A novel magnetic support based on gum Arabic (GA) coated iron oxide magnetic nanoparticles (MNP) has been endowed with affinity properties towards immunoglobulin G (IgG) molecules. The success of the in situ triazine ligand synthesis was confirmed by fluorescence assays. Two synthetic ligands previously developed for binding to IgG, named as ligand 22/8 (artificial Protein A) and ligand 8/7 (artificial Protein L) were immobilized on to MNPs coated with GA (MNP_GA). The dimension of the particles core was not affected by the surface functionalization with GA and triazine ligands. The hydrodynamic diameters of the magnetic supports indicate that the coupling of GA leads to the formation of larger agglomerates of particles with about 1 µm, but the introduction of the triazine ligands leads to a decrease on MNPs size. The non‐functionalized MNP_GA bound 28 mg IgG/g, two times less than bare MNP (60 mg IgG/g). MNP_GA modified with ligand 22/8 bound 133 mg IgG/g support, twice higher than the value obtained for ligand 8/7 magnetic adsorbents (65 mg/g). Supports modified with ligand 22/8 were selected to study the adsorption and the elution of IgG. The adsorption of human IgG on this support followed a Langmuir behavior with a Q_máx of 344 mg IgG/g support and K_a of 1.5 × 10⁵ M. The studies on different elution conditions indicated that although the 0.05 M citrate buffer (pH 3) presented good recovery yields (elution 64% of bound protein), there was occurrence of iron leaching at this acidic pH. Therefore, a potential alternative would be to elute bound protein with a 0.05 M glycine‐NaOH (pH 11) buffer. Copyright © 2010 John Wiley & Sons, Ltd.     

Photosynthetic rate,needle longevity,and nutrient contents in <Emphasis Type="Italic">Picea glehnii</Emphasis> growing on strongly acidic volcanic ash soil in northern Japan

Picea glehnii Masters can grow in strongly acidic volcanic ash soil (pH 3.6) in northern Japan. We compared needle longevity, photosynthetic rate, and concentrations of elements in needles, in mature trees of P. glehnii growing in volcanic ash soil and in brown forest soil (pH 5.4). P. glehnii growing in volcanic ash soil showed suppressed photosynthetic rate and growth by the deficiency in nitrogen compared with its growth in brown forest soil. However, the younger needles of P. glehnii growing in volcanic ash soil maintained a high photosynthetic rate, as a result of large amounts of remobilized nitrogen from senesced needles. Needles of P. glehnii growing in volcanic ash soil did not show deficiencies in Ca, Mg, or K. Moreover, Al was at low levels in the needles, suggesting that P. glehnii was able to avoid Al toxicity by Al exclusion. P. glehnii thus exhibits great ability to adapt to an acidic environment.     

AMMONIA PRODUCTION IN UREA-GROWN CULTURES OF CHLORELLA ELLIPSOIDEA12

Production of ammonia by urea-grown Chlorella ellipsoidea was investigated. Ammonia was produced during the stationary growth phase in cultures with urea as sole nitrogen source and glucose as supplementary carbon source. Ammonia was produced only in medium containing excess urea and limiting amounts of glucose. Ammonia production was accompanied by increase in pH. In cultures with nitrate as sole nitrogen source and glucose as supplementary carbon source, growth and pH changes were similar to those in urea-glucose medium, but no ammonia was detected. Cultures grown in urea-acetate medium were similar to those grown in urea medium without additional organic carbon source. No ammonia was produced under these circumstances and growth was significantly lower than that achieved in glucose-supplemented cultures. C. ellipsoidea evidently produces an enzyme or enzyme system which forms ammonia from urea. This organism was reportedly urease-free because previous workers did not detect ammonia formation from urea. Our findings indicate that special circumstances are required to produce detectable amounts of ammonia from urea. These findings are in agreement with a recent report of urea-splitting, cofactor-requiring enzyme in cell-free extracts of Chlorella.     

Sources of Nitrogen Supporting Growth and Embryogenesis in Cultured Wild Carrot Tissue

This paper seeks to calarify conflicting reports on the nitrogen requirements for in vitro embryogenesis in Daucus carota. Tissue derived from petiole explants of the wild strain of this species were tested with a variety of sources of cellular nitrogen under conditions otherwise favorable for in vitro embryogenesis. The use of very small, sieved and well-washed inocula reduced the carry-over of soluble materials with the inoculum. Embryo yield was quantified by direct counting of samples. Nitrate at concentrations ranging from 5 to 95 mM KNO₃ supportes only weak growth and very low embryogenesis under the exacting conditions of these experiments. As little as 0.1 mM NH₄Cl added to a nitrate medium allows some embryogenesis and 10 mM NH₄Cl is near optimal when KNO₃ is in the range of 12 to 40 mM concentration. Glutamine, glutamic acid, urea and alanine can individually partially replace NH₄Cl as a supplement to KNO₃. Glutamine, alanine, and possibly glutamic acid can serve as sole sources of nitrogen supporting both good growth and embryogenesis. It was concluded that a reduced nitrogen source is required, at least as a supplement to nitrate, for rapid growth and for in vitro embryogenesis of cultured wild carrot tissue. The relationship of pH of the culture medium to growth and embryogenesis was explored and optima observed at approximately pH 5.4 for both processes.