Microbial transformation of azacarbazoles. IV. Conversion of chloro-substituted alpha-carbolines by Kitasatosporia setae strain

Biotransformation of alpha-carboline derivatives substituted at positions C-5, C-6, C-7 and C-8 with chlorine, carried out with Kitasatosporia setae strain yielded corresponding 1-methyl-alpha-iso-carbolines. The formation of products is dependent on the position of chlorine in substrate molecule. When chlorine is introduced at C-6, the yield of N-1 methylation is low, about 5%. Derivatives of alpha-carboline substituted with chlorine at C-7 and C-8 form corresponding alpha-iso-carbolines with yield up to 20% and 30%, respectively, whereas 5-chloro-alpha-carboline is converted into 5-chloro-1-methyl-alpha-iso-carboline with 60% yield. Apparently, additional pathway of microbial transformation of 2-chloro-alpha-carboline has been found. Primarily formed 2-chloro-1-methyl-alpha-iso-carboline subjected to complex enzymic conversion yields quantitatively 2-methoxy-1-methyl-alpha-iso-carboline-9-N-oxide. It has been found that 2-chloro-1-methyl-alpha-iso-carboline exhibit strong cytotoxic activity, against KB cells tissue culture, ID50 = 0.01 microM/ml and inhibits growth of Kitasatosporia setae strain, MIC = 0.5 microM/ml. Toxicity of formed 2-methoxy-1-methyl-alpha-iso-carboline-N-9-oxide is markedly lower, ID50 = 0.3 microM/ml and MIC = 3.5 microM/ml. The remaining C-5, C-6, C-7 and C-8 chloroderivatives of alpha-iso-carboline occur to be less active than 2-chloro-1-methyl-alpha-iso-carboline.     

Microbial transformation of azacarbazoles. II. Conversion of methyl-substituted alpha-carbolines to corresponding alpha-iso-carbolines by Kitasatosporia setae strain

alpha-Carboline derivatives substituted with methyl groups at C-2, C-4, C-2,4, C-6 and C-8 position were subjected to N-1 methylation performed by Kitasatosporia setae. The yield of formed corresponding alpha-iso-carbolines varied, depending on the position of methyl substituent. It has been observed that the methyl group introduced in position C-2 and C-6 of alpha-carboline molecule markedly diminishes the susceptibility of nitrogen for enzymic attack in comparison with methylation reaction performed with methyl iodide. On the contrary, derivatives possessing methyl groups in positions C-4 and C-8 are methylated by chemical and microbial means with approximately the same yield. The obtained alpha-iso-carbolines appeared to be toxic to the transforming Kitasatosporia setae strain at the concentration range of 0.2-2.5 microM/ml. Taking into account that the MIC values of parent alpha-carbolines estimated for Kitasatosporia setae were much higher than 2.5 microM/ml, this finding seems to be out of the accordance with the general belief that the microbial conversion of xenobiotics is only a detoxification process. Apparently, 4-methyl and 2 -dimethyl substituted alpha-iso-carbolines besides of their antimicrobial properties, displayed strong cytotoxic activity against KB cells line at the concentration range 0.03-0.05 microM/ml.     

Microbial transformation of azacarbazoles. III. Conversion of methoxy- and phenyl- substituted alpha-carbolines to corresponding alpha-iso-carbolines by Kitasatosporia setae strain

Microbial N-1 methylation of alpha-carboline derivatives substituted at position C-2, C-6 and C-8 with methoxy- and at position C-2 and C-6 with phenyl groups conducted with Kitasatosporia setae resulted in corresponding alpha-iso-carboline formation. The yield of obtained products is significantly dependent on the position of substituent in alpha-carboline molecule. Compounds, as 2- and 6-methoxy-alpha-carboline undergo N-1 microbial methylation with strikingly low yields of 5%, whereas products of biotransformation of 8-methoxy-alpha-carboline was formed in markedly higher amount, about 50%. Similar correlations were observed for subjected to bioconversion C-2 and C-6 phenyl-alpha-carbolines. The yields of formed products were estimated as 3% and 5%, respectively. All obtained alpha-iso-carbolines were found to be toxic to the transforming strain of Kitasatosporia setae at the range of 1.2-2.5 microM/ml as well as against KB tumor culture cells (ID50 0.04-0.6 microM/ml).     

Microbial transformation of azacarbazoles. I. N-methylation of alpha-, beta-, and gamma-carbolines by Kitasatosporia setae strain

Microbial transformation of selected azacarbazoles, compounds noted for their activity as antitumor agents, conducted with Kitasatosporia setae strain resulted in N-methylation of pyridine nucleus of alpha-, beta- and gamma-carboline molecule. Formed quaternized intermediates were converted in alkaline conditions into corresponding iso-carbolines. The structure of the final products, alpha-, beta- and gamma-iso-carbolines have been established on the basis of spectral data and confirmed by chemical synthesis.     

Microbial transformation of azacarbazoles. V. Studies on N-1 methylation of 4-methyl-alpha-carboline by Kitasatosporia setae strain

N-1 methylation of an azacarbazole, 4-methyl-alpha-carboline by Kitasatosporia setae was associated with antifungal metabolite production. Incubation of 4-methyl-alpha-carboline with intact cells or homogenates of Kitasatosporia setae and L-/14CH3/-methionine or adenosyl-S-/14CH3/-L-methionine yielded radioactive products, 1,4-dimethyl-alpha-iso-carboline. The presence of 14C-labelled methyl groups in antifungal metabolite has been also observed. Mutants of Kitasatosporia setae with lost abilities to produce antifungal metabolite did not convert 4-methyl-alpha-carboline into its methyl derivative.     

Microbial transformation of azacarbazoles. VIII. Production of 14C-labelled benzo-alpha-iso-carbolines by permeabilized cells of Kitasatosporia setae strain

N-1 methylation of benzo-alpha-carbolines performed with Kitasatosporia setae strain yields corresponding benzo-alpha-iso-carbolines, compounds demonstrating relatively strong cytostatic and antitumor properties. To obtain radioactive benzo-alpha-iso-carbolines required for the further studies of their mode of antitumor action, a simple and useful method of the preparation of 14C-labelled compounds by microbial means has been elaborated.     

Microbial transformation of azacarbazoles. VII. Antitumor properties of benzo-alpha-iso-carbolines formed by Kitasatosporia setae strain from corresponding benzo-alpha-carbolines

2,3-Benzo-alpha-carboline, 7,8-benzo-alpha-carboline and their 4-methyl derivatives were subjected to microbial conversion yielding corresponding benzo-alpha-iso-carbolines. All obtained products showed significant antimicrobial and cytotoxic properties. ID50 values were found to be at range 0.01-0.001 microM/ml, regarding in vitro KB tumor cells system data. It has been found that introduction of methyl group at para-position to the nitrogen of pyridine nucleus strongly increases cytotoxic and microbial activity of benzo-alpha-iso-carbolines. Apparently it has been indicated that antitumor activity of benzo-alpha-iso-carbolines is strongly dependent on shape and size of the molecule. Of all the compounds tested only 2,3-benzo-1,4-dimethyl-alpha-iso-carboline increases life span of leukemia P388 bearing mice up to 160%.     

Microbial transformation of azacarbazoles. IX. Preliminary studies on hydroxylation of 2,3-benzo-1,4-dimethyl-alpha-iso-carboline by Paecilomyces flavinosus

Microbial transformation of 2,3-benzo-1,4-dimethyl-alpha-iso-carboline performed with several strains of fungi Beauveria bassiana, Verticillum lecani and Paecilomyces flavinosus yielded common products which were expected to be hydroxylated derivatives of starting compound. Among the microorganisms tested, strain Paecilomyces flavinosus P-5 was selected to perform quantitative bioconversion of 2,3-benzo-1,4-dimethyl-alpha-iso-carboline for preparative scale.     

Antineoplastic activity of azacarbazoles. I. Synthesis and antitumor properties of alpha-carboline and its selected derivatives

alpha-Carboline and its several derivatives have been synthesized and evaluated for their antitumor activity against L1210 lymphoid leukemia, P388 lymphocytic leukemia and Sarcoma 180. It was found that of these compounds only alpha-carboline and its derivatives substituted in C-4 position with a methyl group or in 6-C position with a methyl group and fluorine or chlorine atoms caused moderate inhibition of the tumor growth of Sarcoma 180. The introduction of bromide, iodide atoms, hydroxy-, amino-groups or some other substituents in C-6 position of alpha-carboline molecule reduced significantly the biological activity of the tested compounds against Sarcoma 180. Additionally, the introduction of an ethyl or ethoxycarbonyl group to the pyrrole ring at N-9 also obliterated antitumor properties of these analogues. None of the tested compounds displayed a significant activity against murine leukemias. In the cytotoxicity test of KB cells all the compounds were inactive.     

Antineoplastic activity of azacarbazoles. II. Effect of alpha-carboline and its derivatives on transplantable animal neoplasms

The antineoplastic properties of alpha-carboline, alpha-carboline hydrochloride, alpha-carboline N-1 methyl iodide and c-6 substituted fluoro-, chloro, nitro- and phenyl-alpha-carboline were studied. None of the compounds proved to be active when tested against i.p. transplanted B16 melanoma or i.m. implanted Lewis lung carcinoma. In addition, alpha-carboline was assessed against i.p. inoculated plasmacytoma MP26 and colon carcinoma 26, and solid tumors of mammary carcinoma 16/C and Walker carcinosarcoma 256. Under conditions tested these neoplasms did not respond to alpha-carboline.     

Use of Shewanella oneidensis for Energy Conversion in Microbial Fuel Cells

Anaerobes are a unique class of organisms, they were able to survive the heavy bombardment of the Archean period by reducing various inorganic compounds to support their metabolism and drive respiration in the early Earth's oxygen poor environment. Of all the bacteria that evolved during this period, some of the most interesting ones were the exoelectrogens, which were able to transfer electrons using extracellular routes to a chemical that is not necessarily the immediate electron acceptor. This property allows them to function in microbial fuel cells, a sustainable technology that is being developed for simultaneous wastewater treatment and electrical power generation. There are still many limitations to overcome before microbial fuel cells can be implemented in real applications and fundamental knowledge of the biological processes behind the extra‐cellular electron transfer mechanisms are still lacking. In this work, we will be presenting current research pertaining to Shewanella oneidensis MR‐1. We also introduce the use of extreme conditions as a tool to investigate the mechanisms of biofilm formation and bacterial redox behavior of high pressure‐adapted Shewanella oneidensis.

     

Microbial transformation of isoprenoid systems by means of fungi of Zoophthora genus — Microbial transformation of compounds with α-campholenic system by means of fungus Zoophthora (Neopandora) sp.

Unsaturated ketones ( 4 and 10 ) and epoxides ( 2–3 and 8–9 ) were the main products of biotransformation performed by means of Zoophthora (Neopandora) phyllobii. Enantiospecificity of both reactions leading to these compounds strongly depends on the distance of reacting fragment of molecule from the chiral center at C-1′.     

Heterogeneity of phenol oxidases in Cryptococcus neoformans.

Phenol oxidase enzymes, linked to virulence in Cryptococcus neoformans, were prepared from broken cells. More enzyme activity was found in the ultracentrifugation supernatant; less was found in the membrane fraction. Phenol oxidases were located in acrylamide gel electropherograms by activity staining with L-dihydroxyphenylalanine (DOPA). Mobility differences between soluble and solubilized membrane-bound phenol oxidases were not found. Comparison of enzymes produced at 25 and 37 degrees C revealed that the enzyme had lower activity and lower mobility at 37 degrees C. The mobility of 25 degrees C phenol oxidases from strains of C. neoformans var. gattii was lower than that of those from C. neoformans var. neoformans. Half of the phenol oxidase produced at 25 degrees C was bound by concanavalin A, while that produced at 37 degrees C was not bound. However, glucose starvation of cultures at 25 degrees C overnight resulted in increased amounts of enzyme which did not bind to concanavalin A. A given strain of C. neoformans produces different species of phenol oxidase under different culture conditions.     

Antineoplastic activity of azacarbazoles. III. Synthesis and antitumor evaluation of selected 2-, 3- aza- and diazaanalogues of carbazole

Preliminary screening of the antitumor properties of selected azacarbazoles revealed that of all the compounds tested only 2,7-diazacarbazole (compound IX) and 3,6-diazacarbazole (compound XI) caused the inhibition of Sarcoma 180 growth up to 70%. beta- and gamma-Carbolines and their derivatives in presented testing system were inactive. None of the tested compounds displayed marked activity against murine leukemias and was active in the cytotoxicity test of KB cells.     

Microbial transformation of bile acids. A unified scheme for bile acid degradation,and hydroxylation of bile acids

Through the isolation and identification of a wide variety of degradation products formed from bile acids by microorganisms, a unified scheme for the complete degradation of bile acids to carbon dioxide and water has been proposed and discussed. The proposed degradative pathways mainly consist of the following steps: natural C₂₄ 3-hydroxy bile acids → 3-oxo bile acids → Δ⁴-3-oxo bile acids → → C₁₆ or C₁₈ perhydroindane derivative (at least in two ways) → → (4→)-4-methyl-5-oxo-octanedioic acid (at least in three ways) → → CO₂ and H₂O. A microbial hydroxylation method for the preparation of bile acid samples was investigated which could be used as reference standards in the analysis of bile acids in biological materials and also as materials for studying the function of bile acids. The particular fungi, Curcularia lunata NRRL-2380, Helicostylum piriforme ATTC-8992 and Pestalotia foedans ATCC-11817 effected the 1β-, 11β-, 12β-, 15α- or 15β-hydroxylation of certain bile acids and gave the following products: 1β,3α-, 3α,12β- and 3α,15β-dihydroxy-5β-cholan ?24-oic acids, 3α,12β,15α- and 3α,12β, 15β-trihydroxy-5β-cholan-24-oic acids and 12β,15β-dihydroxy-3-oxo-5β-cholan-24-oic acid from-lithocholic acid; 1β,3α,12α- and 3α,12α,15β-trihydroxy-5β-cholan-24-oic acids and 3α, 11β-dihydroxy-12-oxo-5β-cholan-24-oic acid from deoxycholic acid; 3α, 7α, 12β-trihydroxy-5β-cholan-24-oic acid 3α,7α,12β, 15α-tetrahydroxy-5β-cholan-24-oic acid from chenodeoxycholic acid; 3α-6α,12β- and 3α,6α, 15β-trihydroxy-5β-cholan-24-oic acids from hyodeoxycholic acid; 3α, 7β, 12β trihydroxy-5β-cholan-24-oic acid from ursodeoxycholic acid; 3α,12β-dihydroxy-7-oxo-5β-cholan-24-oic acid from 3α-hydroxy-7-oxo-5β-cholan-24-oic acid. Some of these products were new compounds and their structures were determine.     

Microbial diversity of mine water at Zhong Tiaoshan copper mine, China

Microbial diversity of mine water at Zhong Tiaoshan copper mine, Shanxi province, China, was analyzed using a culture-independent 16S rRNA gene (rDNA) based on cloning approach. A total of 59 Operational Taxonomic Units (OTUs) were obtained from 226 clones from all three samples (8 OTUs from sample SX1, 25 from SX2 and 26 from SX3). 46 of them were representative OTUs and were sequenced. 93.5% of the total clones had sequences that were less than 5% difference from those in the nucleic acids database. The percentage of overlapping OTUs among samples was from 12.1% to 35.3%. Phylogenetic analysis indicated that 60.62% of the clones were affiliated with members of the Proteobacteria (alpha -3.10%, beta -24.78%, gamma -31.41%, delta -1.33%), whereas 29.20% of the clones were closely related to the Nitrospira (Leptospirillum ferrooxidans 20.80%, Leptospirillum ferriphilum 0.88% and Leptospirillum group III 7.52%, respectively). The rest clones were affiliated with the Firmicutes (2.65%) and the Bacteroidetes (7.52%). The results of Principal Component Analysis (PCA) based on the percentages of OTUs and biogeochemical data revealed that biogeochemical properties affected the diversity of microbial communities in mine water. Especially, the pH value, temperature and different concentrations of elements such as lead, zinc, sulfur, iron and copper seemed to be key factors affecting the composition and structure of microbial communities in this study.     

Changes with age in copper and superoxide dismutase levels in brains of C57BL/6J mice.

Superoxide dismutase activity in brains of male C57BL/6J mice decreased with age by 36% on a protein base and by 32% on a DNA base between 50 and 900 days of age. Brain copper increased by 45% up to 600 days of age and appeared to increase slightly from 600 to 900 days of age. Feeding of copper gluconate (5 x 10(-3)M) failed to change the level of copper in either young or old brain, and superoxide dismutase activity was not significantly increased or inhibited by copper feeding. Cupric chloride (2.35 x 10(-6)M) was found to inhibit purified superoxide dismutase (2 x 10(-8)M) by 50%. These observations indicate that brain copper concentrations do not directly determine the activity levels of the copper containing enzyme, superoxide dismutase, under normal ageing conditions.     

Monoamine oxidases and alcoholism. I. Studies in unrelated alcoholics and normal controls

Low platelet MAO activity has been associated with alcoholism. In order to evaluate the role of MAO genes in susceptibility to alcoholism, we have taken a biochemical and molecular genetic approach. The sample consisted of 133 alcoholic probands who were classified by subtypes of alcoholism and 92 normal controls. For those subjects typed for platelet MAO activity, alcoholics (N = 74) were found not to differ from the non-alcoholics controls (N = 34). Neither was there a significant difference between type I and type II alcoholics or between either subtype and normal controls. However, we do find significant differences between male and female alcoholics, but not between male and female controls. The allele frequency distribution for the MAO-A and MAO-B dinucleotide repeats is different between the alcoholic sample (N = 133) and the normal control sample (N = 92). In a two-way analysis of variance of MAO-B activity as a function of the allelic variation of each marker locus and diagnosis, there is no evidence for mean differences in activity levels for the different alleles. Our findings do not rule out a role for the MAO-B gene in controlling the enzyme activity because the dinucleotide repeats are located in introns. © 1995 Wiley-Liss, Inc.     

Structure and function of copper-containing amine oxidases

Inflammation Research -