Sister-chromatid exchanges induced in rabbit lymphocytes by 2-aminofluorene and 2-acetylaminofluorene after in vitro and in vivo metabolic activation

Because short in vitro treatments of Chinese hamster cells with 2-aminofluorene and 2-acetylaminofluorene did not increase SCEs even in the presence of a metabolic activation system, experiments were carried out with rabbits to see if in vivo activation occurred. Rabbits injected with 2-AF could activate the compound and a transient dose-dependent increase in SCEs was found in peripheral lymphocytes cultured at various times after the injection. With 2-AAF, however, the response was more variable: some rabbits showed an increase immediately, but one showed an increase only after a subsequent injection. This indicated that among rabbits differences exist in their ability to detoxify 2-AAF. Because rabbits could activate the compounds, in vitro experiments were carried out to see if their lymphocytes responded differently from Chinese hamster cells and to see if the metabolic changes brought about by PHA stimulation affect the ability of the cells to activate the chemicals. The addition of PHA and the consequent metabolic stimulation do affect the induction of enzymes involved in the activation of 2-AF and 2-AAF. Benzo[a]pyrene, in contrast, can be activated by rabbit lymphocytes independently of PHA stimulation.     

Monoclonal antibodies to tumor necrosis factor-alpha influence metabolic activity in lymphocytes under conditions of chronic ethanol consumption

In Wistar rats consuming ethanol for 10 weeks (the Lieber-De Carli liquid diet), dose-dependent effects of infliximab (monoclonal antibody to tumor necrosis factor alpha, TNFα) have been demonstrated. The 10-day course of intraperitoneal injections of infliximab (1 or 10 mg/kg body weight) to rats exposed to chronic ethanol consumption caused changes in the concentrations of free amino acids and their nitrogencontaining metabolites, and also promoted the development of amino acid imbalance in liver lymphocytes. Treatment of ethanol consuming animals with infliximab increased in a dose-dependent manner the pool of free amino acids and the amount of their nitrogen-containing metabolites in lymphocytes. Adaptive changes in the system of the transport of amino acids and other nutrients in the liver lymphocytes are one of the possible mechanisms associated with TNFα inactivation.     

The timing of UV mutagenesis in yeast: continuing mutation in an excision-defective (rad1-1) strain.

Summary Mutation induction by ultraviolet light was studied in excision-defective (rad1-1) strains of Saccharomyces cerevisiae. Information about the timing of mutations in relation to postirradiation DNA replication was obtained. The experimental system involved pedigree analysis of G1 diploid cells and subsequent tetrad analysis of the mitotic segregants to detect mutations.The mutation pattern of rad1-1 strains differed from that of wild type in two respects: (1) few or none of the mutations affected both strands of the DNA, (2) mutations appeared as frequently in the second postirradiation mitotic generation as in the first.The data have led to the following conclusions about the mutation process in excision-defective (rad1-1) yeast: (a) Mutations are not fixed prior to the first postirradiation round of DNA replication. (b) Unexcised thymine dimers persist as mutagenic lesions through repeated postirradiation cell divisions. (c) Heteroduplex repair is involved in the mutation process. (d) Overlapping daughter-strand gaps are not a prerequisite for mutation. (e) The results provide no evidence that error-prone repair in this strain is inducible rather than constitutive. The data also show that (f) all 2-strand mutations (whole-colony mutants) induced in yeast by exposure to low levels of ultraviolet light are associated with excision repair, and that (g) essentially all lesions induced in excision-proficient strains have been excised at the time of the second round of postirradiation DNA synthesis.On leave of absence from the Department of Genetics, University of Edinburgh, West Mains Road, Edinburgh, Scotland EH9 3JN     

Endogenous 5-HT2B receptor activation regulates neonatal respiratory activity in vitro

An implication of 5-HT(2B) receptors in central nervous system has not yet been clearly elucidated. We studied the role of different 5-HT(2) receptor subtypes in the medullary breathing center, the pre-B?tzinger complex, and on hypoglossal motoneurons in rhythmically active transversal slice preparations of neonatal rats and mice. Local microinjection of 5-HT(2) receptor agonists revealed tonic excitation of hypoglossal motoneurons. Excitatory effects of the 5-HT(2B) receptor agonist BW723C86 could be blocked by bath application of LY272015, a highly selective 5-HT(2B) receptor antagonist. Excitatory effects of the 5-HT(2A/B/C) receptor agonist alpha-methyl 5-HT could be blocked by the preferential 5-HT(2A) receptor antagonist ketanserin. Therefore, 5-HT-induced excitation of hypoglossal motoneurons is mediated by convergent activation of 5-HT(2A) and 5-HT(2B) receptors. Local microinjection of BW723C86 in the pre-B?tzinger complex increased respiratory frequency. Bath application of LY272015 blocked respiratory activity, whereas ketanserin had no effect. Therefore, endogenous 5-HT appears to support tonic action on respiratory rhythm generation via 5-HT(2B) receptors. In preparations of 5-HT(2B) receptor-deficient mice, respiratory activity appeared unaltered. Whereas BW723C86 and LY272015 had no effects, bath application of ketanserin disturbed and blocked rhythmic activity. This demonstrates a stimulatory role of endogenous 5-HT(2B) receptor activation at the pre-B?tzinger complex and hypoglossal motoneurons that can be taken up by 5-HT(2A) receptors in the absence of 5-HT(2B) receptors. The presence of functional 5-HT(2B) receptors in the neonatal medullary breathing center indicates a potential convergent regulatory role of 5-HT(2B) and -(2A) receptors on the central respiratory network.     

Synthesis and in vitro antimycobacterial activity of N1-nicotinoyl-3-(4'-hydroxy-3'-methyl phenyl)-5-[(sub)phenyl]-2-pyrazolines

A series of N1-nicotinoyl-3- (4'-hydroxy-3'-methyl phenyl)-5-(substituted phenyl)-2-pyrazolines were synthesized by the reaction between isoniazid (INH) and chalcones and were tested for their antimycobacterial activity in vitro against Mycobacterium tuberculosis H37Rv (MTB) and INH-resistant M. tuberculosis (INHR-MTB) using the agar dilution method. Among the synthesized compounds, compound (i) N1-nicotinyl-3-(4'-hydroxy-3'-methyl phenyl)-5-(1'-chlorophenyl)-2-pyrazoline was found to be the most active agent against MTB and INHR-MTB, with minimum inhibitory concentration of 0.26 microm. When compared to INH-compound i was found to be 2.8- and 43.7-fold more active against MTB and INHR-MTB, respectively.     

Role and activity of jasmonates in plants under in vitro conditions

Jasmonates (JAs), such as jasmonic acid and its methyl ester, are lipid-derived compounds with signal functions in plant growth and development, as well as in responses to stress. JAs are widely distributed in plants as natural plant growth regulators. JAs do not work independently but work as a part of a complex signaling network with other phytohormones. They are deployed to induce response during wounding and are often used for elicitation and stimulation of secondary metabolites production in different in vitro culture systems. Application of JAs seems to be promising during different steps of the micropropagation system for different species. JAs stimulate proliferation rate of shoots, roots, callus and induce microtubers and bulblets formation. However, negative effects of JAs on the condition of plant tissues are also reported, e.g. leaf senescence, reduced growth and inhibited somatic embryogenesis. This review summarizes the current knowledge of the application and properties of jasmonates under in vitro conditions in terms of cell division, explant growth, proliferation ability, storage organ formation and stress response.

     

Synthesis and biological activity of 1beta-methyl-2-[5'-isoxazoloethenylpyrrolidin-3'-ylthio]carbapenems

A new series of 1beta-methylcarbapenems 1a-i bearing isoxazoloethenyl groups on the pyrrolidine ring has been prepared and evaluated for in vitro antibacterial activity and stability to DHP-I. Most compounds showed excellent antibacterial activity and high stability to DHP-I superior to that of meropenem. Of these new carbapenems, 1a,b,h exhibited the best combination of antibacterial activity and DHP-I stability.     

Synthesis and in vitro leishmanicidal activity of 2-(5-nitro-2-furyl) and 2-(5-nitro-2-thienyl)-5-substituted-1,3,4-thiadiazoles

A series of 2-(5-nitro-2-furyl) and 2-(5-nitro-2-thienyl)-5-substituted-1,3,4-thiadiazoles (5a-d and 6a-j) were synthesized and evaluated against Leishmania major promastigotes using (3)H-thymidine incorporation. Most of the compounds showed activity better than the reference drug sodium stibogluconate (Pentostam). The most active compound was 6c (IC(50)=0.1 microM).     

DNA replication in a diploid strain of Saccharomyces cerevisiae homozygous for the rad6-1 mutation

The generation time of a diploid strain homozygous for the rad6-1 mutation was 160 min, and the duration of the S phase was 80 min; in the parental heterozygote, these values were 90 and 40 min, respectively. Analysis of DNA sedimentation in an alkaline sucrose gradient revealed that heterozygote high-molecular-weight DNA appeared after 60 min, and homozygote high-molecular weight DNA only after a 100-min pulse.     

Induction of -2 frameshift mutations by 2-nitrofluorene, N-hydroxyacetylaminofluorene, and N-2-acetylaminofluorene in reversion assays in Escherichia coli strains differing in permeability and acetyltransferase activity

The mutagenicity of 2-nitrofluorene (NF), N-hydroxyacetylaminofluorene (N-OH-AAF), and N-2-acetylaminofluorene (AAF) was measured in strains of Escherichia coli that contain a lacZ allele that reverts by -2 frameshift mutations from CG(5) to CG(4). Mutagenesis was compared in a strain having wild-type permeability and metabolism, a strain with increased permeability caused by a lipopolysaccharide-defective (LPS(d)) mutation, a strain with N- and O-acetyltransferase (NAT/OAT) activity conferred by the Salmonella nat gene on plasmid pYG219, and a strain carrying both an LPS(d) mutation and pYG219. The LPS(d) mutation facilitated the measurement of mutagenicity but was not absolutely required, in that lower levels of mutagenicity were detected in LPS(+) strains. The NAT/OAT activity conferred by pYG219 strongly potentiated the mutagenicity of NF and N-OH-AAF. Surprisingly, AAF was mutagenic in the NAT/OAT LPS(d) strain without an exogenous P450 metabolic activation system. Its activity may be ascribable to the detection of a directly mutagenic impurity by the highly sensitive strain or to a low level of metabolic activation by the bacteria under the assay conditions. The findings add to the evidence that the lacZ allele derived from E. coli strain CC109 is an effective indicator of -2 frameshift mutagenesis and that strains expressing high levels of NAT/OAT activity are highly sensitive in monitoring the mutagenicity of nitroarenes and aromatic amides.     

Optically active antifungal azoles: synthesis and antifungal activity of (2R,3S)-2-(2,4-difluorophenyl)-3-(5-[2-[4-aryl-piperazin-1-yl]-ethyl]-tetrazol-2-yl/1-yl)-1-[1,2,4]-triazol-1-yl-butan-2-ol

A series of (2R,3S)-2-(2,4-difluorophenyl)-3-(5-[2-[4-aryl-piperazin-1-yl]-ethyl]-tetrazol-2-yl)-1-[1,2,4]-triazol-1-yl-butan-2-ol (11a-n) and (2R,3S)-2-(2,4-difluorophenyl)-3-(5-[2-[4-aryl-piperazin-1-yl]-ethyl]-tetrazole-1-yl)-1-[1,2,4]-triazol-1-yl-butan-2-ol (12a-n) has been synthesized. The antifungal activity of compounds was evaluated by in vitro agar diffusion and broth dilution assay. Compounds 11d and its positional isomer 12d having 3-trifluoromethyl substitution on the phenyl ring of piperazine demonstrated significant antifungal activity against variety of fungal cultures (Candida spp. C. neoformans and Aspergillus spp.). The compound 12d showed MIC value of 0.12 microg/mL for C. albicans, C. albicans V-01-191A-261 (resistant strain); 0.25 microg/mL for C. tropicalis, C. parapsilosis ATCC 22019 and C. krusei and MIC value of 0.5 microg/mL for C. glabrata, C. krusei ATCC 6258, which is comparable to itraconazole and better than fluconazole. Further, compound 11d showed significant activity (MIC; 0.25-0.5 microg/mL) against Candida spp. and strong anticryptococcal activity (MIC; 0.25 microg/mL) against C. neoformans.     

Synthesis of new 1-[2-Azido-2-(2,4-dichlorophenyl)ethyl]-1H/-imidazoles and in vitro evaluation of their antifungal activity

New 1-[2-azido-2-(2,4-dichlorophenyl)ethyl]-1H/-imidazole were synthesized by nucleophilic substitution of various tertiary alcohols with azide anion in presence of boron trifluoride-diethyl etherate. Their antifungal activity was evaluated against Candida albicans, Candida glabrata, Aspergillus fumigatus and an azole-resistant petite mutant of C. glabrata. Preliminary SAR results are discussed.     

Synthesis of new 1-[2-Azido-2-(2,4-dichlorophenyl)ethyl]-1H/-imidazoles and in vitro evaluation of their antifungal activity

New 1-[2-azido-2-(2,4-dichlorophenyl)ethyl]-1H/-imidazole were synthesized by nucleophilic substitution of various tertiary alcohols with azide anion in presence of boron trifluoride-diethyl etherate. Their antifungal activity was evaluated against Candida albicans, Candida glabrata, Aspergillus fumigatus and an azole-resistant petite mutant of C. glabrata. Preliminary SAR results are discussed.     

Synthesis and in vitro anti-HCV activity of beta-D- and 1-2'-deoxy-2'-fluororibonucleosides

Based on the discovery of beta-D-2'-deoxy-2'-fluorocytidine as a potent anti-hepatitis C virus (HCV) agent, a series of beta-D- and L-2'-deoxy-2'-fluoroibonucleosides with modifications at 5 and/or 4 positions were synthesized and evaluated for their in vitro activity against HCV and bovine viral diarrhea virus (BVDV). The introduction of the 2'-fluoro group was achieved by either fluorination of 2,2'-anhydronucleosides with hydrogen fluoride-pyridine or potassium fluoride, or a fluorination of arabinonucleosides with DAST. Among the 27 analogues synthesized, only the 5-fluoro compounds, namely beta-D-2'-deoxy-2',5-difluorocytidine (5), had anti-HCV activity in the subgenomic HCV replicon cell line, and inhibitory activity against ribosomal RNA. As beta-D-N4-hydroxycytidine (NHC) had previously shown potent anti-HCV activity, the two functionalities of the N4-hydroxyl and the 2'-fluoro were combined into one molecule, yielding beta-D-2'-deoxy-2'-fluoro-N4-hydroxycytidine (12). However, this nucleoside showed neither anti-HCV activity nor toxicity. All the L-forms of the analogues were devoid of anti-HCV activity. None of the compounds showed anti-BVDV activity, suggesting that the BVDV system cannot reliably predict anti-HCV activity in vitro.     

Synthesis and antitumor activities of 5-methyl-1- and 2-[[2-dimethylaminoethyl]amino]-aza-thiopyranoindazoles

The synthesis of 1- and 2-substituted aza-benzothiopyranoindazoles has been accomplished. The comparisons of the in vitro antitumor activities of the 2-substituted analogues with the benzothiopyranoindazole chemotypes indicate that the positioning of the nitrogen atom at C-9 (9-aza analogue 4d) leads to a substrate with potent antitumor activity. The 1-substituted aza-benzothiopyranoindazoles, in comparison with the corresponding 2-substituted analogues, exhibit a much lower potency.