Actinomycin D in low concentrations was suggested to inhibit ribosomal RNA (rRNA) transcription via an extranucleolar mechanism. Actinomycin D was proposed to inhibit unique messenger RNAs (mRNAs) coding for proteins needed for the maintenance of rRNA transcription. According to this hypothesis actinomycin D would bind to specific nonribosomal DNA with high affinity. This hypothesis was investigated by isolating high molecular weight rat liver DNA, digesting it with restriction endonuclease EcoRI, adding [³H] actinomycin D in low concentration, performing RPC-5 chromatography to separate the restriction fragments and subsequent hybridization to rRNA. It was observed that actinomycin D bound to nonribosomal DNA with high affinity. The same experiment was performed with nucleolar DNA. High affinity actinomycin D binding was not observed in nucleolar DNA. Discrete high affinity binding DNA for actinomycin in rat liver DNA was also observed when another restriction endonuclease BamHI was used to cleave rat liver DNA. However, with rat liver DNA digested with restriction endonuclease HindIII, such a high affinity actinomycin D binding DNA was not observed. Actinomycin D was also demonstrated to bind to discrete site(s) in at least four more eukaryotic species (salmon, calf, herring and human) after DNA from these species were digested by EcoRI, labeled actinomycin D added, and RPC-5 chromatography performed. Labeled actinomycin D bound to its high affinity binding DNA was displaced by unlabeled actinomycin D in a concentration range of biological significance. However, six other antitumor agents, (doxorubicin, aclacinomycin, carminomycin, marcellomycin, musettamycin and pyrromycin) which also intercalate into DNA, did not significantly displace labeled actinomycin D from its high affinity binding DNA. Since this high affinity actinomycin D binding DNA is hypothesized to be involved in the inhibition of rRNA transcription, the actinomycin D binding DNA could have a role in the regulation of rRNA transcription. To date this is the first time that a probable regulatory DNA has been characterized by selective drug binding.

Lavendamycin, an aminoquinolinedione antibiotic is similar to streptonigrin, an antibiotic with known antiretroviral activity. Their applicability as drugs is limited due to their high toxicity to mammalian cells. A series of novel analogs of lavendamycin has been recently synthesized. In initial screening, three of the analogs showed significant inhibitory activity toward the reverse transcriptase (RT) of the avian myeloblastosis virus (AMV) and exhibited little or no animal toxicity and relatively low cellular cytotoxicity.In this study, we determined the anti-retroviral activity of nine analogs by assessing their anti-reverse transcriptase(anti-RT) activity in comparison to streptonigrin. Using both the human immunodeficiency virus (HIV) and AMV reverse transcriptase in vitro we found that the analogs exhibited significant anti-RT activity. The inhibitory activity of the analogs was dose dependent, and they had different effects on the two enzymes. At 30 µM seven of the analogs inhibited HIV-RT activity by 50% or more. At this concentration, two of the analogs were significantly more effective than streptonigrin. AMV-RT was more sensitive toward both streptonigrin and several of the analogs than was HIV-RT. Furthermore, combination of azidothymidine (AZT)-triphosphate (TP) and several of analogs showed synergistic inhibitory effects at low doses. / Department of Biology

Capes / É crescente a presença de compostos persistentes, tais como fármacos, produtos de higiene pessoal, agrotóxicos, produtos de limpeza e derivados do petróleo, em efluentes industriais, por serem tóxicos causam uma série de danos ao meio ambiente, ao serem descartados de forma inadequada sem passar por um tratamento que os torne biodegradáveis. Em consequência, ocorre o aumento da contaminação de águas subterrâneas e de superfície, estações de tratamento de esgoto e de efluentes industriais. Em função disso, novas tecnologias capazes de degradar esta classe de compostos, como os processos de oxidação avançada, estão sendo investigadas, visto que os tratamentos mais comumente utilizados pelas indústrias, os biológicos, são incapazes de degradar compostos persistentes. Assim, o presente trabalho teve como objetivo principal investigar a eficiência da aplicação de um processo de oxidação avançada (Fenton) no tratamento terciário de um efluente de um frigorífico de suínos, e na degradação do antibiótico norfloxacina (NFX). Numa primeira etapa, o efluente agroindustrial foi caracterizado e os parâmetros operacionais do processo Fenton: pH de reação, razão [H2O2]/[DQO] (m/m), razão [Fe+2]/[H2O2] (m/m) e pH de coagulação foram investigados em função da eficiência de remoção de matéria orgânica, por meio da avaliação da demanda química de oxigênio (DQO). Em uma primeira etapa os parâmetros foram investigados por meio dos ensaios definidos em planejamento fatorial fracionário 24-1 com três repetições no ponto central e, após a definição dos parâmetros significativos (p ≤ 0,05), pH de coagulação e razão [Fe+2]/[H2O2] (m/m), esses foram novamente investigados por meio do Delineamento Central Composto Rotacional (DCCR) 22, com três pontos centrais e quatro pontos axiais. A máxima remoção de DQO alcançada foi de 67,71%. Posteriormente realizou-se um estudo, no qual o antibiótico NFX foi submetido ao tratamento pelo processo Fenton. As variáveis [H2O2] e [Fe+2] foram investigadas por meio de um planejamento DCCR, 22, com três pontos centrais e quatro pontos axiais. A eficiência do processo foi avaliada em função da porcentagem de degradação do fármaco, avaliada a partir da sua concentração no decorrer do processo. A máxima degradação do fármaco alcançada foi de 96,41% para tempo reacional de 5 s e, após 10 s de reação a NFX já não foi mais detectada. A avaliação da cinética de degradação da NFX pelo processo Fenton foi investigada e o comportamento de pseudo-segunda ordem foi observado. A partir análise dos resultados obtidos, com remoções satisfatórias de matéria orgânica e NFX pelo processo Fenton, pode-se concluir de forma geral que esse processo é promissor para o tratamento terciário de efluentes agroindustriais com o objetivo de degradação de compostos persistentes ao tratamento biológico normalmente empregado nas indústrias de alimentos. / There is a growing presence of persistent compounds, such as pharmaceuticals, personal care products, pesticides, cleaning products, and petroleum in industrial wastewaters to be toxic cause a lot of damage to the environment, when improperly discarded without undergo a treatment that makes them biodegradable. As a result, is increase of contamination of groundwater and surface water, sewage treatment plants and industrial wastewater . Because of this, new technologies capable of degrading this class of compounds, such as advanced oxidation processes, are being investigated, since the treatments most commonly used by industries, biological, are unable to degrade persistent compounds. Thus, the present study aimed to investigate the effectiveness of applying an advanced oxidation process (Fenton) in the tertiary treatment of wasterwaters from a swine slaughterhouse, and the degradation of the antibiotic norfloxacin (NFX). In a first step, the agro-industrial wasterwater and wherein the operating parameters of the process Fenton reaction pH, ratio [H2O2]/[COD] (w / w) ratio [Fe + 2]/[H2O2] (w/w) pH and coagulation were investigated depending on the removal efficiency of organic matter, through the evaluation of chemical oxygen demand (COD). In a first step the parameters were investigated by means of tests defined in 24-1 fractional factorial design with three replications at the center point and, after the definition of significant parameters (p≤0.05), pH and coagulation ratio [Fe + 2]/[H2O2] (w/w), these were again investigated by means of the rotational central composite design (RCCD) 22 with three center points and four axial points. The maximum COD removal achieved was 67.71%. Subsequently we performed a study in which the antibiotic norfloxacin was subjected to treatment by the Fenton process. The variables [H2O2], and [Fe 2 +] was investigated by means of a RCCD 22design with three center points and four axial points. Process efficiency was evaluated according to the percentage of degradation of the drug assessed from its concentration in the process. The maximum degradation of the drug achieved was 96.41% for reaction time of 5 if, after 10 s of reaction to NFX was no longer detected. The evaluation of the degradation kinetics of norfloxacin by Fenton process was investigated and the behavior of the pseudo-second order was observed. From analysis of the results obtained with satisfactory removal of organic matter and norfloxacin the Fenton process, it can be concluded that in general this process is promising for tertiary treatment of agroindustrial wasterwater for the purpose of degradation of the biological treatment persistent compounds usually employed in the food industries.

Capes / É crescente a presença de compostos persistentes, tais como fármacos, produtos de higiene pessoal, agrotóxicos, produtos de limpeza e derivados do petróleo, em efluentes industriais, por serem tóxicos causam uma série de danos ao meio ambiente, ao serem descartados de forma inadequada sem passar por um tratamento que os torne biodegradáveis. Em consequência, ocorre o aumento da contaminação de águas subterrâneas e de superfície, estações de tratamento de esgoto e de efluentes industriais. Em função disso, novas tecnologias capazes de degradar esta classe de compostos, como os processos de oxidação avançada, estão sendo investigadas, visto que os tratamentos mais comumente utilizados pelas indústrias, os biológicos, são incapazes de degradar compostos persistentes. Assim, o presente trabalho teve como objetivo principal investigar a eficiência da aplicação de um processo de oxidação avançada (Fenton) no tratamento terciário de um efluente de um frigorífico de suínos, e na degradação do antibiótico norfloxacina (NFX). Numa primeira etapa, o efluente agroindustrial foi caracterizado e os parâmetros operacionais do processo Fenton: pH de reação, razão [H2O2]/[DQO] (m/m), razão [Fe+2]/[H2O2] (m/m) e pH de coagulação foram investigados em função da eficiência de remoção de matéria orgânica, por meio da avaliação da demanda química de oxigênio (DQO). Em uma primeira etapa os parâmetros foram investigados por meio dos ensaios definidos em planejamento fatorial fracionário 24-1 com três repetições no ponto central e, após a definição dos parâmetros significativos (p ≤ 0,05), pH de coagulação e razão [Fe+2]/[H2O2] (m/m), esses foram novamente investigados por meio do Delineamento Central Composto Rotacional (DCCR) 22, com três pontos centrais e quatro pontos axiais. A máxima remoção de DQO alcançada foi de 67,71%. Posteriormente realizou-se um estudo, no qual o antibiótico NFX foi submetido ao tratamento pelo processo Fenton. As variáveis [H2O2] e [Fe+2] foram investigadas por meio de um planejamento DCCR, 22, com três pontos centrais e quatro pontos axiais. A eficiência do processo foi avaliada em função da porcentagem de degradação do fármaco, avaliada a partir da sua concentração no decorrer do processo. A máxima degradação do fármaco alcançada foi de 96,41% para tempo reacional de 5 s e, após 10 s de reação a NFX já não foi mais detectada. A avaliação da cinética de degradação da NFX pelo processo Fenton foi investigada e o comportamento de pseudo-segunda ordem foi observado. A partir análise dos resultados obtidos, com remoções satisfatórias de matéria orgânica e NFX pelo processo Fenton, pode-se concluir de forma geral que esse processo é promissor para o tratamento terciário de efluentes agroindustriais com o objetivo de degradação de compostos persistentes ao tratamento biológico normalmente empregado nas indústrias de alimentos. / There is a growing presence of persistent compounds, such as pharmaceuticals, personal care products, pesticides, cleaning products, and petroleum in industrial wastewaters to be toxic cause a lot of damage to the environment, when improperly discarded without undergo a treatment that makes them biodegradable. As a result, is increase of contamination of groundwater and surface water, sewage treatment plants and industrial wastewater . Because of this, new technologies capable of degrading this class of compounds, such as advanced oxidation processes, are being investigated, since the treatments most commonly used by industries, biological, are unable to degrade persistent compounds. Thus, the present study aimed to investigate the effectiveness of applying an advanced oxidation process (Fenton) in the tertiary treatment of wasterwaters from a swine slaughterhouse, and the degradation of the antibiotic norfloxacin (NFX). In a first step, the agro-industrial wasterwater and wherein the operating parameters of the process Fenton reaction pH, ratio [H2O2]/[COD] (w / w) ratio [Fe + 2]/[H2O2] (w/w) pH and coagulation were investigated depending on the removal efficiency of organic matter, through the evaluation of chemical oxygen demand (COD). In a first step the parameters were investigated by means of tests defined in 24-1 fractional factorial design with three replications at the center point and, after the definition of significant parameters (p≤0.05), pH and coagulation ratio [Fe + 2]/[H2O2] (w/w), these were again investigated by means of the rotational central composite design (RCCD) 22 with three center points and four axial points. The maximum COD removal achieved was 67.71%. Subsequently we performed a study in which the antibiotic norfloxacin was subjected to treatment by the Fenton process. The variables [H2O2], and [Fe 2 +] was investigated by means of a RCCD 22design with three center points and four axial points. Process efficiency was evaluated according to the percentage of degradation of the drug assessed from its concentration in the process. The maximum degradation of the drug achieved was 96.41% for reaction time of 5 if, after 10 s of reaction to NFX was no longer detected. The evaluation of the degradation kinetics of norfloxacin by Fenton process was investigated and the behavior of the pseudo-second order was observed. From analysis of the results obtained with satisfactory removal of organic matter and norfloxacin the Fenton process, it can be concluded that in general this process is promising for tertiary treatment of agroindustrial wasterwater for the purpose of degradation of the biological treatment persistent compounds usually employed in the food industries.

Rapidly emerging strains of bacteria resistant to most advanced antibiotics have become issues of very important public health concern. Research currently directed towards marine actinomycetes presents a vast potential for new compounds that could be able to safely and effectively target resistant species. In this regard, ten putative Streptomyces strains isolated from the Nahoon beach were selected and assessed for antibiotic production and activity against a wide range of bacteria including reference strains, environmental strain and clinical isolates. The ethyl acetate extracts of the putative Streptomyces isolates showed activities against at least 6 and up to 26 of the 32 test bacteria. Inhibition zones were found to range between 9-32 mm diameters at a concentration of 10 mg/ml. The minimum inhibitory concentrations (MICs) of the crude extracts ranged from 0.039 - 10 mg/ml and the least minimum bactericidal concentration (MBC) demonstrated was 0.625 mg/ml against a reference strain Staphylococcus aureus ATCC 6538. Time kill kinetics of all extracts revealed bacteristatic and bactericidal activities. Average Log reductions in viable cell counts for all the extracts ranged from 0.86 Log10 and 3.99 Log10 cfu/ml after 3 h interaction and 0.01 Log10 and 4.86 Log10 after 6 h interaction at MIC, 2 × MIC, 3 × MIC and 4 × MIC concentrations. Most of the extracts were speedily bactericidal at 3 × MIC and 4 × MIC resulting in over 50 % elimination of most of the test bacteria within 3 h and 6 h interaction. The partial characterization of the crude extracts by IR spectral analysis revealed possibility of terpenoid, long chain fatty acids and secondary amine derivatives compounds in the extracts. It is therefore recommended that further investigation should address the relationship between the structure of the active component of the extracts and the broad spectrum activity, as well as a rapid method for large scale production and purification and whether this group of antibiotics has any application in managing human infectious disease.

The intra- and extracellular activities of 2 novel tetramethylpiperidine (TMP)-substituted phenazines, B4121 and B4128 against Mycobacterium tuberculosis H37R (ATCC 27294) were determined and compared with those of clofazimine (B663). Clofazimine, together with B4121 and B4128, were also tested for their activities against drug-resistant strains of M.tuberculosis. Both B4121 and B4128 were significantly more active than clofazimine against M.tuberculosis, including multidrug-resistant clinical strains of this microbial pathogen, demonstrating a lack of cross resistance between the riminophenazines and standard anti-tuberculous drugs. Using M.tuberculosis-infected monocyte-derived macrophages both B4121 and B4128 were found to possess intracellular activity, which was superior to that of both clofazimine and rifampicin. The relationship between anti mycobacterial action of the TMP-subsitituted phenazines and clofazimine and the effects of these agents on microbial PLA2 activity, cation (K+, Ca2+) fluxes and energy metabolism (ATP) was also investigated. PLA2 and cation fluxes were measured by radiometric procedures, while microbial ATP was assayed using a luciferin/luciferase chemiluminescence method. All 3 riminophenazines, particularly B4128 caused dose-related enhancement of microbial PLA2 activity, which was associated with inhibition of K+-influx and enhancement of uptake of Ca2+. The results of kinetics studies demonstrated that riminophenazine-mediated enhancement of PLA2 activity and inhibition of K+ uptake in mycobacteria are rapidly-occurring and probably related events that precede, by several minutes, any detectable effects on microbial ATP concentrations and uptake of Ca2+. Inclusion of the extracellular and intracellular Ca2+-chelating agents EGTA and BAPTA, respectively, individually or in combination, did not prevent the effects of the riminophenazines on mycobacterial PLA2 (enhancement) or K+ transport (inhibition), whereas α-tocopherol, which neutralizes PLA2 primary hydrolysis products, antagonized the inhibitory effects of the riminophenazines on microbial K+ uptake. These results demonstrated that the riminophenazine-mediated enhancement of PLA2 is a Ca2+-independent event. The involvement of PLA2 in the antimicrobial activity of the riminophenazines was supported by the observation that added, exogenous Iysophosphotidylcholine (a primary hydrolysis product of PLA2 action on membrane phospholipids) also inhibited K+ transport and growth of mycobacteria. Enhancement of endogenous PLA2 as a mechanism of riminophenazine-mediated disruption of cation transport and antimycobacterial activity was further investigated using the conventional calcium-mobilizing stimuli, calcium ionophore A23187 and thapsigargin. Both agents, but A23187 in particular caused in dose-related enhancement of microbial PLA2 activity, which was associated with inhibition of K+ influx and growth. Influx of Ca2+ into A23187- and thapsigargin-treated mycobacteria was observed using both radiometric and FURA-2-based spectrofluorimetric procedures. Exposure of the mycobacteria to these agents resulted in an immediate increase in uptake of Ca2+, which implies that enhancement of PLA2 activity in calcium-mobilizing stimuli-treated mycobacteria is Ca2+ dependent. In conclusion, the TMP-substituted phenazines possess anti mycobacterial properties which are superior to those of clofazimine, particularly against intraphagocytic M.tuberculosis. The superior anti mycobacterial properties of these agents is paralleled by their potentiating effects on microbial PLA2 and consequent inhibitory action on uptake of K+, particularly in the case of B4128. Mycobacterial PLA2 and K+ transporters may therefore represent novel targets for antimicrobial chemotherapy. / Thesis (DPhil (Medical Immunology))--University of Pretoria, 2007. / Immunology / unrestricted

Susceptibility of 112 strains of Staphylococcus aureus obtained from Dameron Hospital, Stockton, California was tested with 18 antimicrobials . The MIC method was used with the following antimicrobials : tetracycline, oxacillin, penicillin, ampicillin, vancomycin, cefazolin, erythromycin, clindamycin, gentamycin, rifampin, trimethoprimsulfamethoxazole, chloramphenicol, and cefotaxime . The standard Kirby-Bauer disc diffusion method was used to test neomycin, tobramycin, and amikacin . Methicillin, oxacillin, and nafcillin were tested with a modified Kirby-Bauer method, which included the addition of a 4% salt supplement to the media, incubation at 32C, and readings at both 24 and 48 hours. Comparing results of this study with those of Hall (1975), suggested that resistance to the following antibiotics has increased: penicillin, ampicillin, erythromycin, neomycin, gentamycin, methicillin, oxacillin, nafcillin, cefazolin, and clindamycin . Resistance to tetracycline has decreased. No resistance to chloramphenicol or vancomycin was encountered in either study . Of the 112 strains studied, 13 . 4% were susceptible to all antibiotics tested. Twelve patterns of resistance were identified : 0 . 9% were resistant to neomycin only, 1.8% to erythromycin only, 63.9% to both penicillin and ampicillin, and 20 . 0% were multiply- resistant . Nine patterns of multiple-resistance were found, involving a minimum of three antibiotics and a maximum of nine . Three MRSA strains were identified from out-patient isolates; no in-patient isolates were methicillin-resistant . The study suggests that MRSA strains are not a problem at Dameron Hospital, but identification of this group would be more accurate if incubation of the MIC panels is maintained for at least 24 hours at ~35C . It was found that the MIC method of antimicrobial susceptibility testing is more reliable than the Kirby-Bauer disc diffusion method for detection of methicillin-resistance. Problems involved in identification of heteroresistant staphylococci are discussed .