Assessing the Interactions of Statins with Human Adenylate Kinase Isoenzyme 1: Fluorescence and Enzyme Kinetic Studies.

Statins are the most effective cholesterol-lowering drugs. They also exert many pleiotropic effects, including anti-cancer and cardio- and neuro-protective. Numerous nano-sized drug delivery systems were developed to enhance the therapeutic potential of statins. Studies on possible interactions between statins and human proteins could provide a deeper insight into the pleiotropic and adverse effects of these drugs. Adenylate kinase (AK) was found to regulate HDL endocytosis, cellular metabolism, cardiovascular function and neurodegeneration. In this work, we investigated interactions between human adenylate kinase isoenzyme 1 (hAK1) and atorvastatin (AVS), fluvastatin (FVS), pravastatin (PVS), rosuvastatin (RVS) and simvastatin (SVS) with fluorescence spectroscopy. The tested statins quenched the intrinsic fluorescence of hAK1 by creating stable hAK1-statin complexes with the binding constants of the order of 104 M-1. The enzyme kinetic studies revealed that statins inhibited hAK1 with significantly different efficiencies, in a noncompetitive manner. Simvastatin inhibited hAK1 with the highest yield comparable to that reported for diadenosine pentaphosphate, the only known hAK1 inhibitor. The determined AK sensitivity to statins differed markedly between short and long type AKs, suggesting an essential role of the LID domain in the AK inhibition. Our studies might open new horizons for the development of new modulators of short type AKs.

Evaluation of a pulsed xenon ultraviolet light device for reduction of pathogens with biofilm-forming ability and impact on environmental bioburden in clinical laboratories.

BACKGROUND: Biofilm-forming organisms can persist on surfaces in hospital clinical laboratories and potentially lead to nosocomial infections. Therefore, effective decontamination procedures are essential for reducing infections. In this study, we investigated an alternative to often ineffective manual cleaning methods, a pulsed xenon ultraviolet (PX-UV) light device. We evaluated PX-UV effect on biofilm formation ability of pathogens and also evaluated PX-UV effectiveness on environmental bioburden in clinical laboratories. METHODS: We selected and identified P. aeruginosa PA47, Staphylococcus aureus B1, and K. pnenumoniae CR52 from clinic isolates. Biofilm-forming ability and effectiveness of PX-UV in killing these biofilm forming strains on surfaces was evaluated. The central laboratory, the clinical microbiology laboratory, and the clinical immunology laboratory were chosen for testing environmental bioburden. Air samples and high-touch surface specimens in the three laboratories were obtained before and after routine manual cleaning, and after 6 min of PX-UV disinfection. The cultured microbes were then identified with MALDI- TOF-MS. RESULTS: We found that P. aeruginosa PA47, Staphylococcus aureus B1, and K. pnenumoniae CR52 were able to form robust biofilms, and that PX-UV significantly reduced colony counts of these strains on all surfaces tested. PX-UV reduced the bioburden of air samples and eliminated bioburden on surfaces. All microbes identified in the clinical laboratories were pathogenic and consisted of cocci, rods, and fungi. CONCLUSIONS: The PX-UV device effectively reduced pathogens with biofilm-forming ability on surfaces, and the environmental bioburden was also significantly reduced by PX-UV. PX-UV is a viable option for protecting staff and decreasing rates of laboratory-acquired infections.

Combined calorimetric gas- and spore-based biosensor array for online monitoring and sterility assurance of gaseous hydrogen peroxide in aseptic filling machines.

A combined calorimetric gas- and spore-based biosensor array is presented in this work to monitor and evaluate the sterilization efficacy of gaseous hydrogen peroxide in aseptic filling machines. H2O2 has been successfully measured under industrial conditions. Furthermore, the effect of H2O2 on three different spore strains , namely Bacillus atrophaeus, Bacillus subtilis and Geobacillus stearothermophilus, has been investigated by means of SEM, AFM and impedimetric measurements. In addition, the sterilization efficacy of a spore-based biosensor and the functioning principle are addressed and discussed: the sensor array is convenient to be used in aseptic food industry to guarantee sterile packages.

Evaluation of Novel Process Indicators for Rapid Monitoring of Hydrogen Peroxide Decontamination Processes.

Geobacillus stearothermophilus spores on stainless steel discs are routinely used as biological indicators for the validation of hydrogen peroxide bio-decontamination processes. Given ongoing concerns about the reliability and response time of biological indicators, we explored the potential for an enzyme-based approach to decontamination process evaluation. Thermostable adenylate kinase enzyme was coated onto a solid support and exposed to hydrogen peroxide vapour, in parallel with standard commercial 6-log biological indicators, during a series of vapour-phase hydrogen peroxide cycles in a flexible film isolator. The exposed biological indicators were enumerated to define the degree of kill at different time intervals and the results compared to the thermostable adenylate kinase values, as determined by measuring adenosine triphosphate produced by residual active enzyme. Both biological indicators and the thermostable adenylate kinase indicators exhibited a biphasic inactivation profile during the process. There was significant variance between individual cycles, with some cycles showing complete inactivation of the biological indicators to the limit of detection of the assay, within 6 min, whereas biological indicators in some cycles were inactivated at a time greater than 12 min. The log-kill of the biological indicators at intermediate time points were plotted and compared to the fully quantifiable measurements derived from the thermostable adenylate kinase indicators at the same time points. The results demonstrated very similar inactivation profiles for the enzyme and for the biological indicators, thus it was possible to define a relationship between relative light units measurement and biological indicator kill. This indicates that it is possible to use thermostable adenylate kinase measurement as a direct measure of vapour-phase hydrogen peroxide bio-decontamination performance, expressed in terms of log reduction. Because thermostable adenylate kinase measurement can be achieved within a few minutes of vapour-phase hydrogen peroxide cycle completion, compared with a minimum of 7 days for the evaluation of biological indicator growth, this offers a potentially valuable tool for rapid vapour-phase hydrogen peroxide bio-decontamination cycle development and subsequent re-qualification.LAY ABSTRACT: Pharmaceutical product manufacture is performed in controlled cleanroom and closed chamber environments (isolators) to reduce the risk of contamination. These environments undergo regular decontamination to control microbial contamination levels, using a range of methods, one of which is to vaporize hydrogen peroxide (a chemical disinfectant) into a gas or an aerosol and disperse it throughout the environment, killing any microorganisms present. Biological indicators, which consist of a small steel coupon carrying a population of bacterial spores that are more resistant to hydrogen peroxide than are most microorganisms, are placed within the environment, and then tested for growth following treatment to ensure the process was effective. Confirmation of growth/no growth (and therefore hydrogen peroxide cycle efficacy) can take up to 7 days, which significantly increases time and cost of developing and confirming cycle efficacy. This study tests whether a new technology which uses a robust enzyme, thermostable adenylate kinase, could be used to predict biological indicator growth. The study shows this method can be used to confirm hydrogen peroxide cycle efficacy, by predicting whether the BI is killed at a specific time point or not and results are obtained in a few minutes rather than 7 days. This potentially offers significant time and cost benefits.

Nitrogen gas plasma treatment of bacterial spores induces oxidative stress that damages the genomic DNA.

Gas plasma, produced by a short high­voltage pulse generated from a static induction thyristor power supply [1.5 kilo pulse/sec (kpps)], was demonstrated to inactivate Geobacillus stearothermophilus spores (decimal reduction time at 15 min, 2.48 min). Quantitative polymerase chain reaction and enzyme­linked immunosorbent assays further indicated that nitrogen gas plasma treatment for 15 min decreased the level of intact genomic DNA and increased the level of 8-hydroxy-2'-deoxyguanosine, a major product of DNA oxidation. Three potential inactivation factors were generated during operation of the gas plasma instrument: Heat, longwave ultraviolet-A and oxidative stress (production of hydrogen peroxide, nitrite and nitrate). Treatment of the spores with hydrogen peroxide (3x2­4%) effectively inactivated the bacteria, whereas heat treatment (100˚C), exposure to UV-A (75­142 mJ/cm2) and 4.92 mM peroxynitrite (•ONOO­), which is decomposed into nitrite and nitrate, did not. The results of the present study suggest the gas plasma treatment inactivates bacterial spores primarily by generating hydrogen peroxide, which contributes to the oxidation of the host genomic DNA.

Plasma Decontamination: A Case Study on Kill Efficacy of Geobacillus stearothermophilus Spores on Different Carrier Materials.

A new technology to the pharmaceutical field is presented: surface decontamination by plasmas The technology is comparable to established barrier systems like e-beam, volatile hydrogen peroxide, or radiation inactivation of microbiological contaminations. This plasma technology is part of a fully automated and validated syringe filling line at a major pharmaceutical company and is in production operation. Incoming pre-sterilized syringe containers ("tubs") are processed by plasma, solely on the outside, and passed into the aseptic filling isolator upon successful decontamination. The objective of this article is to present the operating principles and develop and establish a validation routine on the basis of standard commercial biological indicators. Their decontamination efficacies are determined and correlated to the actual inactivation efficacy on the pharmaceutical packaging material.The reference setup is explained in detail and a short presentation of the cycle development and the relevant plasma control parameters is given, with a special focus on the in-process monitor determining the cycle validity. Different microbial inactivation mechanisms are also discussed and evaluated for their contribution and interaction to enhance plasma decontamination. A material-dependent inactivation behavior was observed. In order to be able to correlate the tub surface inactivation of Geobacillus stearothermophilus endospores to metallic biological indicators, a comparative study was performed. Through consistently demonstrating the linear inactivation behavior between the different materials, it becomes possible to develop an effective and time-saving validation scheme. LAY ABSTRACT: The challenge in new decontamination systems lies in a thorough validation of the inactivation efficacy under different operating regimes. With plasma, as an ionized gas, a new barrier concept is introduced into pharmaceutical aseptic processing of syringes. The presented system operates in vacuum and only decontaminates the outer surface of pre-sterilized syringe containers ("tubs"), before they are transferred into the aseptic area. The plasma does not penetrate into the tub. This article discusses the phase from development and test germ selection, across the identified sporicidal mechanisms, to a proposal for a validation scheme on the basis of commercially available biological indicators. A special focus is placed on an extensive investigation to establish a link between the tub surface microbial kill (polystyrene and Tyvek(and (2)) ) and biological indicator inactivation (stainless steel). Additionally, a rationale is developed on how an optical in-process monitor can be applied to establish a validatable limit on the base of the predetermined inactivation data of Geobacillus stearothermophilus endospores.

Metronomic chemotherapy in the neoadjuvant setting: results of two parallel feasibility trials (TraQme and TAME) in patients with HER2+ and HER2− locally advanced breast cancer

Neoadjuvant chemotherapy has practical and theoretical advantages over adjuvant chemotherapy strategy in breast cancer (BC) management. Moreover, metronomic delivery has a more favorable toxicity profile. The present study examined the feasibility of neoadjuvant metronomic chemotherapy in two cohorts [HER2+ (TraQme) and HER2− (TAME)] of locally advanced BC. Twenty patients were prospectively enrolled (TraQme, n=9; TAME, n=11). Both cohorts received weekly paclitaxel at 100 mg/m2 during 8 weeks followed by weekly doxorubicin at 24 mg/m2 for 9 weeks in combination with oral cyclophosphamide at 100 mg/day (fixed dose). The HER2+ cohort received weekly trastuzumab. The study was interrupted because of safety issues. Thirty-six percent of patients in the TAME cohort and all patients from the TraQme cohort had stage III BC. Of note, 33% from the TraQme cohort and 66% from the TAME cohort displayed hormone receptor positivity in tumor tissue. The pathological complete response rates were 55% and 18% among patients enrolled in the TraQme and TAME cohorts, respectively. Patients in the TraQme cohort had more advanced BC stages at diagnosis, higher-grade pathological classification, and more tumors lacking hormone receptor expression, compared to the TAME cohort. The toxicity profile was also different. Two patients in the TraQme cohort developed pneumonitis, and in the TAME cohort we observed more hematological toxicity and hand-foot syndrome. The neoadjuvant metronomic chemotherapy regimen evaluated in this trial was highly effective in achieving a tumor response, especially in the HER2+ cohort. Pneumonitis was a serious, unexpected adverse event observed in this group. Further larger and randomized trials are warranted to evaluate the association between metronomic chemotherapy and trastuzumab treatment.

Evaluation of steam penetration and sterilization of natural latex wraps.

OBJECTIVE: To evaluate the efficacy of steam and ethylene oxide (EtO) sterilization of Vetrap™ bandages. STUDY DESIGN: Prospective experimental study. SAMPLE POPULATION: Vetrap™ bandages (n = 70; 35 as supplied by the manufacturer, 35 unwound and tightly rewound). METHODS: Vetrap™ bandage rolls (n = 60) marked with a 1 cm square were inoculated with 0.1 mL Geobacillus stearothermophilus spores, packaged in a pouch together with independent sterilization indicators and assigned into 3 sub-groups for sterilizer type: dynamic air removal, gravity displacement, and bench-top pre-vacuum and further sub-divided into 2 sterilization temperatures. Vetrap™ bandages rolls (n = 10) were inoculated with 0.1 mL Bacillus atrophaeus spores in the same manner and underwent EtO sterilization. After sterilization, the 1 cm marked square was aseptically resected to the level of the cardboard tube and enriched in a flask containing 10 mL tryptic soy broth for 24 hours at 60°C for G. stearothermophilus and 37°C for B. atrophaeus. Aliquots were subsequently plated on a Petri dish of tryptic soy agar and incubated at 60°C for G. stearothermophilus and 37°C for B. atrophaeus for 24 hours. Samples were scored positive if colonies of indicator organism were present on the nutrient agar after 24 hours. RESULTS: Three Vetrap™ bandages yielded post-sterilization growth of G. stearothermophilus: 2 from the dynamic air removal sterilizer at 134°C for 3.5 minutes, and 1 from the bench-top pre-vacuum sterilizer at 121°C for 15 minutes. After EtO sterilization, no positive samples were detected. CONCLUSIONS: Steam sterilization may be incomplete for Vetrap™ bandages whereas EtO showed complete destruction of resistant bacterial spores.

Efficacy, efficiency and safety aspects of hydrogen peroxide vapour and aerosolized hydrogen peroxide room disinfection systems.

BACKGROUND: This was a head-to-head comparison of two hydrogen-peroxide-based room decontamination systems. AIM: To compare the efficacy, efficiency and safety of hydrogen peroxide vapour (HPV; Clarus R, Bioquell, Andover, U.K.) and aerosolized hydrogen peroxide (aHP; SR2, Sterinis, now supplied as Glosair, Advanced Sterilization Products (ASP), Johnson & Johnson Medical Ltd, Wokingham, U.K.) room disinfection systems. METHOD: Efficacy was tested using 4- and 6-log Geobacillus stearothermophilus biological indicators (BIs) and in-house prepared test discs containing approximately 10(6) meticillin-resistant Staphylococcus aureus (MRSA), Clostridium difficile and Acinetobacter baumannii. Safety was assessed by detecting leakage of hydrogen peroxide using a hand-held detector. Efficiency was assessed by measuring the level of hydrogen peroxide using a hand-held sensor at three locations inside the room, 2 h after the start of the cycles. FINDINGS: HPV generally achieved a 6-log reduction, whereas aHP generally achieved less than a 4-log reduction on the BIs and in-house prepared test discs. Uneven distribution was evident for the aHP system but not the HPV system. Hydrogen peroxide leakage during aHP cycles with the door unsealed, as per the manufacturer's operating manual, exceeded the short-term exposure limit (2 ppm) for more than 2 h. When the door was sealed with tape, as per the HPV system, hydrogen peroxide leakage was <1 ppm for both systems. The mean concentration of hydrogen peroxide in the room 2 h after the cycle started was 1.3 [standard deviation (SD) 0.4] ppm and 2.8 (SD 0.8) ppm for the four HPV and aHP cycles, respectively. None of the readings were <2 ppm for the aHP cycles. CONCLUSION: The HPV system was safer, faster and more effective for biological inactivation.

Evaluation of peracetic acid sanitizers efficiency against spores isolated from spoiled cans in suspension and on stainless steel surfaces.

The aim of this study was to determine the inactivation effect of industrial formulations of peracetic acid biocides on bacterial spores adhering to stainless steel surfaces. A standardized protocol was used to validate biocide activity against spores in suspension. To validate sporicidal activity under practical conditions, we developed an additional protocol to simulate industrial sanitization of stainless steel surfaces with a foam sanitizer. Spores of three spore-forming bacteria, Clostridium sporogenes PA3679, Geobacillus stearothermophilus, and Moorella thermoacetica/thermoautotrophica, were sprayed onto stainless steel as bioaerosols. Sporicidal activity was high against the C. sporogenes spore suspension, with more than 5 log CFU ml(-1) destroyed at all liquid biocide contact times. Sporicidal activity also was high against G. stearothermophilus and M. thermoacetica/thermoautotrophica spores after 30 min of contact, but we found no population reduction at the 5-min contact time for the highest sporicide concentration tested. The foam biocide effectively inactivated C. sporogenes spores adhered to stainless steel but had a reduced decontamination effect on other species. For G. stearothermophilus spores, sanitization with the foam sporicide was more efficient on horizontal steel than on vertical steel, but foam sanitization was ineffective against M. thermoacetica/thermoautotrophica whatever the position. These results highlight that decontamination efficiency may differ depending on whether spores are suspended in an aqueous solution or adhered to a stainless steel surface. Biocide efficiency must be validated using relevant protocols and bacteria representative of the microbiological challenges and issues affecting each food industry.

A head-to-head comparison of hydrogen peroxide vapor and aerosol room decontamination systems.

OBJECTIVE: New technologies have emerged in recent years for the disinfection of hospital rooms and equipment that may not be disinfected adequately using conventional methods. There are several hydrogen peroxide-based area decontamination technologies on the market, but no head-to-head studies have been performed. DESIGN: We conducted a head-to-head in vitro comparison of a hydrogen peroxide vapor (HPV) system (Bioquell) and an aerosolized hydrogen peroxide (aHP) system (Sterinis). SETTING: The tests were conducted in a purpose-built 136-m(3) test room. METHODS: One HPV generator and 2 aHP machines were used, following recommendations of the manufacturers. Three repeated tests were performed for each system. The microbiological efficacy of the 2 systems was tested using 6-log Tyvek-pouched Geobacillus stearothermophilus biological indicators (BIs). The indicators were placed at 20 locations in the first test and 14 locations in the subsequent 2 tests for each system. RESULTS: All BIs were inactivated for the 3 HPV tests, compared with only 10% in the first aHP test and 79% in the other 2 aHP tests. The peak hydrogen peroxide concentration was 338 ppm for HPV and 160 ppm for aHP. The total cycle time (including aeration) was 3 and 3.5 hours for the 3 HPV tests and the 3 aHP tests, respectively. Monitoring around the perimeter of the enclosure with a handheld sensor during tests of both systems did not identify leakage. CONCLUSION: One HPV generator was more effective than 2 aHP machines for the inactivation of G. stearothermophilus BIs, and cycle times were faster for the HPV system.

Formaldehyde gas inactivation of Bacillus anthracis, Bacillus subtilis, and Geobacillus stearothermophilus spores on indoor surface materials.

AIMS: To evaluate the decontamination of Bacillus anthracis, Bacillus subtilis, and Geobacillus stearothermophilus spores on indoor surface materials using formaldehyde gas. METHODS AND RESULTS: B. anthracis, B. subtilis, and G. stearothermophilus spores were dried on seven types of indoor surfaces and exposed to approx. 1100 ppm formaldehyde gas for 10 h. Formaldehyde exposure significantly decreased viable B. anthracis, B. subtilis, and G. stearothermophilus spores on all test materials. Significant differences were observed when comparing the reduction in viable spores of B. anthracis with B. subtilis (galvanized metal and painted wallboard paper) and G. stearothermophilus (industrial carpet and painted wallboard paper). Formaldehyde gas inactivated>or=50% of the biological indicators and spore strips (approx. 1x10(6) CFU) when analyzed after 1 and 7 days. CONCLUSIONS: Formaldehyde gas significantly reduced the number of viable spores on both porous and nonporous materials in which the two surrogates exhibited similar log reductions to that of B. anthracis on most test materials. SIGNIFICANCE AND IMPACT OF THE STUDY: These results provide new comparative information for the decontamination of B. anthracis spores with surrogates on indoor surfaces using formaldehyde gas.

Crystal structure of tryptophanyl-tRNA synthetase complexed with adenosine-5' tetraphosphate: evidence for distributed use of catalytic binding energy in amino acid activation by class I aminoacyl-tRNA synthetases.

Tryptophanyl-tRNA synthetase (TrpRS) is a functionally dimeric ligase, which specifically couples hydrolysis of ATP to AMP and pyrophosphate to the formation of an ester bond between tryptophan and the cognate tRNA. TrpRS from Bacillus stearothermophilus binds the ATP analogue, adenosine-5' tetraphosphate (AQP) competitively with ATP during pyrophosphate exchange. Estimates of binding affinity from this competitive inhibition and from isothermal titration calorimetry show that AQP binds 200 times more tightly than ATP both under conditions of induced-fit, where binding is coupled to an unfavorable conformational change, and under exchange conditions, where there is no conformational change. These binding data provide an indirect experimental measurement of +3.0 kcal/mol for the conformational free energy change associated with induced-fit assembly of the active site. Thermodynamic parameters derived from the calorimetry reveal very modest enthalpic changes, consistent with binding driven largely by a favorable entropy change. The 2.5 A structure of the TrpRS:AQP complex, determined de novo by X-ray crystallography, resembles that of the previously described, pre-transition state TrpRS:ATP complexes. The anticodon-binding domain untwists relative to the Rossmann-fold domain by 20% of the way toward the orientation observed for the Products complex. An unexpected tetraphosphate conformation allows the gamma and deltad phosphate groups to occupy positions equivalent to those occupied by the beta and gamma phosphates of ATP. The beta-phosphate effects a 1.11 A extension that relocates the alpha-phosphate toward the tryptophan carboxylate while the PPi mimic moves deeper into the KMSKS loop. This configuration improves interactions between enzyme and nucleotide significantly and uniformly in the adenosine and PPi binding subsites. A new hydrogen bond forms between S194 from the class I KMSKS signature sequence and the PPi mimic. These complementary thermodynamic and structural data are all consistent with the conclusion that the tetraphosphate mimics a transition-state in which the KMSKS loop develops increasingly tight bonds to the PPi leaving group, weakening linkage to the Palpha as it is relocated by an energetically favorable domain movement. Consistent with extensive mutational data on Tyrosyl-tRNA synthetase, this aspect of the mechanism develops high transition-state affinity for the adenosine and pyrophosphate moieties, which move significantly, relative to one another, during the catalytic step.

In Vivo and In Vitro evaluation of the efficacy of a peracetic acid-based disinfectant for decontamination of acrylic resins

O objetivo deste trabalho foi avaliar a eficácia antimicrobiológica de um desinfetante à base de ácido peracético na descontaminação de resinas acrílicas termicamente ativada, quimicamente ativada e polimerizada em forno de microondas. Placas de resina foram contaminadas in vivo por meio do uso intraoral por 10 voluntários durante 7 noites e corpos-de-prova de resina foram contaminados in vitro por meio do contato com microrganismos conhecidos: Bacillus subtilis e Bacillus stearothermophilus. Os espécimes contaminados foram imersos em desinfetante à base de ácido peracético a 0,2% (Sterilife®; Lifemed) durante 5 ou 10 min e então colocados no meio de cultura BHI. Após incubação a 37°C for 48 h, o crescimento bacteriano foi avaliado por meio análise da turvação do meio de cultura. Todos os espécimes imersos em ácido peracético por 5 ou 10 min não apresentaram turvação do meio de cultura, enquanto os espécimes contaminados e colocados diretamente no meio de cultura (grupo controle) apresentaram turvação. Concluiu-se que a imersão em ácido peracético por pelo menos 5 min foi eficaz na desinfecção de resinas acrílicas termicamente ativada, quimicamente ativada e polimerizada em forno de microondas contaminadas tanto com saliva humana quanto com Bacillus subtilis ou Bacillus stearothermophilus.

Comparison of low-temperature hydrogen peroxide gas plasma sterilization for endoscopes using various Sterrad models.

This study compared the effectiveness of sterilizing four types of endoscope using different models of the Sterrad system (Sterrad 50, 100, 100S and 200). Sterilization levels meeting international requirements were attained in all cases with carriers inoculated with Geobacillus stearothermophilus spores. The endoscopes were tested in half cycles ('overkill'). This is the first study to compare the Sterrad models marketed to date in terms of effective sterilization of endoscopes with narrow lumens.

Tamoxifen induces ultrastructural alterations in membranes of Bacillus Stearothermophilus.

Tamoxifen (TAM), a non-steroid antiestrogen, is the mostly used drug for chemotherapy and chemoprevention of breast cancer. However, the mechanisms by which TAM inhibits cell proliferation in breast cancer are not fully understood. TAM strongly incorporates in biomembranes and a variety of effects have been assigned to biophysical and biochemical interactions with membranes. Therefore, a better understanding of the physicochemical basis of interaction of TAM with biomembranes is essential to elucidate the molecular mechanisms of action. A strain of Bacillus stearothermophilus has been used as a model to clarify the interaction of TAM with the cell membrane. TAM effects on the ultrastructure of membranes of this bacterium were evaluated by electron microscopy. Important ultrastructural alterations were observed in B. stearothermophilus treated with TAM, namely change in the geometry of the membrane profile from asymmetric to symmetric, disaggregation of ribosomes, coagulation of the cytoplasmic matrix, occurrence of mesossomes, appearance of fractures in membranes and the alteration of the ultrastructure of cell wall. These ultrastructural alterations confirm that TAM is a membrane-active drug and that membrane damage may be involved in molecular mechanisms of cell death induced by this drug.

Molecular mechanisms of the metabolite 4-hydroxytamoxifen of the anticancer drug tamoxifen: use of a model microorganism.

A strain of the thermophilic eubacterium Bacillus stearothermophilus was used as a model system to identify membrane mediated cytotoxic effects of 4-hydroxytamoxifen, following previous studies with tamoxifen. With this experimental approach we attempted to further clarify tamoxifen and 4-hydroxytamoxifen membrane interactions often evoked as responsible for their multiple cellular effects. Bacterial growth and the oxygen consumption rate provided quantitative data of the cytotoxic action of hydroxytamoxifen. The effects of hydroxytamoxifen on the physical properties of bacterial lipid membrane preparations were also evaluated by fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene. Cultures of B. stearothermophilus grown in a complex medium containing hydroxytamoxifen in the concentration range of 1 to 7 microM exhibited progressively longer lag adapting periods, decreased specific growth rates and lower growth yields, as compared to control cultures. Hydroxytamoxifen also affected the electron redox flow of B. stearothermophilus protoplasts and induced significant perturbation of the structural order of bacterial lipid dispersions. We concluded that the bacterial model provides useful information about the nature and repercussion of membrane physical interactions of this lipophilic drug, on the basis of an easy and economic methodology.

Evaluation of a low-temperature steam and formaldehyde sterilizer.

We evaluated a low-temperature steam and formaldehyde (LTSF) sterilizer based on the draft European Standard prEN 14180. Microbiological tests were conducted on small and full loads using process challenge devices in five programs (P1-P5). With small loads all tests showed no growth of Bacillus stearothermophilus (ATCC7953) spores. However, positive cultures were observed with full-load tests using P5 (sterilization temperature, 50 degrees C). Our data indicated that the load influenced the efficacy of the LTSF sterilizer. Desorption tests were conducted to determine residual formaldehyde in indicator strips. The mean concentrations of formaldehyde in P1-P5 were 31.9, 56.3, 54.9, 82.2 and 180.6 microg, respectively, which are below the limits allowed by the draft Standard. Our results indicate that the LTSF sterilizer is useful for sterilization because of its excellent efficacy, short handling time, and safety.

Expression of Bacillus stearothermophilus LV cadmium resistance genes in Escherichia coli causes hypersensitivity to cadmium chloride.

Determination of the nucleotide sequence of a 4.5-kb chromosomal DNA fragment of Bacillus stearothermophilus LV revealed two open reading frames (ORFs) of 121 and 727 amino acids (aa) that exhibit a high degree of similarity with the cadC and cadA cadmium resistance genes of a number of microorganisms. Transfer and expression of the B. stearothermophilus LV cadA or cadC/ cadA genes in E. coli caused increased cadmium chloride susceptibility in the bacterial host. Transfer of cadC alone did not result in any detectable phenotypic change in E. coli.