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Redesigning metabolic routes: manipulation of TOL plasmid pathway for catabolism of alkylbenzoates.

Increasing quantities of man-made organic chemicals are released each year into the biosphere. Some of these compounds are both toxic and relatively resistant to physical, chemical, or biological degradation, and they thus constitute an environmental burden of considerable magnitude. Genetic manipulation of microbial catabolic pathways offers a powerful means by which to accelerate evolution of biodegradative routes through which such compounds might be eliminated from the environment. In the experiments described here, a catabolic pathway for alkylbenzoates specified by the TOL plasmid of Pseudomonas was restructured to produce a pathway capable of processing a new substrate, 4-ethylbenzoate. Analysis of critical steps in the TOL pathway that prevent metabolism of 4-ethylbenzoate revealed that this compound fails to induce synthesis of the catabolic enzymes and that one of its metabolic intermediates inactivates catechol 2,3-dioxygenase (C23O), the enzyme that cleaves the aromatic ring. Consequently, the pathway was sequentially modified by recruitment of genes from mutant bacteria selected for their production of either an altered pathway operon regulator that is activated by 4-ethylbenzoate or an altered C23O that is less sensitive to metabolite inactivation. The redesigned pathway was stably expressed and enabled host bacteria to degrade 4-ethylbenzoate in addition to the normal substrates of the TOL pathway.

Benzoates↗

Analysis of drug resistance in the archaebacterium Methanococcus voltae with respect to potential use in genetic engineering.

The sensitivity of the methanogenic archaebacterium Methanococcus voltae to 12 inhibitors was tested in liquid medium. Four compounds appeared to be inhibitors of growth. Their MICs were as follows: pseudomonic acid, 0.1 micrograms/ml (0.19 microM); puromycin, 2 micrograms/ml (3.6 microM); methionine sulfoximine, 30 micrograms/ml (170 microM); and fusidic acid, 100 micrograms/ml (170 microM). On solid medium, the MICs were similar and the frequency of spontaneous resistance was found to be 5 X 10(-5) (methionine sulfoximine), 10(-7) (pseudomonic acid), and less than 10(-7) (puromycin and fusidic acid). Pseudomonic acid was found to inhibit isoleucyl-tRNA synthetase activity as measured by the in vitro aminoacylation of M. voltae tRNA with L-[U-14C]isoleucine. Fusidic acid and puromycin were shown to inhibit poly(U)-dependent polyphenylalanine synthesis in S30 extracts. Acetylpuromycin was inhibitory at much higher concentrations both in vivo and in vitro for M. voltae. Thus, the pac gene of Streptomyces alboniger, which is responsible for acetylation of puromycin and which conferred resistance to puromycin when introduced in eubacteria and eucaryotes, is a potential selective marker in gene transfer experiments with M. voltae. The latter was recently shown to be transformable. The same would be true for the cat gene of Tn9, which encodes resistance to fusidic acid in eubacteria in addition to resistance to chloramphenicol.

Anti-Bacterial Agents↗

Use of a systematic risk analysis method to improve safety in the production of paediatric parenteral nutrition solutions.

BACKGROUND: Until recently, the preparation of paediatric parenteral nutrition formulations in our institution included re-transcription and manual compounding of the mixture. Although no significant clinical problems have occurred, re-engineering of this high risk activity was undertaken to improve its safety. Several changes have been implemented including new prescription software, direct recording on a server, automatic printing of the labels, and creation of a file used to pilot a BAXA MM 12 automatic compounder. The objectives of this study were to compare the risks associated with the old and new processes, to quantify the improved safety with the new process, and to identify the major residual risks. METHODS: A failure modes, effects, and criticality analysis (FMECA) was performed by a multidisciplinary team. A cause-effect diagram was built, the failure modes were defined, and the criticality index (CI) was determined for each of them on the basis of the likelihood of occurrence, the severity of the potential effect, and the detection probability. The CIs for each failure mode were compared for the old and new processes and the risk reduction was quantified. RESULTS: The sum of the CIs of all 18 identified failure modes was 3415 for the old process and 1397 for the new (reduction of 59%). The new process reduced the CIs of the different failure modes by a mean factor of 7. The CI was smaller with the new process for 15 failure modes, unchanged for two, and slightly increased for one. The greatest reduction (by a factor of 36) concerned re-transcription errors, followed by readability problems (by a factor of 30) and chemical cross contamination (by a factor of 10). The most critical steps in the new process were labelling mistakes (CI 315, maximum 810), failure to detect a dosage or product mistake (CI 288), failure to detect a typing error during the prescription (CI 175), and microbial contamination (CI 126). CONCLUSIONS: Modification of the process resulted in a significant risk reduction as shown by risk analysis. Residual failure opportunities were also quantified, allowing additional actions to be taken to reduce the risk of labelling mistakes. This study illustrates the usefulness of prospective risk analysis methods in healthcare processes. More systematic use of risk analysis is needed to guide continuous safety improvement of high risk activities.

Child↗

Influence of temperature and plumbing material selection on biofilm formation and growth of Legionella pneumophila in a model potable water system containing complex microbial flora.

Survival and growth of Legionella pneumophila in both biofilm and planktonic phases were determined with a two-stage model system. The model used filter-sterilized tap water as the sole source of nutrient to culture a naturally occurring mixed population of microorganisms including virulent L. pneumophila. At 20 degrees C, L. pneumophila accounted for a low proportion of biofilm flora on polybutylene and chlorinated polyvinyl chloride, but was absent from copper surfaces. The pathogen was most abundant on biofilms on plastics at 40 degrees C, where it accounted for up to 50% of the total biofilm flora. Copper surfaces were inhibitory to total biofouling and included only low numbers of L. pneumophila organisms. The pathogen was able to survive in biofilms on the surface of the plastic materials at 50 degrees C, but was absent from the copper surfaces at the same temperature. L. pneumophila could not be detected in the model system at 60 degrees C. In the presence of copper surfaces, biofilms forming on adjacent control glass surfaces were found to incorporate copper ions which subsequently inhibited colonization of their surfaces. This work suggests that the use of copper tubing in water systems may help to limit the colonization of water systems by L. pneumophila.

Bacterial Adhesion↗

Inhaled diesel engine emissions reduce bacterial clearance and exacerbate lung disease to Pseudomonas aeruginosa infection in vivo.

Despite experimental evidence supporting an adverse role for air pollution in models of human disease, little has been done in the way of assessing the health effects of inhalation of whole mixtures from defined sources at exposure levels relevant to ambient environmental exposures. The current study assessed the impact of inhaled diesel engine emissions (DEE) in modulating clearance of Pseudomonas aeruginosa (P.a.) and the adverse effects of infection to the pulmonary epithelium. At DEE concentrations representing from high ambient to high occupational exposures, mice were exposed to DEE continuously for one week or six months (6 h/day), and subsequently infected with P.a. by intratracheal instillation. At 18 h following P.a. infection, prior exposure to DEE impaired bacterial clearance and exacerbated lung histopathology during infection. To assess the airway epithelial cell changes indicative of lung pathogenesis, markers of specific lung epithelial cell populations were analyzed by immunohistochemistry. Both ciliated and non-ciliated airway epithelial cell numbers were decreased during P.a. infection by DEE exposure in a concentration-dependent manner. Furthermore, the lung transcription regulator, thyroid transcription factor 1 (TTF-1), was also decreased during P.a. infection by prior exposure to DEE concordant with changes in airway populations. These findings are consistent with the notion that environmental levels of DEE can decrease the clearance of P.a. and increase lung pathogenesis during pulmonary bacterial infection.

Animals↗

Neomycin resistance as a dominant selectable marker for selection and isolation of vaccinia virus recombinants.

The antibiotic G418 was shown to be an effective inhibitor of vaccinia virus replication when an appropriate concentration of it was added to cell monolayers 48 h before infection. Genetic engineering techniques were used in concert with DNA transfection protocols to construct vaccinia virus recombinants containing the neomycin resistance gene (neo) from transposon Tn5. These recombinants contained the neo gene linked in either the correct or incorrect orientation relative to the vaccinia virus 7.5-kilodalton gene promoter which is expressed constitutively throughout the course of infection. The vaccinia virus recombinant containing the chimeric neo gene in the proper orientation was able to grow and form plaques in the presence of G418, whereas both the wild-type and the recombinant virus with the neo gene in the opposite polarity were inhibited by more than 98%. The effect of G418 on virus growth may be mediated at least in part by selective inhibition of the synthesis of a subset of late viral proteins. These results are discussed with reference to using this system, the conferral of resistance to G418 with neo as a positive selectable marker, to facilitate constructing vaccinia virus recombinants which contain foreign genes of interest.

Animals↗

Sink flora in a long-stay hospital is determined by the patients' oral and rectal flora.

Sinks in a new long-stay hospital (LSH) were cultured weekly during 4 consecutive months to evaluate the microbial profile before and after occupancy of the hospital. From the elderly patients admitted to the patient care rooms oral and rectal specimens were collected to examine the contribution of the patients' flora to the sink contamination. Isolates were typed biochemically, serologically and by susceptibility pattern. Before occupancy Gram-negative bacilli were not isolated. Once the elderly patients, who were highly colonized on admission, occupied their rooms identical strains gradually contaminated the sinks. Escherichia coli, Klebsiella, Pseudomonas and Acinetobacter species were the major correlating strains. The mean concentration of the correlating isolates was higher in throat and intestines compared to the mean concentration of the non-correlating strains. These strains seem to have a greater chance to be shed and then transferred via the hands of personnel to sinks. This report shows that the major route of environmental contamination is from patient carriers to sinks, and not the reverse way.

Acinetobacter↗

A single amino acid substitution in the antimicrobial defense protein cecropin B is associated with diminished degradation by leaf intercellular fluid.

Degradation is one of several factors that may affect the level of accumulation of transgene products in plants. In plants engineered to secrete antimicrobial proteins to the intercellular compartment of leaves, the degenerative activity of proteases residing in leaf intercellular fluid (IF) could be critical to achieving the expected transgene function. We synthesized a structural analogue (MB39) of the antibacterial protein cecropin B and compared the susceptibility of both proteins to degradation in vitro by IF extracted from leaves of various crops. The half-life of the two proteins in the various IF extracts ranged from 3 min to 25.5 h, with the analogue MB39 displaying the longer half-life in IF from nine of 10 species. Overall, the half-life of MB39 averaged 2.9 times greater than that of cecropin B. Analysis of the peptides produced by endopeptidase activity in potato iF indicated that the 5.7-fold lower degradation rate of MB39 was associated with the substitution of valine for methionine at residue 11 of cecropin B. These findings point to the possibility of tailoring antimicrobial protein genes to reduce the rate of protein degradation in a particular target crop.

Amino Acid Sequence↗

Spore formation in Bacillus subtilis biofilms.

Spore formation by a Bacillus strain (Bacillus subtilis SpoIVFB-GFP) engineered with a green fluorescent protein (GFP) fused to a polytopic membrane protein (SpoIVF) that fluoresces during sporulation was observed. Biofilms of B. subtilis SpoIVFB-GFP containing ca. 8 log CFU/ml vegetative cells and spores below the lower detection limit (i.e., <1 log CFU/ ml) were allowed to develop on glass wool (37 degrees C). These biofilms were subsequently exposed to nutrient limitation to stimulate spore formation, which was monitored for fluorescence by confocal scanning laser microscopy. Sporulation in corresponding planktonic cells was also monitored for comparative purposes. Planktonic B. subtilis SpoIVFB-GFP cells began fluorescing after 5 h, while B. subtilis SpoIVFB-GFP biofilm cells began fluorescing after 30 h. Results suggested that an existing biofilm of vegetative B. subtilis cells may be stimulated to form spores when exposed to conditions of nutrient limitation. From a practical point of view, it may be suggested that a window of time does exist before sporulation occurs in attached Bacillus biofilms highlighting the need for shorter operating runs between cleaning and sanitation of food-processing equipment surfaces.

Bacillus subtilis↗

Cloning and overexpression of the old yellow enzyme gene of Candida macedoniensis, and its application to the production of a chiral compound.

The gene encoding old yellow enzyme (OYE), which catalyzes the conversion of ketoisophorone (KIP; 2,6,6-trimethyl-2-cyclohexen-1,4-dione) to (6R)-levodione (2,2,6-trimethylcyclohexane-1,4-dione), of Candida macedoniensis was cloned and sequenced. A 1212bp nucleotide fragment (oye) was confirmed to be the gene encoding OYE based on the agreement of internal amino acid sequences. Oye encodes a total 403 amino acid residues, and the deduced amino acid sequence shows a high degree of similarity to those of other microbial OYE family proteins. An expression vector, pETOYE, that contains the full length of oye was constructed. Escherichia coli harboring pETOYE exhibited an about six-fold increase in specific KIP-reducing activity under the control of the T7 promoter as compared with that of C. macedoniensis. (6R)-Levodione formed with washed cells of the transformant and a cofactor regeneration system amounted to 638 mM (98.2 mg ml(-1)), the a molar yield being 96.9%. The asymmetric reduction of KIP to (6R)-levodione with E. coli cells, which co-expressed both oye and the glucose dehydrogenase gene (gdh), as a catalyst was investigated. The (6R)-levodione formed amounted to 627 mM (96.6 mg ml(-1)), the a molar yield being 95.4%. Since the use of E. coli BL21 (DE3) cells co-expressing oye and gdh as a catalyst is simple and does not require the addition of glucose dehydrogenase, it is highly advantageous for the practical synthesis of (6R)-levodione.

Amino Acid Sequence↗

Dihydropteroate synthase mutations in Pneumocystis jiroveci can affect sulfamethoxazole resistance in a Saccharomyces cerevisiae model.

Dihydropteroate synthase (DHPS) mutations in Pneumocystis jiroveci have been associated epidemiologically with resistance to sulfamethoxazole (SMX). Since P. jiroveci cannot be cultured, inherent drug resistance cannot be measured. This study explores the effects of these mutations in a tractable model organism, Saccharomyces cerevisiae. Based on the sequence conservation between the DHPS enzymes of P. jiroveci and S. cerevisiae, together with the structural conservation of the three known DHPS structures, DHPS substitutions commonly observed in P. jiroveci were reverse engineered into the S. cerevisiae DHPS. Those mutations, T(597)A and P(599)S, can occur singly but are most commonly found together and are associated with SMX treatment failure. Mutations encoding the corresponding changes in the S. cerevisiae dhps were made in a yeast centromere vector, p414FYC, which encodes the native yeast DHPS as part of a trifunctional protein that also includes the two enzymes upstream of DHPS in the folic acid synthesis pathway, dihydroneopterin aldolase and 2-amino-4-hydroxymethyl dihydropteridine pyrophosphokinase. A yeast strain with dhps deleted was employed as the host strain, and transformants having DHPS activity were recovered. Mutants having both T(597) and P(599) substitutions had a requirement for p-aminobenzoic acid (PABA), consistent with resistance being associated with altered substrate binding. These mutants could be adapted for growth in the absence of PABA, which coincided with increased sulfa drug resistance. Upregulated PABA synthesis was thus implicated as a mechanism for sulfa drug resistance for mutants having two DHPS substitutions.

4-Aminobenzoic Acid↗

Bacterial contamination of a cornea tissue bank: implications for the safety of graft engineering.

PURPOSE: To analyze the difficulties involved in managing an episode of bacterial contamination in a cornea bank. We describe (1) the circumstances of bacterial contamination discovery, (2) the methods used to investigate the outbreak, (3) the corrective measures adopted, and (4) the method introduced to improve the reaction capacity in case of bacterial contamination. METHODS: All the samples collected were cultured in an attempt to identify the environmental reservoir of the contaminated epidemic clone. Bacteria were identified by Gram stain, oxidase test, and biochemical characteristics. The clonality of the strains was assessed by pulsed-field gel electrophoresis. RESULTS: The bacterial contamination was confirmed for 28 corneas, and 70 additional corneas were discarded. The source of the contamination was identified 17 days after the beginning of the episode. It consisted of a clonal bacterial strain that was found in trypan blue, the dye, used to examine all the tissues. The contaminating bacterium was Burkholderia cepacia, a well-known nosocomial pathogen. A total of 169 grafted corneas had been checked with the contaminated reagent. No cases of post-graft infection were recorded. CONCLUSION: Trypan blue played a major role in this outbreak. The mode and chronology of contamination remain unresolved. This exceptional event emphasizes the risk of bacterial contamination in tissue/cell banks, the necessity to improve methods for its prevention, and procedures to limit its consequences.

Burkholderia Infections↗

The influence of dilution rate, temperature, and influent substrate concentration on the efficiency of steady-state biomass production in continuous microbial culture.

The efficiency of steady-state biomass production was defined as the ratio of the biomass produced at steady state to the biomass that would be produced if influent substrate were completely and permanently incorporated. The scope of analysis was confined to microbial growth processes described by a Monod reaction mechanism. A thermo-kinetic analysis of this mechanism with coefficient values estimated from experimental data showed: (table; see text) (1) As the dilution rate was increased, the efficiency passed through a single peak. Raising the temperature did not markedly change the peak efficiency, but did broaden and shift the peak toward higher dilution rates. In contrast, elevating the influent substrate concentration significantly improved the peak efficiency while still broadening and shifting the peak toward higher dilution rates. (2) As the temperature was raised, the efficiency also showed a single peak. Increasing the dilution rate did not significantly alter the peak value or breadth, but did shift the peak to higher temperatures. In contrast, elevating the influent substrate concentration substantially improved the peak efficiency and caused the peak to shift toward lower temperatures without broadening. (3) As the influent substrate concentration was elevated, the peak efficiency improved and asymptotically approached unity. These results suggested that relatively high influent substrate concentrations, low temperatures, and moderate dilution rates would be needed to optimize the efficiency of steady-state biomass production by Monod processes with coefficient values similar to those used in this paper.

Bacteria↗

Molecular biology of stress genes in methanogens: potential for bioreactor technology.

Many agents of physical, chemical, or biological nature, have the potential for causing cell stress. These agents are called stressors and their effects on cells are due to protein denaturation. Cells, microbes, for instance, perform their physiological functions and survive stress only if they have their proteins in the necessary concentrations and shapes. To be functional a protein shape must conform to a specific three-dimensional arrangement, named the native configuration. When a stressor (e.g., temperature elevation or heat shock, decrease in pH, hypersalinity, heavy metals) hits a microbe, it causes proteins to lose their native configuration, which is to say that stressors cause protein denaturation. The cell mounts an anti-stress response: house-keeping genes are down-regulated and stress genes are activated. Among the latter are the genes that produce the Hsp70(DnaK), Hsp60, and small heat protein (sHsp) families of stress proteins. Hsp70(DnaK) is part of the molecular chaperone machine together with Hsp40(DnaJ) and GrpE, and Hsp60 is a component of the chaperonin complex. Both the chaperone machine and the chaperonins play a crucial role in assisting microbial proteins to reach their native, functional configuration and to regain it when it is partially lost due to stress. Proteins that are denatured beyond repair are degraded by proteases so they do not accumulate and become a burden to the cell. All Archaea studied to date possess chaperonins but only some methanogens have the chaperone machine. A recent genome survey indicates that Archaea do not harbor well conserved equivalents of the co-chaperones trigger factor, Hip, Hop, BAG-1, and NAC, although the data suggest that Archaea have proteins related to Hop and to the NAC alpha subunit whose functions remain to be elucidated. Other anti-stress means involve osmolytes, ion traffic, and formation of multicellular structures. All cellular anti-stress mechanisms depend on genes whose products are directly involved in counteracting the effects of stressors, or are regulators. The latter proteins monitor and modulate gene activity. Biomethanation depends on the concerted action of at least three groups of microbes, the methanogens being one of them. Their anti-stress mechanisms are briefly discussed in this Chapter from the standpoint of their role in biomethanation with emphasis on their potential for optimizing bioreactor performance. Bioreactors usually contain stressors that come with the influent, or are produced during the digestion process. If the stressors reach levels above those that can be dealt with by the anti-stress mechanisms of the microbes in the bioreactor, the microbes will die or at least cease to function. The bioreactor will malfunction and crash. Manipulation of genes involved in the anti-stress response, particularly those pertinent to the synthesis and regulation of the Hsp70(DnaK) and Hsp60 molecular machines, is a promising avenue for improving the capacity of microbes to withstand stress, and thus to continue biomethanation even when the bioreactor is loaded with harsh waste. The engineering of methanogenic consortia with stress-resistant microbes, made on demand for efficient bioprocessing of stressor-containing effluents and wastes, is a tangible possibility for the near future. This promising biotechnological development will soon become a reality due to the advances in the study of the stress response and anti-stress mechanisms at the molecular and genetic levels.

Archaea↗

Production of microbial polyesters: fermentation and downstream processes.

Poly(3-hydroxyalkanoates) (PHAs) constitute a large and versatile family of polyesters produced by various bacteria. PHAs are receiving considerable attention because of their potential as renewable and biodegradable plastics, and as a source of chiral synthons since the monomers are chiral. Industrial PHA production processes have been developed for poly(3-hydroxybutyrate) (poly(3HB)) and poly(3-hydroxybutyrate-co-3-valerate) (poly(3HB-co-3HV). More than 100 other poly(3HAMCL)s, characterized by monomers of medium chain length, have been identified in the past two decades. These monomers typically contain 6-14 carbon atoms, are usually linked via-3-hydroxy ester linkages, but can occasionally also exhibit 2-, 4-, 5-, or 6-hydroxy ester linkages. Such polyesters are collectively referred to as medium chain length PHAs poly(3HAMCL)s. The vast majority of these interesting biopolyesters have been studied and produced only on the laboratory scale. However, there have been several attempts to develop pilot scale processes, and these provide some insight into the production economics of poly(3HAMCL)s other than poly(3HB) and poly(3HB-co-3HV). These processes utilize diverse fermentation strategies to control the monomer composition of the polymer, enabling the tailoring of polymer material properties to some extent. The best studied of these is poly(3-hydroxyoctanoate) (poly(3HO)), which contains about 90% 3-hydroxyoctanoate. This biopolyester has been produced on the pilot scale and is now being used in several experimental applications.

Chemical Engineering↗

Interactions between HIV-1 and cytomegalovirus in human osteosarcoma cells carrying both viruses.

Cytomegalovirus (CMV) and the human immunodeficiency virus type 1 (HIV-1) may interact in the pathogenesis of AIDS. We compared CMV replication in human osteosarcoma (HOS) cells to that in HOS cells genetically engineered to contain an envelope-deficient HIV-1 proviral construct (designated HOS-HXG). Following acute CMV infection of each cell line, HOS-HXG cells contained higher numbers of intranuclear CMV nucleocapsids than did HOS cells. Infectious CMV could be persistently detected in culture supernatant fluids of the CMV-infected HOS-HXG cells, whereas CMV was lost over several weeks from HOS cells infected with CMV in parallel. HIV-1 CMV pseudotypes were not detected in supernatant fluids from CMV-infected HOS-HXG cells. On day 119 after CMV infection, these cultures were superinfected with HIV-1. These dually infected HOS-HXG cells produced infectious HIV-1 and exhibited markedly enhanced CMV replication compared to parental CMV-infected HOS-HXG cells. Two different HIV-1 tat gene function antagonists, Ro24-7429 and chemically modified antibodies to the Tat protein, did not inhibit the replication of CMV in either acute or persistent infections of HOS-HXG cells at concentrations that inhibited HIV-1 replication.

Antiviral Agents↗

Synergistic antifungal activities of bafilomycin A(1), fluconazole, and the pneumocandin MK-0991/caspofungin acetate (L-743,873) with calcineurin inhibitors FK506 and L-685,818 against Cryptococcus neoformans.

Cryptococcus neoformans is an opportunistic fungal pathogen that causes life-threatening infections of the central nervous system. Existing therapies include amphotericin B, fluconazole, and flucytosine, which are limited by toxic side effects and the emergence of drug resistance. We recently demonstrated that the protein phosphatase calcineurin is required for growth at 37 degrees C and virulence of C. neoformans. Because calcineurin is the target of potent inhibitors in widespread clinical use, cyclosporine and FK506 (tacrolimus), it is an attractive drug target for novel antifungal agents. Here we have explored the synergistic potential of combining the calcineurin inhibitor FK506 or its nonimmunosuppressive analog, L-685,818, with other antifungal agents and examined the molecular basis of FK506 action by using genetically engineered fungal strains that lack the FK506 target proteins FKBP12 and calcineurin. We demonstrate that FK506 exhibits marked synergistic activity with the H(+)ATPase inhibitor bafilomycin A(1) via a novel action distinct from calcineurin loss of function. FK506 also exhibits synergistic activity with the pneumocandin MK-0991/caspofungin acetate (formerly L-743,873), which targets the essential beta-1,3 glucan synthase, and in this case, FK506 action is mediated via FKBP12-dependent inhibition of calcineurin. Finally, we demonstrate that FK506 and fluconazole have synergistic activity that is independent of both FKBP12 and calcineurin and may involve the known ability of FK506 to inhibit multidrug resistance pumps, which are known to export azoles from fungal cells. In summary, our studies illustrate the potential for synergistic activity of a variety of different drug combinations and the power of molecular genetics to define the mechanisms of drug action, as well as identify a novel action of FK506 that could have profound implications for therapeutic or toxic effects in other organisms, including humans.

Anti-Bacterial Agents↗

A more potent bystander cytocidal effect elicited by tumor cells expressing the herpes simplex virus-thymidine kinase gene than by fibroblast virus-producer cells in vitro.

Retrovirus-mediated herpes simplex virus-thymidine kinase (HSV-tk) gene therapy is a promising approach in the treatment of brain tumors. Previous in vitro and in vivo studies have demonstrated a bystander effect in which nonmodified tumor cells in proximity to HSV-tk-modified tumor cells are killed with the modified cells in the presence of ganciclovir. In the present study the authors assessed the contribution of infectious HSV-tk retrovirus made by producer cells to the bystander cytocidal effect in tissue culture using Walker 256 rat breast carcinosarcoma cells, which represent an established model for carcinomatous meningitis. The authors observed ganciclovir-dependent growth inhibition even when only one HSV-tk-positive Walker cell was mixed with 1000 HSV-tk-negative Walker cells and showed that the bystander cytocidal effect is not mediated by toxic cell lysis products. Walker cells engineered to produce HSV-tk retrovirus with titers ranging from 10(3) to 10(5) colony-forming units/ml exert no greater cytocidal effect than nonviral producer HSV-tk-positive Walker cells in vitro. Murine fibroblast-producer cells with viral titers ranging from 10(6) to 10(7) colony-forming units/ml exerted a stronger cytocidal effect than nonviral producer HSV-tk-positive murine fibroblasts. Despite the high viral titers of fibroblast producer cells, HSV-tk-modified Walker cells performed better than fibroblast producer cells in their cytotoxic effect on wild-type Walker tumor cells. Given that HSV-tk-modified tumor cells can become ganciclovir resistant, we tested gamma-irradiation as a means to overcome resistance. Lethal gamma-irradiation of the HSV-tk-positive Walker cells did not abolish their bystander effect on Walker HSV-tk-negative cells. One can infer from these results that HSV-tk-modified tumor cells, irradiated or not, may be a better alternative to murine fibroblast producer cells in the treatment of central nervous system neoplasia.

3T3 Cells↗