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S N Agathos

Publications and source records attributed to S N Agathos.

At least 37 records · Page 2Linked to original sources
PubMed1998

Effect of specific inhibitors on the anaerobic reductive dechlorination of 2,4,6-trichlorophenol by a stable methanogenic consortium.

The transformation of 2,4,6-trichlorophenol (TCP) into 4-chlorophenol (4CP) was studied using a stable methanogenic enrichment culture derived from an anaerobic fixed bed reactor. Using acetate as a growth substrate, different inhibitors of methanogenesis exhibited distinct effects on TCP dechlorination. Whereas reductive dechlorination activity was not affected by 2% ethylene in the gas phase, 25 mM bromoethanesulfonic acid (BESA) had a direct inhibitory effect on this process. The choice of BESA as a specific inhibitor for identifying the subpopulations involved in reductive dechlorination of chloroaromatics is thus questionable. Inhibitors of sulfate reduction such as molybdate (20 mM) and selenate (20 mM) had a direct inhibitory effect on reductive dechlorination independently of the presence of sulfate in the medium supplemented with acetate as growth substrate. Consequently much more care must also be taken with these inhibitors to prove that reductive chlorination is coupled to sulfate reduction.

Journal Article↗
PubMed1997

Introduction and PCR detection of Desulfomonile tiedjei in soil slurry microcosms.

The aim of this work was to test the feasibility of introducing an anaerobic microbial reductive dechlorination activity into non sterile soil slurry microcosms by inoculation with the pure anaerobic bacterial strain Desulfomonile tiedjei, which is capable of dechlorinating 3-chlorobenzoate to benzoate. To show that the bacterium was established in the microcosms we followed the expression of the reductive dechlorination activity and a molecular probe based on PCR amplification of the 16S rDNA gene was developed. However, the success of PCR amplification of the 16S rDNA gene depends on the DNA extraction and purification methodologies applied, as shown through the use of several protocols. In this study we report a DNA extraction and purification method which generates sufficient and very clean DNA suitable for PCR amplification of the D. tiedjei 16S rDNA gene. The threshold of detection was about 5.10(3) bacteria per gram of soil slurry. Introduction of D. tiedjei in soil slurry microcosms proved successful since 3-chlorobenzoate dechlorination activity was established with this bacterium in microcosms normally devoid of this dechlorination capacity. Indeed, the addition of D. tiedjei to microcosms supplemented with acetate plus formate as cosubstrate, at their respective concentrations of 5 and 6 mM, led to a total biotransformation of 2.5 mM of 3-chlorobenzoate within 12 days. After complete 3-chlorobenzoate dechlorination, the 16S rDNA gene of this bacterium was specifically detected only in the inoculated microcosms as shown by PCR amplification followed by restriction mapping confirmation.

DNA, Bacterial↗
PubMed1997

Introduction of anaerobic dechlorinating bacteria into soil slurry microcosms and nested-PCR monitoring.

Desulfomonile tiedjei and Desulfitobacterium dehalogenans were chosen as model bacteria to demonstrate the introduction of an anaerobic microbia reductive dechlorination activity into nonsterile soil slurry microcosms by inoculation. De novo 3-chlorobenzoate dechlorination activity was established with the bacterium D. tiedjei in microcosms normally devoid of this dechlorination capacity. The addition of D. tiedjei to microcosms supplemented with 20 mM pyruvate as the cosubstrate resulted in total biotransformation of 1.5 mM 3-chlorobenzoate within 7 days. The introduction of the bacterium Desulfitobacterium dehalogenans into nonsterile microcosms resulted in a shortening of the period required for dechlorination activity to be established. In microcosms inoculated with Desulfitobacterium dehalogenans, total degradation of 6 mM 3-chloro-4-hydroxy phenoxyacetic acid (3-Cl-4-OHPA) was observed after 4 days in contrast to the result in noninoculated microcosms, where the total degradation of 3-Cl-4-OHPA by indigenous microorganisms was observed after 11 days. Both externally introduced bacterial strains were detected in soil slurry microcosms by a nested-PCR methodology.

Acetates↗
PubMed1994

Evaluation of monitoring approaches and effects of culture conditions on recombinant protein production in baculovirus-infected insect cells.

The baculovirus infection process of Spodoptera frugiperda (Sf9) insect cells in oxygen-controlled bioreactors in serum-free medium was investigated using a recombinant Autographa californica (AcNPV) virus expressing beta-galactosidase enzyme as a model system. A variety of monitoring techniques including trypan blue exclusion, fluorescent dye staining, oxygen uptake rate (OUR) measurements, and glucose consumption were applied to infected cells to determine the best way of evaluating cell integrity and assessing the course of baculovirus infection. The metabolism of newly-infected cells increased 90% during the first 24 hours, but as infection proceeded, and cells gradually succumbed to the baculovirus infection, the cytopathic effect of the baculovirus on the cells became evident. Oxygen and glucose uptake rate measurements appeared to more accurately assess the condition of infected cells than conventional trypan blue staining, which tended to overestimate cell viability in the mid stages of infection. The optimal harvest time varied, depending on which technique--SDS-PAGE, chromogenic (ONPG) or fluorometric (C12FDG)--was used to monitor beta-galactosidase production. Specific beta-galactosidase production was found to be insensitive to a wide range of culture dissolved oxygen tensions, whereas resuspending cells in fresh medium prior to infection increased volumetric productivity approximately two-fold (800,000 units beta-galactosidase/ml) compared to cultures infected in batch mode and allowed successful infections to occur at higher cell densities.

Animals↗
PubMed1993

Dynamic response of immobilized cells to pulse addition of L-valine in cyclosporin A biosynthesis.

A feeding strategy for L-valine was tested in the production of cyclosporin A (Cy A), a powerful immunosuppressive secondary metabolite, in celite-immobilized cells of the fungus Tolypocladium inflatum. This system has been previously shown to have promise over conventional submerged systems. Significant increase in Cy A biosynthesis was manifested in the immobilized cells when L-valine was added at 108 h (system C) and at 156 h (system D) during the exponential growth phase. However, no clearly stimulating effect of L-valine on Cy A titre was observed when the amino acid was supplemented at hour 60 (lag phase, system B) or when the valine was present from the start (system A), where system A = 100%, system B = 113%, system C = 253%, system D = 302%. The large contribution to the enhanced production of Cy A in systems C and D may be explained by the preferential channeling of L-valine to growth during the lag phase and to secondary metabolism during the late exponential phase of the immobilized cells.

Biotechnology↗
PubMed1991

Dynamics of L-valine in relation to the production of cyclosporin A by Tolypocladium inflatum.

We report the kinetics of endogenous L-valine in the fungus Tolypocladium inflatum, in an effort to understand the enhancing effect of externally supplemented L-valine on the production of the immunosuppressant cyclosporin A (CyA) in chemically defined medium. In a batch laboratory stirred reactor cultivation, the concentration of intracellular L-valine increased by up to four times between the end of the exponential phase and the beginning of the stationary phase when the medium was supplemented externally with 4 g/l L-valine. The final CyA titre under these conditions was 710 mg/l compared to only 130 mg/l attained without L-valine supplementation. In contrast to substantial growth-associated production of CyA in unsupplemented culture, the formation of the immunosuppressant was prolonged during the stationary phase in L-valine-supplemented medium. As a result, the conversion yield of CyA on L-valine remained constant during the stationary phase at 0.27 g CyA/g L-valine.

Cyclosporins↗
PubMed1991

Production scale insect cell culture.

Insect cells in culture are currently commanding great interest as superior hosts for the efficient production of biologicals with applications in health care and in agriculture. Insect cell culture is ripe for scale-up technologies, in order to meet future projected production requirements of (a) insect viruses used as bioinsecticides and (b) recombinant proteins of therapeutic potential for humans and animals. The single most prominent system used in research-based and in commercial insect cell culture today involves lepidopteran cells transfected with baculovirus expression vectors for abundant formation of recombinant biologicals. However, dipteran insect cell lines also are beginning to emerge as useful tools in biotechnology. Current practices in bioprocess development using insect cell culture, advances in media formulation and in insect cell bioreactor design, and emerging trends are presented and critically evaluated.

Journal Article↗
PubMed1990

Pigmented variants of Tolypocladium inflatum in relation to cyclosporin A production.

We have isolated four distinct colony types of the fungus Tolypocladium inflatum, the producer of the immunosuppressive agent cyclosporin A: morphologically normal white, red, and orange colonies and morphologically diverse tiny brown colonies. In liquid cultures, white normal and brown colonies developed into yellow broths. The broth of the brown colony had a low final pH and low cyclosporin production, whereas orange and red colonies had dark brown and even black broths with higher final pH and high cyclosporin production. The specific production of cyclosporin A by the red colony was three times that of the white normal colonies.

Cyclosporins↗
PubMed1990

Enhancement of cyclosporin production in a Tolypocladium inflatum strain after epichlorohydrin treatment.

Following treatment of conidia of the cyclosporin producer fungus, Tolypocladium inflatum, with 0.15 M epichlorohydrin, strain M6 was isolated. The new strain exhibited a similar growth rate to the parent organism but more extensive conidiation and several-fold higher overall cyclosporin production. Strain M6 reached titres of 318 mg l-1 cyclosporin A in agar cultures, whereas in liquid medium it produced 140 mg l-1 cyclosporin A and 68 mg l-1 cyclosporin C. It also maintained a steady volumetric productivity of 0.48 mg l-1 h-1 cyclosporin A over 2 weeks of submerged cultivation in maltose-based semisynthetic medium. The new strain holds potential for improved cyclosporin production due to the superior titres and demonstrated capacity to sustain elevated production of cyclosporin for periods greater than the wild type.

Chlorohydrins↗
PubMed1986

Studies on in vivo inactivation of gramicidin S synthetase and its retardation.

The oxygen-dependent in vivo inactivation of gramicidin S synthetase was investigated in Bacillus brevis ATCC 9999. Inhibitors of energy metabolism and of protein synthesis added to aerated cell suspensions did not provide any protection against inactivation, thus indicating that the process does not depend on energy-yielding metabolism nor on de novo protein synthesis. Organic thiols added to anaerobic long-term incubations retarded synthetase inactivation for several hours, whereas in short-term incubations of previously air-exposed cells they resulted in partial restoration of activity. The in vivo inactivation of the enzyme was found to be accompanied by a parallel drop in intracellular thiols. These results implicate enzyme SH oxidation as a mechanism of in vivo inactivation. Retardation of inactivation was achieved upon addition of utilizable carbon sources (glycerol, fructose, inositol) to aerated cell suspensions in buffer, the degree of stabilization being proportional to the ease of uptake and to the concentration of C source. This effect involves actual consumption of the exogenous C source and is accompanied by lower dissolved oxygen levels in the cell suspension. Pulsed additions of C source could retard inactivation but could not restore partly or fully lost activity. The C-source effect was blocked by the uncoupler dinitrophenol, while dissolved oxygen levels in the suspension remained low. C-source-supplemented cell suspensions incubated under air had a decreased intracellular redox state, as revealed by intracellular SH concentration.

2,4-Dinitrophenol↗
PubMedDate not supplied

Copolymers of lysine and polyethylene glycol: a new family of functionalized drug carriers.

Poly(PEG-Lys), a new, water-soluble poly(ether urethane), derived from L-lysine and poly(ethylene glycol) was investigated as a precursor for the preparation of polymeric drug conjugates. To facilitate a wide variety of coupling chemistries, the pendent carboxyl groups of poly(PEG-Lys) were converted to other reactive functional groups (amino, hydroxyl, active ester, and aldehyde) in high yield. These reactive pendent chains were then used as anchors for the covalent attachment of penicillin V and cephradine, two clinically used antimicrobial agents. Coupling to the carrier was achieved in good yields and the chemical versatility of this system was demonstrated by the preparation of conjugates having antibiotic ligands linked via biostable or biodegradable linkages to the carrier, either directly or via a spacer. Conjugate 4, poly(PEG-Lys-penicillin V ester), was obtained by linking penicillin V to the polymer backbone via hydrolytically labile ester bonds. This conjugate exhibited activity similar to that of the parent drug against three clinically important strains of bacteria. Drug activity coincided with the release of the drug from the carrier. Hydrolytically stable cephradine-containing conjugates were prepared by three different coupling methods but showed no antibiotic activity. 14C-labeled poly(PEG-Lys) was injected into mice and its biodistribution was monitored for 48 h. The carrier showed no preferential uptake by liver, spleen, or kidney. No signs of acute toxicity were evident in mice or rats when poly(PEG-Lys) was administered iv and ip at doses up to 10 g/kg. These results indicate that poly(PEG-Lys) is a promising precursor for the preparation of soluble drug conjugates.

Animals↗
PubMedDate not supplied

Mathematical modeling of the production of cyclosporin A by Tolypocladium inflatum: effect of L-valine.

A mathematical model was developed to describe the kinetics of submerged fungal growth, consumption of nutrients, and production of the immunosuppressant drug, cyclosporin A (CyA). Special emphasis was placed on the experimentally observed stimulatory effect of L-valine on CyA production. The proposed model was based on kinetic information and emerging mechanistic data on CyA biosynthesis. It was assumed that L-valine acts as both precursor and inducer of the CyA-synthesizing multienzyme (CyA synthetase), and an unmethylated intermediate of CyA was postulated in the biosynthetic process. The model consisted of two parts: one describing cell growth and substrate consumption and the other addressing the kinetics of internal properties such as endogenous valine, biosynthetic enzyme, CyA intermediate, and CyA. The success of this extensive model was confirmed by its ability to simulate adequately the kinetic profiles of both external and internal variables. For instance, the model correctly predicted the time course of intracellular L-valine accumulation. In addition, both the optimal level and timing of exogenous L-valine addition could be predicted for maximum drug production. This study suggests rational new ways of improving the fungal production process of this important immunosuppressant.

Cell Division↗