Frank DiCosmo

Plant cell and tissue culture: Alternatives for metabolite production

Plant cell culture systems represent a potential renewable source of valuable medicinals, flavours, essences and colourants that cannot be produced by microbial cells or chemical syntheses. However, only a few cultures produce these compounds in commercially useful amounts. The low productivities are associated with our poor understanding of the biochemistry of these systems. Recent advances in molecular biology, enzymology, physiology and fermentation technology of plant cell cultures suggest that these systems will become a viable source of important natural products. This review examines the sate of the art of production of medicinal plant secondary metabolites by plant cell cultures.

A liposomal hydrogel for the prevention of bacterial adhesion to catheters

The adhesion of bacteria to medical implants and the subsequent development of a biofilm frequently results in the infection of surrounding tissue and may require removal of the device. We have developed a liposomal hydrogel system that significantly reduces bacterial adhesion to silicone catheter material. The system consists of a poly (ethylene glycol)-gelatin hydrogel in which liposomes containing the antibiotic ciprofloxacin are sequestered. A poly (ethylene glycol)-gelatin-liposome mixture was applied to a silicone surface that had been pre-treated with phenylazido-modified gelatin. Hydrogel cross-linking and attachment to surface-immobilized gelatin was accomplished through the formation of urethane bonds between gelatin and nitrophenyl carbonate-activated poly (ethylene glycol). Liposomal hydrogel-coated catheters were shown to have an initial ciprofloxacin content of 185+/-16 microg cm(-2). Ciprofloxacin was released over seven days with an average release rate of 1.9+/-0.2 microg cm(-2) h(-1) for the first 94 h. In vitro assays using a clinical isolate of Pseudomonas aeruginosa established the antimicrobial efficacy of the liposomal hydrogel. A modified Kirby-Bauer assay produced growth-inhibition zone diameters of 39+/-1 mm, while bacterial adhesion was completely inhibited on catheter surfaces throughout a seven-day in vitro adhesion assay. This new antimicrobial coating shows promise as a prophylactic and/or treatment for catheter-related infection.

Purification and characterization of α‐3′,4′‐anhydrovinblastine synthase (peroxidase‐like) from Catharanthus roseus (L.) G. Don

An H2O2‐dependent enzyme capable of coupling catharanthine and vindoline into α‐3′,4′‐anhydrovinblastine (AVLB) was purified to apparent homogeneity from Catharanthus roseus leaves. The enzyme shows a specific AVLB synthase activity of 1.8 nkat/mg, and a molecular weight of 45.40 kDa (SDS‐PAGE). In addition to AVLB synthase activity, the purified enzyme shows peroxidase activity, and the VIS spectrum of the protein presents maxima at 404, 501 and 633 nm, indicating that it is a high spin ferric heme protein, belonging to the plant peroxidase superfamily. Kinetic studies revealed that both catharanthine and vindoline were substrates of the enzyme, AVLB being the major coupling product.

Stress and Secondary Metabolism in Cultured Plant Cells

Grime1 defines “stress” in plants as the external constraints which limit the rate of dry matter production of all or part of the vegetation, e.g. shortages of water, light, mineral nutrients and suboptimal temperatures. These shortages may be an inherent characteristic of the environment, or they may be induced or intensified by the vegetation itself. The use of the word “stress” to describe an external constraint on dry matter production differs from that of many plant physiologists however, who have used the word to describe the physiological state of the plant. Grime’s definition could be applied to cultured plant cells; stress in this case would be any type of constraint which reduces the dry matter of the cultures. This otherwise excellent and precise definition is not very useful, however, to phytochemists who are generally not interested in dry matter production. We will define stress as the external constraints on cultured cells which limit the normal production of secondary metabolites. Let us call this phytochemical stress to be absolutely specific.

Antibiotic hydrogel coated Foley catheters for prevention of urinary tract infection in a rabbit model.

PURPOSE We developed an antibiotic liposome (ciprofloxacin liposome) containing hydrogel for external coating of silicone Foley catheters and evaluated its efficacy in a rabbit model. Our goal was to create a catheter that would hinder the development of catheter associated nosocomial urinary tract infections. MATERIALS AND METHODS We inserted either an untreated, liposomal hydrogel coated or a liposome hydrogel with ciprofloxacin coated 10F silicone Foley catheter into New Zealand White rabbits. We challenged the system with 5x10(6) virulent Escherichia coli at the urethral meatus twice daily for 3 days. Urine cultures were evaluated twice daily for 7 days. When urine cultures became positive, the rabbits were sacrificed and urine, urethral catheter and urethral tissue were cultured. RESULTS The time to bacteriuria detection in 50% of the specimens was double for hydrogel with ciprofloxacin coated catheters versus untreated and hydrogel coated catheters. A significant (p = 0.04) improvement in average time to positive urine culture from 3.5 to 5.3 days and a 30% decrease in the bacteriuria rate for hydrogel with ciprofloxacin coated catheters were noted compared to untreated catheters. CONCLUSIONS A significant benefit was realized by coating the extraluminal catheter surface with a ciprofloxacin liposome impregnated hydrogel. We believe this procedure will provide a significant clinical advantage, while reducing health care costs substantially.

Secondary metabolite biosynthesis in cultured cells of Catharanthus roseus (L.) G. Don immobilized by adhesion to glass fibres

SummarySuspension-cultured cells of Catharanthus roseus (L.) G. Don were immobilized on glass fibre mats and cultivated in shake flasks. The highly-aggregated immobilized cells exhibited a slower growth rate and accumulated reduced levels of tryptamine and indole alkaloids, represented by catharanthine and ajmalicine, in comparison to cells in suspension. The increased total protein synthesis in immobilized cells suggests a diversion of the primary metabolic flux toward protein biosynthetic pathways and away from other growth processes. In vitro assays for the specific activity of tryptophan decarboxylase (TDC) and tryptophan synthase (TS) suggest that the decreased accumulation of tryptamine in immobilized cells was due to reduced tryptophan biosynthesis. The specific activity of TDC was similar in immobilized and suspension-cultured cells. However, the expression of TS activity in immobilized cells was reduced to less than 25% of the maximum level in suspension-cultured cells. The reduced availability of a free tryptophan pool in immobilized cells is consistent with the reduced TS activity. Reduced tryptamine accumulation, however, was not responsible for the decreased accumulation of indole alkaloids in immobilized cells. Indole alkaloid accumulation increased to a similar level in immobilized and suspension-cultured cells only after the addition of exogenous secolaganin to the culture medium. The addition of tryptophan resulted in increased accumulation of tryptamine, but had no effect on indole alkaloid levels. Reduced biosynthesis of secologanin, the monoterpenoid precursor to indole alkaloids, in immobilized cells is suggested. Immobilization does not appear to alter the activity of indole alkaloid biosynthetic enzymes in our system beyond, and including, strictosidine synthase.

Localized drug delivery using crosslinked gelatin gels containing liposomes: Factors influencing liposome stability and drug release

We describe a drug-delivery vehicle that combines the sustained release properties of liposomes with the structural advantages of crosslinked gelatin gels that can be implanted directly or coated onto medical devices. Liposome inclusion in gelatin gels does not compromise thermal stability nor does it interfere with the resiliency of gels to tensile force. However, electron spin resonance analysis of sequestered DPPC liposomes revealed a slight depression (ca. 1.0 degrees C) of the gel-to-fluid phase transition relative to liposomes in suspension. The level of liposome release from gels was determined by liposome concentration, liposome size, and the presence of poly(ethylene oxide) chains in the gel matrix or in the liposome membrane. Both neutral and charged liposomes displayed relatively high affinities for poly(ethylene glycol)gelatin gels, with only 10-15% release of initially sequestered liposomes while liposomes in which poly(ethylene glycol) was included within the membrane were not as well retained (approximately 65% release). The in vitro efflux of ciprofloxacin from liposomal gels immersed in serum was nearly complete after 24 h compared to 38% release of liposomal chlorhexidine after 6 days. The serum-induced destabilization of liposomal ciprofloxacin depended on the accessibility of serum components to gels as partly immersed gels retained approximately 50% of their load of drug after 24 h. In vivo experiments using a catheterized rabbit model of urinary tract infection revealed the absence of viable Escherichia coli on coated catheter surfaces in seven out of nine cases while all untreated catheter surfaces examined (n = 7) were contaminated.

Increased synthesis of ajmalicine and catharanthine by cell suspension cultures of Catharanthus roseus in response to fungal culture-filtrates

The ammonium sulfate-precipitated fraction from mycelia and culture-filtrates and the crude, cell-free culture filtrates from the growth medium of the fungiChrysosporium palmorum, Eurotium rubrum, Micromucor isabellina, andPythium aphanidermatum when aseptically added to cell suspensions ofCantharanthus roseus caused a rapid and dramatic increase in indole alkaloid biosynthesis. Up to 400 μg/L ajmalicine and 600 μg/L catharanthine were detected in C.roseus cell suspension grown in the presence of theM. isabellina fungal culture filtrate for 3 d. Untreated cells produced only trace levels of ajmalicine and catharanthine per liter of cell suspension after 15 d of culture.

Immobilization of cultured Catharanthus roseus cells using a fibreglass substratum

SummaryCultured Catharanthus roseus cells were immobilized using geometrically identical needled fibreglass mats prepared with a range of surface coatings. The phenyl (PS), polyglycol (PG), aldehyde (CHO), alkyl (CTMS), and silanol (AW) coatings, along with the untreated glass (HC) surface, produced surfaces with a range of surface tensions. The immobilization efficiency of the substratum, measured as the percentage of cells immobilized, increased with increasing substratum surface tension in the order PS < PG < CHO < CTMS < AW < HC. The dependence of immobilization efficiency on substratum surface tension can be described using a thermodynamic model of adhesion that considers the extent of plant cell adhesion to be a function of the surface tensions of the substratum, the suspending liquid, and the plant cells. In addition, this dependence also demonstrates the fundamental role of adhesion in the immobilization process involving a glass fibre matrix. However, cell entrapment processes are also implicated. The untreated glass fibre substratum (HC), which demonstrated the greatest immobilization efficiency, was used for further characterization of the immobilization strategy. Maximum inoculum biomass was determined to be approximately 1.9 g cells (fresh weight)/g substratum (dry weight) to achieve greater than 90% immobilization efficiency. The growth rate of immobilized cultures was slower than suspension cultures, probably due to mass transfer limitations. Production of the indole alkaloids, tryptamine, catharanthine, and ajmalicine, was also suppressed relative to suspension-cultured cells. These results are considered in relation to other immobilization strategies and their apparent effects on cellular processes.

Thermodynamic aspects of plant cell adhesion to polymer surfaces

SummaryA thermodynamic model of particle adhesion from a suspension onto a solid surface is used to predict the extent of adhesion of suspension-cultured Catharanthus roseus cells to the following polymer substrates: fluorinated ethylene-propylene (FEP), polystyrene (PS), polyethylene terephthalate (PET), sulphonated polystyrene (SPS), and glass. According to this model, the extent of adhesion is determined by the surface tensions of the plant cells, the polymer substrates, and the suspending liquid medium. Experimentally, adhesion of the washed plant cells was found to decrease with increasing substrate surface tension, following the sequence FEP>PS>PET>SPS>glass, when the surface tension of the liquid was greater than that of the plant cells, in agreement with the model. However, adhesion increased with increasing substrate surface tension when the liquid surface tension was lower than the cellular surface tension, also in agreement with the model. When the liquid and cellular tensions were equal the extent of adhesion was independent of the substrate surface tension. This also agrees with model predictions and leads to a value for the surface tension of C. roseus cells of approximately 54 ergs/cm2 which is in agreement with a value obtained from contact angle measurements on layers of cells and sedimentation volume analysis. The cellular surface tension determined by the sedimentation volume method showed a biphasic alteration during growth cycles of C. roseus cell cultures. These variations (between 55 and 58 ergs/cm2) agree with the pattern of adhesion previously described.