Biosynthesis of Streptomycin

A method for the accumulation of the streptomycin precursor (L) in the culture broth of Streptomyces griseus was developed and the precursor was successfully isolated from the broth.

When the microorganism was cultured under shaking in the glucose-meat extract-peptone medium (0.5% glucose, 0.2% yeast extract, 0.2% meat extract, 0.4% peptone, 0.5% sodium chloride, 0.025% magnesium sulfate, pH 7.0), the accumulation of the precursor in the broth was induced by the addition of supplementary glucose (e.g., 2 g glucose per 100 ml broth) 24 hr after inoculation followed by further cultivation for 48 hr. Increased accumulation of L component was obtained merely by increasing glucose content in the culture medium (e.g., 5% glucose-containing medium in the above-indicated one) instead of glucose supplement on the way of fermentation. For the accumulation of a large amount of L component in a culture broth, it looked to be necessary for pH value of the broth to be maintained between 6 and 7 during fermentation.

L component was isolated from the culture broth by adsorption on Amberlite IRC-50 and elution with 2% NaCl solution. The L component was separated on this column from contaminated streptomycin which requires 5% NaCl solution to be eluted. The L component in the 2% NaCl eluate was adsorbed on active carbon at neutral or slightly alkaline pH and eluted with 95% methanol at acidic pH, Partially purified L component precipitated as hydrochloride by addition of acetone to the methanol extract which had been concentrated in vacuo.     

In situ near infrared spectroscopy for analyte‐specific monitoring of glucose and ammonium in streptomyces coelicolor fermentations

There are many challenges associated with in situ collection of near infrared (NIR) spectra in a fermentation broth, particularly for highly aerated and agitated fermentations with filamentous organisms. In this study, antibiotic fermentation by the filamentous bacterium Streptomyces coelicolor was used as a model process. Partial least squares (PLS) regression models were calibrated for glucose and ammonium based on NIR spectra collected in situ. To ensure that the models were calibrated based on analyte‐specific information, semisynthetic samples were used for model calibration in addition to data from standard batches. Thereby, part of the inherent correlation between the analytes could be eliminated. The set of semisynthetic samples were generated from fermentation broth from five separate fermentations to which different amounts of glucose, ammonium, and biomass were added. This method has previously been used off line but never before in situ. The use of semisynthetic samples along with validation on an independent batch provided a critical and realistic evaluation of analyte‐specific models based on in situ NIR spectroscopy. The prediction of glucose was highly satisfactory resulting in a RMSEP of 1.1 g/L. The prediction of ammonium based on NIR spectra collected in situ was not satisfactory. A comparison with models calibrated based on NIR spectra collected off line suggested that this is caused by signal attenuation in the optical fibers in the region above 2,000 nm; a region which contains important absorption bands for ammonium. For improved predictions of ammonium in situ, it is suggested to focus efforts on enhancing the signal in that particular region. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Direct extraction of astaxanthin from <Emphasis Type="Italic">Haematococcus</Emphasis> culture using vegetable oils

A green, downstream process using common vegetable oils was used for the direct extraction of astaxanthin from Haematococcus. The process consists of a single integrated unit to extract astaxanthin with subsequent separation of the astaxanthin-containing oil extract. Without a cell harvest process step, the culture broth was directly mixed with the vegetable oils; the astaxanthin inside the cell was extracted into the vegetable oil phase by hydrophobic interactions, with recovery yields of 88% and above. The oil extracts were simply separated from the culture medium containing cell debris by gravity settling only.     

A new paradigm for producing astaxanthin from the unicellular green alga Haematococcus pluvialis

The unicellular green alga Haematococcus pluvialis has been exploited as a cell factory to produce the high‐value antioxidant astaxanthin for over two decades, due to its superior ability to synthesize astaxanthin under adverse culture conditions. However, slow vegetative growth under favorable culture conditions and cell deterioration or death under stress conditions (e.g., high light, nitrogen starvation) has limited the astaxanthin production. In this study, a new paradigm that integrated heterotrophic cultivation, acclimation of heterotrophically grown cells to specific light/nutrient regimes, followed by induction of astaxanthin accumulation under photoautotrophic conditions was developed. First, the environmental conditions such as pH, carbon source, nitrogen regime, and light intensity, were optimized to induce astaxanthin accumulation in the dark‐grown cells. Although moderate astaxanthin content (e.g., 1% of dry weight) and astaxanthin productivity (2.5 mg L^?1 day^?1) were obtained under the optimized conditions, a considerable number of cells died off when subjected to stress for astaxanthin induction. To minimize the susceptibility of dark‐grown cells to light stress, the algal cells were acclimated, prior to light induction of astaxanthin biosynthesis, under moderate illumination in the presence of nitrogen. Introduction of this strategy significantly reduced the cell mortality rate under high‐light and resulted in increased cellular astaxanthin content and astaxanthin productivity. The productivity of astaxanthin was further improved to 10.5 mg L^?1 day^?1 by implementation of such a strategy in a bubbling column photobioreactor. Biochemical and physiological analyses suggested that rebuilding of photosynthetic apparatus including D1 protein and PsbO, and recovery of PSII activities, are essential for acclimation of dark‐grown cells under photo‐induction conditions. Biotechnol. Bioeng. 2016;113: 2088–2099. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.

     

High concentrations of biotechnologically produced astaxanthin by lowering pH in a <Emphasis Type="Italic">Phaffia rhodozyma</Emphasis> bioprocess

Astaxanthin additions to animal diets predominantly serve as colorization aid to satisfy consumer expectations and desire for a consistent product with familiar coloration, e.g. the characteristic pink colorization of the flesh of species being produced by aquaculture. The heterobasidiomycetous yeast Phaffia rhodozyma (Xanthophyllomyces dendrorhous) can be used as natural feed source of astaxanthin. However, currently, the majority of astaxanthin used for the feed market is produced by chemical synthesis. We present a further step in direction of a competitive production of natural astaxanthin in an optimized bioprocess with non-genetically modified Phaffia rhodozyma. After medium optimization AXJ-20, a mutant strain of P. rhodozyma wild-type strain ATCC 96594, was able to grow to a cell dry weight concentration of over 114 g per kg of culture broth in a fed-batch process. In this bioprocess, where pH was lowered from 5.5 to 3.5 during the maturation phase, AXJ-20 produced the highest value reported for astaxanthin production with P. rhodozyma up to now: 0.7 g astaxanthin per kg of culture broth with a space-time-yield of 3.3 mg astaxanthin per kg of culture broth per hour. Lowering the pH during the bioprocess and increasing trace element and vitamin concentrations prevented loss of cell dry weight concentration in the maturation phase and proved to be critical for astaxanthin concentration and purity.     

ACCUMULATION OF ASTAXANTHIN IN HAEMATOCOCCUS LACUSTRIS (CHLOROPHYTA)1

In N-limited continuous chemostat cultures of the green alga Haematococcus lacustris (Gir.) Rostaf. (UTEX 16), the steady-state astaxanthin content of the cells was determined by the specific growth rate of the cultures. The highest, pigment content was obtained at the lowest dilution rate. The specific rate of astaxanthin accumulation was, however, a function of the photon flux density measured at the illuminated culture surface. In nongrowing Haematococcus cultures, the specific rate of astaxanthin accumulation was determined by the growth rate of the culture during growth phase. The highest possible cellular astaxanthin content of all cultures was comparable and independent of the culture parameters.     

Control of glucose feeding using exit gas data and its application to the production of PHB from tapioca hydrolysate byAlcaligenes eutrophus

Summary A method to estimate the glucose concentration in the culture broth using CO₂ evolution rate (CER) data from a mass spectrometer was developed.Alcaligenes eutrophus was cultivated to produce poly(3-hydroxybutyric acid) (PHB) from tapioca hydrolysate using this method. Thek value (g glucose/mol CO₂), defined as the glucose consumption per CO₂ evolution, decreased with culture time and was automatically changed using CER data. The glucose concentration in the culture broth could be controlled at 10 to 20 g/L. A final cell concentration of 106 g/L, PHB concentration of 61 g/L. and PHB content of 58 % of dry cell weight were obtained after 59 h of cultivation.     

Growth-associated biosynthesis of astaxanthin in heterotrophic Chlorella zofingiensis (Chlorophyta)

The green microalga Chlorella zofingiensis can produce the ketocarotenoid astaxanthin under heterotrophic culture conditions. Here we report the growth-associated biosynthesis of astaxanthin in this biotechnologically important alga. With glucose as sole carbon and energy source, C. zofinginesis grew fast in the dark with rapid exhaustion of nitrogen and carbon sources from media, leading to a high specific growth rate (0.034 h⁻¹). Cultures started at a cell concentration of about 3.4 × 10⁹ cells l⁻¹ reached, after 6 days, standing biomass values of 1.6 × 10¹¹ cells or 8.5 g dry weight l⁻¹. Surprisingly, the biosynthesis of astaxanthin was found to start at early exponential phase, independent of cessation of cell division. A general trend was observed that the culture conditions benefiting cell growth also benefited astaxanthin accumulation, indicating that astaxanthin was a growth-associated product in this alga. The maximum cell dry biomass and astaxanthin yield were 11.75 g l⁻¹ and 11.14 mg l⁻¹ (about 1 mg g⁻¹), simultaneously obtained in the fed-batch culture with a combined glucose–nitrate mixture addition, which were the highest ever reported in dark-heterotrophic algal cultures. The possible reasons why dark-heterotrophic C. zofingiensis could produce astaxanthin during the course of cell growth were discussed.     

Optimum Culture Conditions for Production of Exfoliative Toxin by Staphylococcus hyicus

Optimum culture conditions for the production of exfoliative toxin by Staphylococcus hyicus (shET) were examined. High shET activity was obtained from the culture filtrate of HI and TY broth inoculated with S. hyicus. The pH in these two media ranged from 7 to 8.5 during bacterial culture, while the lowest pH in TS and BHI broth was less than 6. shET activity in the culture filtrate from TY broth inoculated with 10⁷ CFU of S. hyicus per ml was higher than that in TY broth inoculated with 10⁶ and 10⁸ CFU of bacteria per ml. When shET activity in the culture filtrate was measured under various shaking conditions, the culture filtrate shaken at 75 oscillations per min had the highest shET activity of the five shaking conditions. shET activity of the culture filtrate of TY broth to which protease inhibitor had been added was the same as that of TY broth without inhibitor. shET activity in a shaking culture in an Erlenmeyer flask was also the same as that in sac culture and that in shaking culture using a shaking (Sakaguchi) flask. shET activity in TY broth supplemented with 100 mM glucose was significantly lower than that in TY broth without glucose. Based on the above results, the optimum culture conditions for the production of shET were as follows: inoculation of 3 × 10⁹ CFU of S. hyicus strain P-1 into 300 ml of TY broth in a 2,000-ml Erlenmeyer flask, and incubation at 37 C with shaking at 75 oscillations per min. Then shET activity of the culture filtrate under appropriate culture conditions was measured after various incubation periods. shET activity was detected 6 hr after inoculation, reached the maximum (2⁵³ exfoliative unit/0.1 ml) at 16 hr and decreased between 20 and 48 hr. Thus, the optimum incubation period was determined to be 16 hr. Then the optimum concentration of ammonium sulfate for isolation of shET from the culture filtrate under appropriate culture conditions was examined. The greatest shET activity was obtained from the fraction salted out with 90% saturated ammonium sulfate. Thus, the optimum concentration of ammonium sulfate for the isolation of shET was determined to be 90% saturation.     

Reduction of fatty acid flux results in enhancement of astaxanthin synthesis in a mutant strain of Phaffia rhodozyma

A moderate-temperature mutant strain of the yeast Phaffia rhodozyma, termed MK19, was selected by 1-methyl-3-nitro-1-nitrosoguanidine (NTG) and Co60 mutagenesis. MK19 displayed fast cell growth and elevated astaxanthin content at 25°C, whereas optimal temperature for growth and astaxanthin synthesis of wild-type P. rhodozyma was 17–21°C. Optimized astaxanthin yield for MK19 after 4 days culture in shaking flask at 25°C, determined by response surface methodology, was 25.8 mg/l, which was 17-fold higher than that of the wild-type. MK19 was tolerant of high initial concentration of glucose (>100 g/l) in optimized medium. Total fatty acid content of MK19 was much lower than that of the wild-type. Acetyl-CoA is a common precursor of fatty acid and terpenoid biosynthesis, and it is possible that decreased fatty acid synthesis results in transfer of acetyl-CoA to the carotenoid biosynthetic pathway. Our results indicate that astaxanthin content is negatively correlated with fatty acid content in P. rhodozyma. Nutrient analysis showed that MK19 cells are enriched in lysine, vitamin E, and other rare nutrients, and have potential application as fish food without nutritional supplementation. This moderate-temperature mutant strain is a promising candidate for economical industrial-scale production.     

Effect of acetic acid on astaxanthin production by Phaffia rhodozyma

Summary Low concentrations of acetic acid decreased the growth rate of and astaxanthin production by Phaffia rhodozyma on glucose, with growth completely inhibited by 2 g acetic acid/l. Using H₂SO₄ for pH control after sugar depletion caused a decline in the biomass concentration, whereas using acetic acid as titrant resulted in an increase in the biomass with a high astaxanthin content of 1430 g/g cells. An extended culture with a continuous glucose feed failed to maintain a high astaxanthin content.     

Production of astaxanthin by Haematococcus pluvialis in a sequential heterotrophic-photoautotrophic culture

Production of astaxanthin by sequential heterotrophic-photoautotrophiccultivation of a green alga, Haematococcus pluvialis was investigated.This involved cultivating the cells heterotrophically to high cellconcentration, followed by illumination of the culture for astaxanthinaccumulation. The optimum pH and temperature for heterotrophic biomassproduction were 8 and 25 ^°C, respectively. There was no significantdifference in the specific growth rate of the cells when acetateconcentration was varied between 10 mM and 30 mM. However, cellgrowth was inhibited at higher acetate concentrations. A pH stat methodwas then used for fed-batch heterotrophic culture, using acetate as theorganic carbon source. A cell concentration of 7 g L^-1 wasobtained. Higher cell concentration could not be obtained because the cellschanged from vegetative to cyst forms during the heterotrophic cultivation.However, by using repeated fed-batch processes, the cells could bemaintained in the vegetative form, leading to more than two times increasein cell number output rate. When the vegetative cells were transferred tophotoautotrophic phase, there was a sharp decrease in the cell number andonly very few cells encysted and accumulated astaxanthin. On the otherhand, when the shift from heterotrophic to photoautotrophic condition wasdone when most of the cells had encysted, there was still a decrease in cellnumber but astaxanthin accumulation was very high. The astaxanthinconcentration (114 mg L^-1) and productivity (4.4 mg L^-1d^-1) obtained by this sequential heterotrophic-photoautotrophiccultivation method are very high compared to the data in the literature.     

Role of media composition in biomass and astaxanthin production of Haematococcus pluvialis under two-stage cultivation

In the present study, the effects of four different culture media on the growth, astaxanthin production and morphology of Haematococcus pluvialis LUGU were studied under two-step cultivation. The interactions between astaxanthin synthesis and secondary messengers, reactive oxygen species (ROS) and mitogen-activated protein kinases (MAPK) were also investigated. In the first green vegetative cell stage, maximal biomass productivity (86.54 mg L⁻¹ day⁻¹) was obtained in BBM medium. In the induction stage, the highest astaxanthin content (21.5 mg g⁻¹) occurred in BG-11 medium, which was higher than in any other media. The expressions of MAPK and astaxanthin biosynthetic genes in BG-11 were higher than in any other media, whereas the ROS content was lower. Biochemical and physiological analyses suggested that the ROS, MAPK and astaxanthin biosynthetic gene expression was involved in astaxanthin biosynthesis in H. pluvialis under different culture media conditions. This study proposes a two-step cultivation strategy to efficiently produce astaxanthin using microalgae.

     

Effect of culture conditions on astaxanthin production by a mutant of Phaffia rhodozyma in batch and chemostat culture

Temperature and pH had only a slight effect on the astaxanthin content of a Phaffia rhodozyma mutant, but influenced the maximum specific growth rate and cell yield profoundly. The optimum conditions for astaxanthin production were 22°C at pH 5.0 with a low concentration of carbon source. Astaxanthin production was growth-associated, and the volumetric astaxanthin concentration gradually decreased after depletion of the carbon source. The biomass concentration decreased rapidly during the stationary growth phase with a concomitant increase in the cellular content of astaxanthin. Sucrose hydrolysis exceeded the assimilation rates of D-glucose and D-fructose and these sugars accumulated during batch cultivation. D-Glucose initially delayed D-fructose uptake, but D-fructose utilization commenced before glucose depletion. In continuous culture, the highest astaxanthin content was obtained at the lowest dilution rate of 0.043 h^–1. The cell yield reached a maximum of 0.48 g cells·g^–1 glucose utilized between dilution rates of 0.05 h^–1 and 0.07 h^–1 and decreased markedly at higher dilution rates. Correspondence to: J. C. Du Preez     

Hydrogen peroxide-induced astaxanthin biosynthesis and catalase activity in Xanthophyllomyces dendrorhous

Xanthophyllomyces dendrorhous (formerly Phaffia rhodozyma) in shake-flask cultures was exposed to 10–20 mmol/L H₂O₂ at various culture stages, and the astaxanthin production was significantly increased by H₂O₂ fed at 0 or 24 h (exponential phase), but only slightly at 48 h (near stationary phase). The astaxanthin production was enhanced most significantly with double feeding of 10 mmol/L H₂O₂ at 0 and 24 h, reaching a cellular content of 1.30 mg/g cell and a volumetric yield of 10.4 mg/L, which were 83 and 65% higher, respectively, than those of the control (0.71 mg/g cell and 6.3 mg/L). The intracellular catalase (CAT) activity was also increased after H₂O₂ treatment. The increases in CAT and astaxanthin of cells could be detected within 4 h of H₂O₂ treatment. The increase in the astaxanthin content of cells was concomitant with a notable decrease in the β-carotene content. The older yeast cells at late culture stage (120 h), due perhaps in part to their higher astaxanthin contents, were more tolerant to H₂O₂ toxicity than the younger cells (24 h). No enhancement of the astaxanthin biosynthesis was attained when H₂O₂ was added to the yeast culture together with a sufficient amount of exogenous CAT. The results suggest that astaxanthin biosynthesis in X. dendrorhous can be stimulated by H₂O₂ as an antioxidative response.     

In‐situ monitoring of Saccharomyces cerevisiae ITV01 bioethanol process using near‐infrared spectroscopy NIRS and chemometrics

The application feasibility of in‐situ or in‐line monitoring of S. cerevisiae ITV01 alcoholic fermentation process, employing Near‐Infrared Spectroscopy (NIRS) and Chemometrics, was investigated. During the process in a bioreactor, in the complex analytical matrix, biomass, glucose, ethanol and glycerol determinations were performed by a transflection fiber optic probe immersed in the culture broth and connected to a Near‐Infrared (NIR) process analyzer. The NIR spectra recorded between 800 and 2,200 nm were pretreated using Savitzky‐Golay smoothing and second derivative in order to perform a partial least squares regression (PLSR) and generate the calibration models. These calibration models were tested by external validation and then used to predict concentrations in batch alcoholic fermentations. The standard errors of calibration (SEC) for biomass, ethanol, glucose and glycerol were 0.212, 0.287, 0.532, and 0.296 g/L and standard errors of prediction (SEP) were 0.323, 0.369, 0.794, and 0.507 g/L, respectively. Calibration and validation criteria were defined and evaluated in order to generate robust and reliable models for an alcoholic fermentation process matrix. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:510–517, 2016     

Improvement of astaxanthin production by a newly isolated Phaffia rhodozyma mutant with low-energy ion beam implantation

Aims: Isolation, characterization and identification of Phaffia sp. ZJB 00010, and improvement of astaxanthin production with low‐energy ion beam implantation. Methods and Results: A strain of ZJB 00010, capable of producing astaxanthin, was isolated and identified as Phaffia rhodozyma, based on its physiological and biochemical characteristics as well as its internal transcribed spacer (ITS) rDNA gene sequence analysis. With low‐energy ion beam implantation, this wild‐type strain was bred for improving the yield of astaxanthin. After ion beam implantation, the best mutant, E5042, was obtained. The production of astaxanthin in E5042 was 2512 μg g^?1 (dry cell weight, DCW), while the wild‐type strain was about 1114 μg g^?1 (DCW), an increase of 125·5%. Moreover, the fermentation conditions of mutant E5042 for producing astaxanthin were optimized. The astaxanthin production under the optimized conditions was upscaled and studied in a 50‐l fermentor. Conclusions: A genetically stable mutant strain with high yield of astaxanthin was obtained using low‐energy ion beam implantation. This mutant may be a suitable candidate for the industrial‐scale production of astaxanthin. Significance and Impact of the Study: Astaxanthin production in Phaffia rhodozyma could be fficiently improved by low‐energy ion beam implantation, which is a new technology in the mutant breeding of micro‐organisms. The mutant obtained in this work could potentially be utilized in industrial production of astaxanthin.     

Use of alfalfa residual juice as a substrate for propagation of the red yeast Phaffia rhodozyma

Summary Alfalfa residual juice (ARJ) supported good growth of the yeast Phaffia rhodozyma but formation of astaxanthin was inhibited. Supplementary nutrients did not reverse the inhibition, indicating that the the juice probably contained some inhibitor of astaxanthin biosynthesis. Six strains of P. rhodozyma were tested and found to be susceptible to the inhibitory effects of the juice. Concentrations of ARJ above 1.25% (v/v) were inhibitory to pigmentation of the yeast. Above approximately 3.7%, total inhibition of astaxanthin formation was observed but some chromogenic components of the juice were adsorbed on Phaffia cells and appeared as artefacts in astaxanthin analyses. Phaffia biomass produced in ARJ showed greater susceptibility to autolysis than that produced in a peptone-glucose-salts medium. Supplementation of ARJ with glucose enhanced yield of cell mass and minimised the autolytic phenomenon, and is potentially useful for producing Phaffia biomass for use as a source of single cell protein.Unsupplemented brewer's malt wort and molasses, separately and in a suitable combination, were compared with ARJ and were found suitable for growth and pigmentation of P. rhodozyma.     

Comparison of heterotrophic and photoautotrophic induction on astaxanthin production by Haematococcus pluvialis

During light induction for astaxanthin formation in Haematococcus pluvialis, we substituted photoautotrophic induction for heterotrophic induction using acetate, both to prevent contamination by heterotrophs due to addition of organic carbon and to enhance carbon assimilation in the induced cells. Strong photoautotrophic induction was performed by N-deprivation of photoautotrophically grown Haematococcus cells followed by supplementation with bicarbonate (HCO₃^–) or CO₂. Bicarbonate-induced cells contained more astaxanthin than acetate-induced cells, and even further enhancement of astaxanthin accumulation was achieved by continuous CO₂ supply. The maximum astaxanthin content (77.2 mg g^–1 biomass, 3.4-fold higher than with heterotrophic induction) was obtained under conditions of 5% CO₂, yielding astaxanthin concentration and productivity of 175.7 mg l^–1 and 6.25 mg l^–1 day^–1, respectively. The results indicate that photoautotrophic induction is more effective than heterotrophic induction for astaxanthin synthesis in H. pluvialis.     

Effect of process parameters on the production and drying of Leuconostoc mesenteroides cultures

Leuconostoc mesenteroides BLAC was grown on MRS broth or on a carrot juice medium, and the effects of sugar concentration, type of pH control, aeration and fermentor size on viable counts were examined. The effect on viability of the type of centrifuge used to concentrate the bacterial culture was also examined. When the MRS broth had the traditional 110 mM glucose, pH control did not increase the final population. However, using a zone pH control mode, increasing the glucose content of MRS both from 110 to 220 mM almost doubled the population. In MRS broth, the amount of acetic acid produced was the same for all treatments, and was proportional to the amount of citrate consumed. There was a significantly lower cell yield in the carrot juice medium when the pH was not regulated. In the carrot juice medium, pH had a more pronounced effect on the final population level than did aeration, even though the quantity of viable cells was greater when the culture was aerated. In MRS broth, glucose was completely consumed during fermentation, but this was not the case in carrot juice medium. Aeration resulted in increased acetic acid content of the fermented medium. Viable counts were not affected by scaling the volume of the fermentation from 2 to 15 l ,or by the type of centrifuge used to concentrate the cells. Cells were concentrated by a factor of 10, but in both centrifuge types, viable counts showed only an eightfold average increase. However, freeze-dried powders obtained from the continuous pilot-plant-centrifuged cultures had, on the average, 33% lower populations than those obtained from the laboratory unit. Journal of Industrial Microbiology & Biotechnology (2002) 28, 291–296 DOI: 10.1038/sj/jim/7000245 Received 09 July 2001/ Accepted in revised form 25 January 2002