Deoxyribonucleoside triphosphate pools in Neurospora crassa: effects of histidine and hydroxyurea

An effective HPLC method for detecting deoxyribonucleoside triphosphates in hyphae from the fungus Neurospora crass has been developed. In rapidly growing cells the nucleotide levels vary from 11.8 pmoles/μg DNA for dGTP to 24.2 pmoles/μg DNA for dTTP. These levels fall by approximately one half in stationary-phase cultures but the ration of each pool to dGTP remains the same. The dNTP pools in conidia are at least 5-fold lower than in rapidly growing cells. The pool sizes are the same in static and shaking cultures. When the ribonucleotide reductase inhibitor, hydroxyurea (30 mM), is added to rapidly growing cultures, DNA synthesis is stopped and the dGTP pool is reduced by 39%, while the size of the other poolds remains the same. In the presence of 11 mM histadine, DNA synthesis is also stopped and the size of the dGTP pool reduced by 46% while the deoxypyrimidine pools are somewhat increased. This suggests that the toxicity of excess histidine in Neurospora may be due to its ability to interact with the ribonucleotide reductase, inactivating the enzyme. Histidine may react with free radical at the active sites, as does hydroxyurea.     

Adenine nucleotide pool variations in intact Nitrobacter winogradskyi cells

1. The ATP pool in Nitrobacter winogradskyi cells was determined by means of the luciferin-luciferase enzyme system and the ADP and AMP pools were measured after enzymatic conversion into ATP.
2. In the fist 10 min after addition of nitrite to endogenously respiring cells, which had stood for 5–16 days after completion of the nitrite oxidation, the ATP pool dropped about 60%.
3. During the log phase the ATP pool was approx. 20–40 pmoles/5 μg cell-N. During growth it increased exponentially by 3–4 times the amount until the nitrite had been used up. Subsequently the ATP pool decreased at first rapidly and then more slowly without sinking to 0 in the first 2 months after nitrification.
4. Nitrite oxidizing cells had an energy charge of 0.37 during the log-phase. After approx. 90% of the substrate had been used up the energy charge had reached 0.57.
5. If the CO₂ assimilation was inhibited in growing cultures by increased oxygen partial pressure, nitrite oxidation continued but the ATP pool increased.
6. The ATP pool and the activity of the endogenous respiration decreased by more than 50% during the first hours after the substrate had been used up.

     

Bromodeoxyuridine (BrdU) template and thymodine pool effects on high frequencies of sister-chromatid exchange (SCE) in Bloom syndrome cells and a mutant cell line (AsHa) originated from ataxia telangiectasia

The effect of bromodeoxyuridine (BrdU)-substituted DNA template and thymidine (dT) pool on excess sister-chromatid exchanges (SCEs) was studied in Bloom syndrome (BS) cells and an ataxia telangiectasia (AT)-derived mutant cell line (AsHa). When BS endomitotic cells were labeled with low and high (or high and low) BrdU concentrations during S₁ and S₂, only the BrdU concentration during S₁ phase affected the observed SCE. In BS cells about a 10-fold increase in SCEs occurs during or following replication on a BrdU-substituted template (high-high and high-low BrdU labeling) relative to the normal DNA template. SCEs decreased to about half in AsHa cells labeled with various BrdU doses (40, 60, 80 and 100 μg/ml) during only S₁, compared with those labeled during S₁ and S₂. Co-cultivation of AsHa and BS cells resulted in a significant reduction in SCE level from 70 to 13–17 in BS cells, lowered the BrdU concentrations necessary for sister-chromatid differential (SCD) staining from 40 to 10 μg/ml with normal SCE level and resulted in decreased level of SCEs at high BrdU concentrations (80–100 μg/ml) 12–14 SCE) in AsHa cells, compared with the originally increased SCE level (36.65 SCE at 100 μg/ml) without co-culture. However, co-cultivation between AsHa and normal cells lowered the BrdU dose necessary for SCD staining from 40 to 30 μg/ml; the dT pool possibly balanced at this level, which is clearly higher than that at co-cultivation between AsHa and BS cells. The reason for the very high BrdU doses needed to achieve SCD would seem to be that AsHa cells have high levels of thymidylate (TMP) synthetase, which maintain a large endogenous thymidine pool. This has been confirmed by direct measurement. These findings strongly support that excess and decreased dT pools are closely related to the condition necessary for high SCE induction.     

The pool of ADP and ATP regulates anaerobic product formation in resting cells of Lactococcus lactis

Lactococcus lactis grows homofermentatively on glucose, while its growth on maltose under anaerobic conditions results in mixed acid product formation in which formate, acetate, and ethanol are formed in addition to lactate. Maltose was used as a carbon source to study mixed acid product formation as a function of the growth rate. In batch and nitrogen-limited chemostat cultures mixed acid product formation was shown to be linked to the growth rate, and homolactic fermentation occurred only in resting cells. Two of the four lactococcal strains investigated with maltose, L. lactis 65.1 and MG1363, showed more pronounced mixed acid product formation during growth than L. lactis ATCC 19435 or IL-1403. In resting cell experiments all four strains exhibited homolactic fermentation. In resting cells the intracellular concentrations of ADP, ATP, and fructose 1,6-bisphosphate were increased and the concentration of P(i) was decreased compared with the concentrations in growing cells. Addition of an ionophore (monensin or valinomycin) to resting cultures of L. lactis 65.1 induced mixed acid product formation concomitant with decreases in the ADP, ATP, and fructose 1,6-bisphosphate concentrations. ADP and ATP were shown to inhibit glyceraldehyde-3-phosphate dehydrogenase, lactate dehydrogenase, and alcohol dehydrogenase in vitro. Alcohol dehydrogenase was the most sensitive enzyme and was totally inhibited at an adenine nucleotide concentration of 16 mM, which is close to the sum of the intracellular concentrations of ADP and ATP of resting cells. This inhibition of alcohol dehydrogenase might be partially responsible for the homolactic behavior of resting cells. A hypothesis regarding the level of the ATP-ADP pool as a regulating mechanism for the glycolytic flux and product formation in L. lactis is discussed.     

The involvement of ecto-ATPase activity in the phosphorylation of intracellular proteins by the addition of extracellular [P]ATP in PC12 cells

We have investigated the possibility that ecto-phosphorylation by extracellular ATP may play a role in the development of PC12 cells. To test this model and to identify putative target membrane proteins, intact PC12 cells were radiolabeled by the addition of 20 μM [γ-³²P]ATP. An analysis of the labeled proteins revealed that a 57 kDa protein was the most abundant phosphorylated protein even within time periods as short as 3 min and continued to be labeled over and above the level of other proteins. This protein was identified as tyrosine hydroxylase by immunoprecipitation with antiserum to tyrosine hydroxylase. When intact cells were incubated with either [γ-³²P]ATP or ³²P_i of comparable specific radioactivity, the overall protein labeling pattern and the degree of phosphorylation of tyrosine hydroxylase were similar. There were no discrete proteins that were labeled by [γ-³²P]ATP and not by ³²P_i that would provide evidence for ecto-kinase activity in PC12 cells. Also, the addition of nonradioactive P_i reduced the incorporation of radioactivity into the protein from extracellular [γ-³²P]ATP. These results suggested that the phosphorylation of tyrosine hydroxylase by extracellular [γ-³²P]ATP required the initial hydrolysis of ATP and the subsequent incorporation of the ³²P_i into the intracellular ATP pool. To support this interpretation, we have demonstrated directly the presence of ecto-ATPase activity in intact PC12 cells by measuring the hydrolysis of extracellular [γ-³²P]ATP. Nearly 50% of the total ATP added (20 μM) was hydrolyzed within 10 min under conditions identical to those used to demonstrate intracellular protein phosphorylation. PC12 cells express both a Ca²⁺-dependent ecto-ATPase activity and a Mg²⁺-dependent ecto-ATPase activity. In addition, extracellular ATP is degraded enzymatically not only to ADP, but sequentially to adenosine. Our results also point out the difficulties inherent in attempts to identify ecto-kinase activity in cells that also contain ecto-ATPase activities.     

Enhancement of growth and gymnodimine production by the marine dinoflagellate, Karenia selliformis

Because of its novel bioactive properties the production of gymnodimine for use as a pharmaceutical precursor has aroused interest. The dinoflagellate, Karenia selliformis produces gymnodimine when grown in bulk culture using GP + selenium medium but the growth rates (μ) and levels of gymnodimine are low (μ, 0.05 days⁻¹; gymnodimine 250 μg L⁻¹ max). We describe the effects of organic acid additions (acetate, glycolate, alanine and glutamate additions and combinations of these) in enhancing growth and gymnodimine production in axenic cultures. The most effective organic acid combinations in decreasing order were: glycolate/alanine > acetate > glycolate. Glycolate/alanine optimised gymnodimine production by prolonging growth (maximum cell yield, 1.76 × 10⁵ cells mL⁻¹; gymnodimine, 1260 μg L⁻¹; growth rate (μ), 0.2 days⁻¹) compared to the control (growth maximum cell yield, 7.8 × 10⁴ cells mL⁻¹; gymnodimine, 780 μg L⁻¹; μ, 0.17 days⁻¹). Acetate enhanced gymnodimine by stimulating growth rate (μ, 0.23 days⁻¹) and the large concentration of gymnodimine per cell (16 pg cell⁻¹ cf. 9.8 pg cell⁻¹ for the control) suggests a role for this compound in gymnodimine biosynthesis. Amending culture media with Mn²⁺ additions resulted in slightly decreased growth in control cultures and increased the gymnodimine while in glycolate/alanine cultures growth was stimulated but gymnodimine production decreased. The results suggest that the organic acid can enhance gymnodimine production by either enhancing growth maximum or the biosynthetic pathway.     

Deoxyribunucleoside triphosphate pools in Neurospora crassa: effects of histadine and hydroxyurea

An effective HPLC method for detecting deoxyribonucleoside triphosphates in hyphae from the fungus Neurospora crass has been developed. In rapidly growing cells the nucleotide levels vary from 11.8 pmoles/μg DNA for dGTP to 24.2 pmoles/μg DNA for dTTP. These levels fall by approximately one half in stationary-phase cultures but the ration of each pool to dGTP remains the same. The dNTP pools in conidia are at least 5-fold lower than in rapidly growing cells. The pool sizes are the same in static and shaking cultures. When the ribonucleotide reductase inhibitor, hydroxyurea (30 mM), is added to rapidly growing cultures, DNA synthesis is stopped and the dGTP pool is reduced by 39%, while the size of the other poolds remains the same. In the presence of 11 mM histadine, DNA synthesis is also stopped and the size of the dGTP pool reduced by 46% while the deoxypyrimidine pools are somewhat increased. This suggests that the toxicity of excess histidine in Neurospora may be due to its ability to interact with the ribonucleotide reductase, inactivating the enzyme. Histidine may react with free radical at the active sites, as does hydroxyurea.     

The neurotoxic effect of sickle cell hemoglobin

A growing body of experimental evidence suggests that the oxidative neurotoxicity of hemoglobin A may contribute to neuronal loss after CNS hemorrhage. Several hemoglobin variants, including hemoglobin S, are more potent oxidants in cell-free systems. However, despite the increased incidence of hemorrhagic stroke associated with sickle cell disease, little is known of the effect of hemoglobin S on cells of neural origin. In the present study, its toxicity was quantified and directly compared with that of hemoglobin A in murine cortical cell cultures. Reactive oxygen species production, as assessed by cellular fluorescence after treatment with dihydrorhodamine 123, was significantly increased by exposure to 10 μM hemoglobin S for 2-4 h. Neuronal death, as measured by propidium iodide staining and lactate dehydrogenase release, commenced at 4 h; for a 20-h exposure, the EC₅₀ was approximately 0.71 μm. Glial cells were not injured. Cell death was completely blocked by iron chelation with deferoxamine or phenanthroline. Direct comparison of sister cultures exposed to either hemoglobin A or hemoglobin S revealed a similar amount of cell injury in both groups. A significant difference was consistently observed only after treatment with 1 μM hemoglobin for 20 h, which resulted in death of approximately one third more neurons with hemoglobin S than with hemoglobin A. The results of this study suggest that sickle cell hemoglobin is neurotoxic at physiologically relevant concentrations. This toxicity is iron-dependent, oxidative, and quantitatively similar to that produced by hemoglobin A.     

Photosynthetic response of Myriophyllum salsugineum A.E. orchard to photon irradiance, temperature and external free CO2

The photosynthetic capacity of Myriophyllum salsugineum A.E. Orchard was measured, using plants collected from Lake Wendouree, Ballarat, Victoria and grown subsequently in a glasshouse pond at Griffith, New South Wales. At pH 7.00, under conditions of constant total alkalinity of 1.0 meq dm⁻³ and saturating photon irradiance, the temperature optimum was found to be 30–35°C with rates of 140 μmol mg⁻¹ chlorophyll a h⁻¹ for oxygen production and 149 μmol mg⁻¹ chlorophyll a h⁻¹ for consumption of CO₂. These rates are generally higher than those measured by other workers for the noxious Eurasian water milfoil, Myriophyllum spicatum L., of which Myriophyllum salsugineum is a close relative. The light-compensation point and the photon irradiance required to saturate photosynthetic oxygen production were exponentially dependent on water temperature. Over the temperature range 15–35°C the light-compensation point increased from 2.4 to 16.9 μmol (PAR) m⁻² s⁻¹ for oxygen production while saturation photon irradiance increased from 41.5 to 138 μmol (PAR) m⁻² s⁻¹ for oxygen production and from 42.0 to 174 μmol (PAR) m⁻² s⁻¹ for CO₂ consumption. Respiration rates increased from 27.1 to 112.3 μmol (oxygen consumed) g⁻¹ dry weight h⁻¹ as temperature was increased from 15 to 35°C. The optimum temperature for productivity is 30°C.     

Microencapsulated Chromatophores for the Production of ATP

Chromatophores, organelles for photophosphorylation in non-sulfur purple photosynthetic bacteria, were microencapsulated and utilized in ATP production. The microcapsules were formed by photocrosslinking with trimethylolpropane triacrylate (TMPTA). In batch experiments chromatophores microencapsulated in TMPTA capsules were repeatedly used in ATP production for more than 5 times. Continuous ATP production was then undertaken. ATP was produced at a production rate of 14 μmol h^-1 L^-1 over 200 hrs. The yield (from ADP to ATP) was 35%. The total amount of ATP produced was 0.7 mM (μM Bchl)^-1. Therefore, this microencapsulation method was found to be suitable for the continuous ATP production using chromatophores.     

Mitochondrial superoxide production during oxalate-mediated oxidative stress in renal epithelial cells

Crystals of calcium oxalate monohydrate (COM) in the renal tubule form the basis of most kidney stones. Tubular dysfunction resulting from COM-cell interactions occurs by mechanism(s) that are incompletely understood. We examined the production of reactive oxygen intermediates (ROI) by proximal (LLC-PK1) and distal (MDCK) tubular epithelial cells after treatment with COM (25–250 μg/ml) to determine whether ROI, specifically superoxide (O₂^•−), production was activated, and whether it was sufficient to induce oxidative stress. Employing inhibitors of cytosolic and mitochondrial systems, the source of ROI production was investigated. In addition, intracellular glutathione (total and oxidized), energy status (ATP), and NADH were measured. COM treatment for 1–24 h increased O₂^•− production 3–6-fold as measured by both lucigenin chemiluminescence in permeabilized cells and dihydrorhodamine fluorescence in intact cells. Using selective inhibitors we found no evidence of cytosolic production. The use of mitochondrial probes, substrates, and inhibitors indicated that increased O₂^•− production originated from mitochondria. Treatment with COM decreased glutathione (total and redox state), indicating a sustained oxidative insult. An increase in NADH in COM-treated cells suggested this cofactor could be responsible for elevating O₂^•− generation. In conclusion, COM increased mitochondrial O₂^•− production by epithelial cells, with a subsequent depletion of antioxidant status. These changes may contribute to the reported cellular transformations during the development of renal calculi.     

Nitrogenase activity and regeneration of the cellular ATP pool in Azotobacter vinelandii adapted to different oxygen concentrations.

The in vivo activity of nitrogenase under aerobiosis was studied with diazotrophic chemostat cultures of Azotobacter vinelandii grown under glucose- or phosphate-limited conditions at different dilution rates (Ds, representing the growth rate mu) and different dissolved oxygen concentrations. Under steady-state conditions, the concentration as well as the cellular level of ATP increased in glucose-limited cultures when D was increased. Irrespective of the type of growth limitation or the dissolved oxygen concentration, the steady-state concentrations of ATP and of dinitrogen fixed by nitrogenase increased in direct proportion to each other. Specific rates of dinitrogen fixation as well as of the regeneration of the cellular ATP pool were compared with specific rates of cellular respiration. With glucose-limited cultures, the rate of regeneration of the ATP pool and the rate of respiration varied in direct proportion to each other. This relationship, however, was dependent on the dissolved oxygen concentration. As compared to the phosphate-sufficient control, phosphate-limited cultures exhibited the same nitrogenase activity but significantly increased respiratory activities. Rates of ATP regeneration and of cellular respiration of phosphate-limited cultures did not fit into the relationship characteristic of glucose-limited cultures. However, a linear relationship between the rates of dinitrogen fixation and ATP regeneration was identified irrespective of the type of growth limitation and the dissolved oxygen concentration. The results suggest that the ATP supply rather than cellular oxygen consumption is of primary importance in keeping nitrogenase activity in aerobic cultures of A. vinelandii.     

Effect of diabetic sera on the conversion of eicosapentaenoic acid (EPA) to prostaglandin I3 by cultured bovine aortic endothelial cells

The effects of sera from diabetic patients and healthy donors on the synthesis of prostaglandin I₂ (PGI₂) and PGI₃in vitro were studied in confluent bovine aortic endothelial cells cultured with EPA. The products 6-keto-PGF₁ and Δ¹⁷-6-keto-PGF₁ were measured by GC/SIM as markers of PGI₂ and PG7₃ formation in growth medium after 60 min of incubation. PGI₂ and PG7₃ synthesis with 10% diabetic sera were less than with sera from healthy donors (p < 0.05). However, the total prostacyclin production (PGI₂ and PGI₃) in the cell cultures incubated with 10 μM EPA and 10% diabetic sera approximated that of the cultures incubated with the sera of healthy donors without EPA. These results suggest that the diabetic sera inhibits PGI₂ and PGI₃ synthesis in the cultured endothelial cells, and that EPA intake may reduce the complications of diabetes mellitus, such as microangiopathy and vaso-occlusive diseases, and enhances the production of PGI₃ which seems to exert a strong anti-aggregatory effect.     

Turning point in apoptosis/necrosis induced by hydrogen peroxide

The turning point between apoptosis and necrosis induced by hydrogen peroxide (H₂O₂) have been investigated using human T-lymphoma Jurkat cells. Cells treated with 50 μM H₂O₂ exhibited caspase-9 and caspase-3 activation, finally leading to apoptotic cell death. Treatment with 500 μM H₂O₂ did not exhibit caspase activation and changed the mode of death to necrosis. On the other hand, the release of cytochrome c from the mitochondria was observed under both conditions. Treatment with 500 μM H₂O₂, but not with 50 μM H₂O₂, caused a marked decrease in the intracellular ATP level; this is essential for apoptosome formation. H₂O₂-reducing enzymes such as cellular glutathione peroxidase (cGPx) and catalase, which are important for the activation of caspases, were active under the 500 μM H₂O₂ condition. Prevention of intracellular ATP loss, which did not influence cytochrome c release, significantly activated caspases, changing the mode of cell death from necrosis to apoptosis. These results suggest that ATP-dependent apoptosome formation determines whether H₂O₂-induced cell death is due to apoptosis or necrosis.     

Microcalorimetry studies of energy changes during the growth of Klebsiella aerogenes in simple salts/glucose media; correlation of specific power and size of the ATP pool

A parallel study has been made of the variation of the ATP pool and the specific power of cells of Klebsiella aerogenes during aerobic growth in glucose-limited medium under carefully defined conditions of growth and test. During the early part of exponential growth there was a marked increase in both the ATP pool and the specific power to near constant values during the later stages of growth; oscillations about the mean values were observed for each parameter. With the exhaustion of glucose and the cessation of growth both and ATP pool and the specific power decreased rapidly, the ATP pool to a low constant value and the specific power to zero. Changes in the values of these parameters during growth are discussed and it is concluded that the specific power is more dependent on the rate of catabolism rather than on the degree of coupling while the opposite is true for the ATP pool. Both parameters are, however, indicators of similar metabolic processes.     

Effect of growth hormone and γ-aminobutyric acid on Brachionus plicatilis (Rotifera) reproduction at low food or high ammonia levels

Growth hormone (GH, 0.0025 and 0.025 I.U. ml⁻¹) and γ-aminobutyric acid (GABA, 50 μg ml⁻¹) enhance rotifer population growth in batch cultures. In order to further understand the mechanism of their actions, we conducted experiments culturing isolated females at low food and high free ammonia levels. At an optimum food level of 7×10⁶ Nannochloropsis oculata cells ml⁻¹ or at low free ammonia level of 2.4 μg ml⁻¹, the F₁ offspring of rotifers treated with GH at 0.0025 I.U. ml⁻¹ had significantly higher population growth rate (r) and net reproduction rate (Ro), and shorter generation time than untreated rotifers. At a lower food level of 7×10⁵ cells ml⁻¹ or at high free ammonia level of 3.1 μg ml⁻¹, rotifers treated with GABA at 50 μg ml⁻¹ had significantly higher r and Ro, and shorter generation time. These results indicate that GABA is effective in enhancing rotifer reproduction when rotifers are cultured under stress whereas GH enhances rotifer reproduction when culture conditions are optimal. Significant effects were also observed in F¹ and F² generations which were not treated with hormones. These data may be useful for treating rotifer mass cultures to mitigate the effects of stress caused by high population densities.     

The prostaglandins of articular cartilage I. Correlates of prostaglandin activity in a chondrocyte culture system

Suspensions of aggregated chondrocytes display active prostaglandin (PG) production. Radioimmunoassay of culture media and thin layer chromatographic analysis suggests that PGE₂ is the primary PG synthesized. In order of decreasing concentration, the following PG were tentatively identified; PGE> PGI> PGA + PGB PGF₁₊₂ > T×B. An inverse logarithmic relationship was identified between PG synthesis and cells cultured at densities of 1.5 to 7.5 × 10⁶ cells/ml. Little or no change in the PG distribution profile was seen at these high cell densities. Maximum PG synthesis was attained after 36 hours of incubation with persistence of high synthetic levels up to 48 hours. PGE₂ production measured at various post-isolation intervals indicated an initial high rate of synthesis during the first 4 hours which decreased with time up to 24 hours. Cartilage explant organ cultures demonstrated a similar level of PG synthesis suggesting minimal effect of matrix on cellular PG production. Indomethacin (5 μg/ml) inhibited PG synthesis by 70% within 4 hours and 85% after 24 hours of exposure. Arachidonic acid supplementation (10 μM) stimulated PG synthesis by 300%.     

In vivo and in vitro studies on sex steroid binding protein (SBP) regulation in rainbow trout (oncorhynchus mykiss): Influence of sex steroid hormones and of factors linked to growth and metabolism

The respective roles of sex steroids and hormones related to growth and metabolism, on SBP regulation have been studied in rainbow trout. In vivo, oestradiol (E2) supplementation induces a slow but significant increase of plasma SBP concentration. Testosterone or cortisol injections have no effect. In vitro, the steroid binding protein that accumulates in incubation medium of hepatic cell primary cultures has been characterized and found to be similar to blood SBP. Its production is increased by addition of E2 (maximum: + 300%). This effect develops slowly over several days of culture and is dose dependent; as little as 1–10 nM E2 is effective.

Recombinant rainbow trout GH (rtGH)—0.01 to 1 μg/ml—also increases SBB accumulation as compared to control cells and seems to maintain SBP production over culture duration. In preliminary experiments, (1) insulin-like growth factor (IGF) and SBP concentrations were found to change inversely after a 4 days stimulation with increasing concentrations of GH; (2) recombinant human IGF₁ (250 ng/ml) tended to be inhibitory when SBP production was expressed per mg of total cellular protein, and a micromolar concentration of bovine insulin was clearly inhibitory.

Other hormones tested in vitro: triiodothyronine (10–1000 nM), thyroxine (100 nM), 17,20β-dihydroprogesterone (10–2000 nM), and testosterone (1–1000 nM) did not influence SBP concentration in hepatic cells culture media.     

ATP pool and bioluminescence in psychrophilic bacteria Photobacterium phosphoreum

Bioluminescence activity and ATP pool were investigated in the cells of psychrophilic bacteria Photobacterium phosphoreum collected from the exponential and stationary growth phases and immobilized in polyvinyl alcohol (PVA) cryogel. In liquid culture, ATP pool remained at an almost constant level throughout the luminescence cycle (over 100 h). The ATP pool in the stationary-phase and PVA-immobilized cells remained constant throughout their incubation in the medium (over 200 h) and in 3% NaCl solution (over 100 h). Quantitative assessment of integral photon yield and ATP pool indicated that bioluminescence decay in growing or stationary cells was not caused by limitation from the energy substrates of the luciferase reaction. Kinetic and quantitative parameters of emission activity and ATP pool excluded the possibility of formation of the aldehyde substrate for luciferase via reduction of the relevant fatty acids in NADPH and ATP-dependent reductase reaction and its oxidation in the monooxygenase reaction. Our results indicate that the aliphatic aldehyde is not utilized in the process of light emission.     

Vanilla Flavour Production Through Biotransformation Using Capsicum frutescens Root Cultures

Normal roots of Capsicum frutescens were excised from tissue-cultured plants into half strength Murashige and Skoog's medium with 2.23 μM naphthalene acetic acid. Maximum growth of cultured roots was 6.5 g fresh weight 40 ml^-1, as recorded on day 20. Even though normal roots were unable to accumulate capsaicin, they contained other phenylpropanoid intermediates and vanillylamine, as detected by HPLC analysis. Normal roots of Capsicum frutescens were treated with ferulic acid and protocatechuic aldehyde in order to study their biotransformation ability. Ferulic acid, which is the nearest precursor to vanillin, when fed at concentrations of 1 and 2 mM led to the accumulation of vanilla flavour metabolites, vanillin being the major one. In cultures treated with 1 and 2 mM ferulic acid, maximum vanillin accumulation of 12.3 and 16.4 μM was observed, on day 6 after precursor addition, respectively. Feeding of ferulic acid and β-cyclodextrin complex (2 mM each) enhanced the accumulation of biotransformed products. Moreover, vanillin accumulation was recorded as 24.7 μM on day 6 after precursor addition, which was 1.5 times higher than in cultures fed with ferulic acid (2 mM) alone. When ferulic acid was fed along with β-cyclodextrin (1 mM each) to cultures growing in a three-litre bubble column bioreactor, the maximum vanillin production of 10.7 μM was obtained; other vanilla flavour metabolites were also formed after 9 days of precursor addition. Root cultures could also biotransform protocatechuic aldehyde wherein a maximum vanillin production of 7.9 μM was recorded on day 6 after precursor addition. The bioconversion efficiency was observed to be 5-7% in case of ferulic acid fed cultures and 3.2% in case of protocatechuic aldehyde fed cultures suggesting the possible channelling of precursors to alternate biosynthetic pathways such as lignin.