Genomic cloning,expression and recombinant protein purification of α and β subunits of the allophycocyanin gene <Emphasis Type="Italic">(apc)</Emphasis> from the cyanobacterium <Emphasis Type="BoldItalic">Anacystis nidulans</Emphasis> UTEX 625

A genomic fragment encoding α^APC and β^APC (i.e., α and β units of the allophycocyanin, APC) from Anacystis nidulans UTEX 625 was cloned and sequenced. This fragment, containing a non-coding sequence of 56 nucleotides in between, was then subcloned into the expression vector pMal-c2 downstream from and in frame with the malE gene of E. coli encoding MBP (maltose binding protein). The fusion protein was purified by amylose affinity chromatography and cleaved by coagulation factor Xa. α^APC and β^APC were then separated from MBP and MBP fusion proteins, respectively, and concentrated by membrane centrifugation. The study provides a method to produce recombinant allophycocyanin subunits for biomedical and biotechnological applications.     

High-level production of arachidonic acid by fed-batch culture of <Emphasis Type="Italic"> Mortierella alpina</Emphasis> using NH<Subscript>4</Subscript>OH as a nitrogen source and pH control

Mortierella alpina was grown in a fed-batch culture using a 12-l jar fermenter with an initial 8-l working volume containing 20 g glucose l⁻¹ and 10 g corn-steep powder l⁻¹. Glucose was intermittently fed to give 32 g l⁻¹ at each time. The pH of culture was maintained using 14% (v/v) NH₄OH, which also acted as a nitrogen source. A final cell density of 72.5 g l⁻¹ was reached after 12.5 days with a content of arachidonic acid (ARA) at 18.8 g l⁻¹. These values were 4 and 1.8 times higher than the respective values in batch culture. Our results suggest that the combined feeding of glucose and NH₄⁺ to the growth of M. alpina could be applied for the industrial scale production of ARA.     

Dissolved oxygen and nutrient budgets in a phytotreatment pond colonised by <Emphasis Type="Italic">Ulva</Emphasis> spp.

Net daily budgets of dissolved oxygen (O₂), dissolved inorganic carbon (DIC), dissolved inorganic nitrogen (DIN = NH₄⁺+NO₂⁻+NO₃⁻) and soluble reactive phosphorus (SRP) were determined in a pond colonised by Ulva spp. This pond received wastewater from a land-based fish farm and was used as a phytotreatment plant. Three consecutive 24-h cycles of measurements were performed with 8–14 samplings per day. Water samples were collected at the inlet and outlet of the pond and budgets were estimated from differences between inlet and outlet loadings. The first cycle was started when Ulva biomass was 8 kg m⁻², as wet weight. The second cycle was performed after the harvest of ~20% of the macroalgal biomass and the third after the harvest of another ~20% of the remaining biomass. Ulva removal was very fast (<1 h) and samplings for cycles 2 and 3 were started two hours after harvesting, so that the whole experiment lasted ~80 h. When Ulva biomass was at its maximum, the aquatic system was heterotrophic with an O₂ demand of 519 mol d⁻¹ and a net regeneration of DIC (2686 mol d⁻¹), NH₄⁺ (49 mol d⁻¹) and SRP (2.5 mol d⁻¹). The DIC to O₂ ratio was an indicator of persistent anaerobic metabolism. Following the first harvest intervention, this system displayed a prompt response and shifted toward a lower O₂ demand (from −519 to −13 mol d⁻¹), with a lesser regeneration degree of NH₄⁺ (11.4 mol d⁻¹) and DIC (1066 mol d⁻¹). After the second Ulva removal the net budget of SRP became negative (−1.0 mol d⁻¹). By integrating these results over the three days cycle we estimated that in order to operate an efficient nutrient control and maintain macroalgal mats in a healthy status the optimal Ulva biomass should be well below ~4 kg m⁻² as wet weight. Above this threshold, self-limitation would render most of the algal mat unable to exploit light and nutrients. An efficient removal of nitrogen and phosphorus could be attained through the management of macroalgal biomass only with an optimisation of recipient surface to nutrient loading ratio.     

Growth characters and photosynthetic capacity of <Emphasis Type="BoldItalic">Gracilaria lemaneiformis</Emphasis> as a biofilter in a shellfish farming area in Sanggou Bay,China

Experiments on growth characters and ecological functions of the macroalgae Gracilaria lemaneiformis, collected from south China, were conducted in polyculture areas of kelp and filter-feeding bivalve in Sanggou Bay in Weihai City, Shandong, in north China from May 2002 to May 2003. The results of 116 days cultivation showed that the average wet weight of alga increased 89 times from 0.1 to 8.9 kg rope⁻¹, with an average specific growth rate (based on wet weight) of 3.95% per day. The most favorable water layer for its growth was 1.0–1.8 m below the surface in July and August, with an average specific growth rate of 8.2% per day in 30-day experiments. Photosynthetic activity changed seasonally, with an average of 7.3 mg O₂ g dw⁻¹ h⁻¹. The maximum rate (14.4 mg O₂ g dw⁻¹ h⁻¹) was recorded in July, or 19.3 mg CO₂ g dw⁻¹ h⁻¹, while the minimum (0.40 mg CO₂ g dw⁻¹ h⁻¹) was in April. This study indicated that the culture of G. lemaneiformis is an effective way to improve water quality where scallops are cultivated intensively.     

Effects of Growth Regulators on Direct Flowering of Isolated Ginseng Buds <Emphasis Type="Italic">in vitro</Emphasis>

An in vitro method for obtaining gingseng inflorescences directly from explants of gingseng (Panax ginseng) is reported. Isolated shoot-buds of somatic embryo-derived plantlets ginseng were used as explants and incubated in B5 medium supplemented with 1 mg l⁻¹ benzyladenine (BA) and 1 mg l⁻¹ gibberellic acid (GA₃). About 15% of the buds flowered directly without developing vegetative organs. Cytokinin was found to be the key factor for inducing these isolated buds to proliferate and flower, but both these processes also occurred when benzyladenine (BA) was replaced by thidiazuron (TDZ). The optimal concentration of TDZ for obtaining the best ratios of bud proliferation and total flowering was 0.1 mg l⁻¹, while the highest number of vegetative shoots was obtained in medium supplemented with 1 mg l⁻¹ GA₃ and 0.1 mg l⁻¹ TDZ. The explant elongated abnormally in the presence of 10 mg l⁻¹ GA₃. Although a low concentration (1 mg l⁻¹) of NAA increased the bud proliferation ratio in the medium supplemented with 0.1 mg l⁻¹ TDZ and 1 mg l⁻¹ GA₃, a high concentration (5 mg l⁻¹) of NAA reduced the bud proliferation ratio and inhibited the flowering.     

Effect of 28-homobrassinolide on antioxidant capacity and photosynthesis in Brassica juncea plants exposed to different levels of copper

A sand culture experiment was conducted to study ameliorative role of 28-homobrassinolide (HBL) in Brassica juncea seedlings raised from the seeds treated with water, or 10⁻¹⁰, 10⁻⁸ and 10⁻⁶ M 28-homobrassinolide (HBL) and grown in the presence of copper (50, 100 and 150 mg kg⁻¹ sand) and sampled at 30 days after sowing. The plants grown in the presence of copper exhibited a significant decline in growth, chlorophyll and photosynthetic parameters. However, the activity of antioxidant enzymes: catalase (E.C. 1.11.1.6), peroxidase (E.C. 1.11.1.7) and superoxide dismutase (E.C. 1.15.1.1) and the content of proline increased in the plants grown under copper stress and/or raised from treatment with HBL. However, H₂O₂ content increased significantly in copper-treated plants and decreased in plants given HBL treatment. Treatment of seeds with HBL improved the growth, photosynthetic parameters and antioxidant enzymes and also improved in the plants grown under copper stress. The elevated antioxidant enzyme and proline might be responsible to overcome the toxic effects of copper in B. juncea.     

Characteristics of fed-batch cultures of recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis> containing human-like collagen cDNA at different specific growth rates

Fed-batch cultures of recombinant Escherichia coli BL21 for producing human-like collagen were performed at different specific growth rates (0.1~0.25 h⁻¹) before induction and at a constant value of 0.05 h⁻¹ after induction by the method of pseudo-exponential feeding. Although the final biomass (around 69 g l⁻¹) was almost the same in all fed-batch cultures, the highest product concentration (13.6 g l⁻¹) was achieved at the specific growth rate of 0.15 h⁻¹ and the lowest (9.6 g l⁻¹) at 0.25 h⁻¹. The mean productivity of human-like collagen was the highest at 0.15 h⁻¹ (0.57 g l⁻¹ h⁻¹) and the lowest at 0.1 h⁻¹ (0.35 g l⁻¹ h⁻¹). In the phase before induction, the cell yield coefficient (Y_X/S) decreased when the specific growth rate increased, while the formation of acetic acid increased upto 2.5 g l⁻¹ at 0.25 h⁻¹. The mean product yield coefficient (Y_P/S) also decreased with specific growth rate increasing. The respiration quotient (RQ) increased slightly with specific growth rate increasing before induction, and the mean value of RQ was around 72%. The optimum growth rate for human-like collagen production was 0.15~0.2 h⁻¹.     

Physico-chemical characterization of exomannan from <Emphasis Type="Italic">Rhodotorula acheniorum</Emphasis> MC

The batch fermentation of Rhodotorula acheniorum MC on a culture medium containing 5% sucrose, mineral salts and yeast extract at 26 °C for 96 h, with aeration at 0.75 v/v/m and agitation at 500 rev min ⁻¹ resulted in the synthesis of an exopolysaccharide (6.2 g l ⁻¹) which formed two fractions upon precipitation. The fractions were purified to a carbohydrate content of 98.2% for fraction I and 87.3% for fraction II. Mannose was the main monosaccharide component in a 92.8% concentration in fraction I and a 90.6% concentration in Fraction II. The exopolysaccharide was thus a mannan. The gel chromatograms confirmed the chemical composition of both fractions. The molecular weight of mannan I was 310 kD, whereas that of mannan II was 249 kD. The mannan I intrinsic viscosity [η]=6.23 dl g⁻¹ was higher than that of mannan II [η]=2.73 dl g⁻¹. The water-binding capacity of the mannan samples was established within the 1.2–3.5 g g⁻¹ range. The multiplicative model [η]=387.22. D_r^−0.1913. T^−1.095. C^1.814 describing the effect of the velocity gradient D_r, the exomannan concentration C and the temperature T on the dynamic viscosity values η of polymer solutions was obtained.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of Perilla (<Emphasis Type="Italic">Perilla frutescens</Emphasis>)

Agrobacterium tumefaciens-mediated transformation system for perilla (Perilla frutescens Britt) was developed. Agrobacterium strain EHA105 harboring binary vector pBK I containing bar and γ-tmt cassettes or pIG121Hm containing nptII, hpt, and gusA cassettes were used for transformation. Three different types of explant, hypocotyl, cotyledon and leaf, were evaluated for transformation and hypocotyl explants resulted in the highest transformation efficiency with an average of 3.1 and 2.2%, with pBK I and pIG121Hm, respectively. The Perilla spp. displayed genotype-response for transformation. The effective concentrations of selective agents were 2 mg l⁻¹ phosphinothricin (PPT) and 150 mg l⁻¹ kanamycin, respectively, for shoot induction and 1 mg l⁻¹ PPT and 125 mg l⁻¹ kanamycin, respectively, for shoot elongation. The transformation events were confirmed by herbicide Basta spray or histochemical GUS staining of T₀ and T₁ plants. The T-DNA integration and transgene inheritance were confirmed by PCR and Southern blot analysis of random samples of T₀ and T₁ transgenic plants.     

The routine metabolic rate of mulloway (Argyrosomus japonicus: Sciaenidae) and yellowtail kingfish (Seriola lalandi: Carangidae) acclimated to six different temperatures

This study compared the mass-specific routine metabolic rate (RMR) of similar sized mulloway (Argyrosomus japonicus), a sedentary species, and yellowtail kingfish (Seriola lalandi), a highly active species, acclimated at one of several temperatures ranging from 10–35 °C. Respirometry was carried out in an open-top static system and RMR corrected for seawater–atmosphere O₂ exchange using mass-balance equations. For both species RMR increased linearly with increasing temperature (T). RMR for mulloway was 5.78T − 29.0 mg O₂ kg^− 0.8 h^− 1 and for yellowtail kingfish was 12.11T − 39.40 mg O₂ kg^− 0.8 h^− 1. The factorial difference in RMR between mulloway and yellowtail kingfish ranged from 2.8 to 2.2 depending on temperature. The energetic cost of routine activity can be described as a function of temperature for mulloway as 1.93T − 9.68 kJ kg^− 0.8 day^− 1 and for yellowtail kingfish as 4.04T − 13.14 kJ kg^− 0.8 day^− 1. Over the full range of temperatures tested Q₁₀ values were approximately 2 for both species while Q₁₀ responses at each temperature increment varied considerably with mulloway and yellowtail kingfish displaying thermosensitivities indicative of each species respective niche habitat. RMR for mulloway was least thermally dependent at 28.5 °C and for yellowtail kingfish at 22.8 °C. Activation energies (E_a) calculated from Arrhenius plots were not significantly different between mulloway (47.6 kJ mol^− 1) and yellowtail kingfish (44.1 kJ mol^− 1).     

In situ measurement of fine root water absorption in three temperate tree species—Temporal variability and control by soil and atmospheric factors

Miniature heat balance-sap flow gauges were used to measure water flows in small-diameter roots (3–4 mm) in the undisturbed soil of a mature beech–oak–spruce mixed stand. By relating sap flow to the surface area of all branch fine roots distal to the gauge, we were able to calculate real time water uptake rates per root surface area (J_s) for individual fine root systems of 0.5–1.0 m in length. Study aims were (i) to quantify root water uptake of mature trees under field conditions with respect to average rates, and diurnal and seasonal changes of J_s, and (ii) to investigate the relationship between uptake and soil moisture θ, atmospheric saturation deficit D, and radiation I. On most days, water uptake followed the diurnal course of D with a mid-day peak and low night flow. Neighbouring roots of the same species differed up to 10-fold in their daily totals of J_s (<100–2000 g m⁻² d⁻¹) indicating a large spatial heterogeneity in uptake. Beech, oak and spruce roots revealed different seasonal patterns of water uptake although they were extracting water from the same soil volume. Multiple regression analyses on the influence of D, I and θ on root water uptake showed that D was the single most influential environmental factor in beech and oak (variable selection in 77% and 79% of the investigated roots), whereas D was less important in spruce roots (50% variable selection). A comparison of root water uptake with synchronous leaf transpiration (porometer data) indicated that average water fluxes per surface area in the beech and oak trees were about 2.5 and 5.5 times smaller on the uptake side (roots) than on the loss side (leaves) given that all branch roots <2 mm were equally participating in uptake. Beech fine roots showed maximal uptake rates on mid-summer days in the range of 48–205 g m⁻² h⁻¹ (i.e. 0.7–3.2 mmol m⁻² s⁻¹), oak of 12–160 g m⁻² h⁻¹ (0.2–2.5 mmol m⁻² s⁻¹). Maximal transpiration rates ranged from 3 to 5 and from 5 to 6 mmol m⁻² s⁻¹ for sun canopy leaves of beech and oak, respectively. We conclude that instantaneous rates of root water uptake in beech, oak and spruce trees are above all controlled by atmospheric factors. The effects of different root conductivities, soil moisture, and soil hydraulic properties become increasingly important if time spans longer than a week are considered.     

Comparative study of the pulmonary effects of intravenous leukotrienes and other bronchoconstrictors in anaesthetized guinea pig

Pulmonary responses to intravenous leukotrienes C₄, D₄ and E₄ administered as a bolus injection and by continuous infusion were studied in anesthetized guinea pigs. LTD₄, LTC₄ and LTE₄ (respective ED₅₀ of 0.21 ± .1, 0.64 ± .2 and 2.0 ± .1 μg kg⁻¹) produced dose-dependent increases in insufflation pressure when given as a bolus injection to anesthetized guinea pigs (Konzett-Rössler). Bronchoconstriction was antagonized by FPL-55712 (50–200 μg kg⁻¹), and indomethacin (50–200 μg kg⁻¹) but was not significantly altered by mepyramine (1.0 mg kg⁻¹), methysergide (0.1 mg kg⁻¹), intal (10 mg kg⁻¹) mepacrine (5 mg kg⁻¹) or dexamethasone (10 mg kg⁻¹). The beta adrenoceptor blocker, timolol (5 μg kg⁻¹) produced a significantly greater potentiation of the responses to the leukotrienes than to arachidonic acid, histamine and acetylcholine. Responses to bolus injection of LTE₄ but not LTD₄ or LTC₄ were partially antagonized by atropine (100 μg kg⁻¹) and bilateral vagotomy. In experiments of a different design, continuous infusion of LTD₄ and LTE₄ (2.8–3.2 μg kg⁻¹ min⁻¹) into indomethacin-treated animals produced slowly developing increases in pulmonary resistance and decreases in compliance. The increase in resistance produced by LTE₄ and LTD₄ was partly reversed by intravenous FPL-55712 (1.0 mg kg⁻¹) and atropine (100 μg kg⁻¹) but was almost completely reversed by FPL-55712 (3 – 10 mg kg⁻¹). These findings indicate that leukotrienes can produce bronchoconstriction in guinea pigs through cyclooxygenase-dependent and cyclooxygenase independent mechanisms both of which are blocked by FPL-55712. Cholinergic mechanisms are involved in the mediation of part of the response to bolus injection of LTE₄ as well as a small part of the initial response to continuous infusion of LTD₄ and LTE₄. Intrinsic beta adrenoceptor activation serves to down modulate responses to the leukotrienes to a greater extent than responses to arachidonic acid, histamine and acetylcholine.     

Purification and characterization of lignin peroxidases from <Emphasis Type="Italic">Penicillium decumbens</Emphasis> P6

Summary Peroxidases are essential enzymes in biodegradation of lignin and lignite which have been investigated intensively in the white-rot fungi. This is the first report of purification and characterization of lignin peroxidase from Penicillium sp. P6 as lignite degradation fungus. The results indicated that the lignin peroxidase of Penicillium decumbens P6 had physical and chemical properties and a N-terminal amino acid sequence different from the lignin peroxidases of white-rot fungi. The lignin peroxidase was isolated from a liquid culture of P. decumbens P6. This enzyme had a molecular weight of 46.3 KDa in SDS-PAGE and exhibited greater activity, temperature stability and wider pH range than those previously reported. The isolation procedure involved (NH₄)₂SO₄ precipitation, ion-exchange chromatography on DEAE-cellulose and CM-cellulose, gel filtration on Sephadex G-100, and non-denaturing, discontinuous polyacrylamide gel electrophoresis. The K _m and V _max values of this enzyme using veratryl alcohol as substrate were 0.565 mmol L ⁻¹ and 0.088 mmol (mg protein) ⁻¹ min ⁻¹ respectively. The optimum pH of P6 lignin peroxidase was 4.0, and 70.6 of the relative activity was remained at pH 9.0. The optimum temperature of the enzyme was 45 °C.     

A high cell density fermentation strategy to produce recombinant malarial antigen in <Emphasis Type="Italic">E. coli</Emphasis>

A high cell density cultivation method was developed to produce recombinant PvRII, a malaria vaccine candidate, in E. coli for use in vaccine studies. Cells were grown in completely defined media and glucose was fed to achieve a specific growth rate of 0.12 h^–1 until cells reached 55 g dry wt l^–1. Culture was then induced with 1 mm IPTG and cells were further grown for 4 h to reach 85 g dry wt l^–1 at 0.1 h^–1. Recombinant PvRII was purified from inclusion bodies under denaturing conditions using metal affinity chromatography which yielded 10 mg PvRII g^–1 dry wt. After refolding, PvRII was greater than 98% pure, homogeneous and functionally active in that it specifically bound Duffy positive human red cells.Revisions requested 21 September 2004; Revisions received 29 October 2004     

Long-Term Temperature Acclimation of Photosynthesis in Steady-State Cultures of the Polar Diatom <Emphasis Type="Italic">Fragilariopsis cylindrus</Emphasis>

Cultures of the obligate psychrophilic diatom Fragilariopsis cylindrus (Grunow) were grown for 4 months under steady-state conditions at −1 °C and +7 °C (50 μmol photons m⁻² s⁻¹) prior to measurements in order to investigate long-term acclimation of photosynthesis to both temperatures. No differences in maximum intrinsic quantum yield of PS II (F_V/F_M) and relative electron transport rates could be detected at either temperature after 4 months of acclimation. Measurements of photosynthesis (relative electron transport rates) vs. irradiance (P vs. E curves) revealed similar values for relative light utilization efficiency (α = 0.57 at −1 °C, α = 0.60 at +7 °C) but higher values for irradiance levels at which photosynthesis saturates (E_K) at −1 °C and, therefore, higher maximum photosynthesis (P_MAX = 54 (relative units) at −1 °C, P_MAX = 49 at +7 °C). Nonphotochemical quenching (NPQ) measurements at 385 μmol photons m⁻² s⁻¹ indicated higher (37%) NPQ for diatoms grown at −1 °C compared to +7 °C, which was possibly related to a 2-fold increase in the concentration of the pigment diatoxanthin and a 9-fold up-regulation of a gene encoding a fucoxanthin chlorophyll a,c-binding protein. Expression of the D1 protein encoding gene psbA was ca. 1.5-fold up-regulated at −1 °C, whereas expression levels of other genes from Photosystem II (psbC, psbU, psbO), as well as rbcL, the gene encoding the Rubisco large subunit were similar at both temperatures. However, a 2-fold up-regulation of a plastid glyceraldehyde-P dehydrogenase at −1 °C indicated enhanced Calvin cycle activity. This study revealed for the first time that a polar diatom could efficiently acclimate photosynthesis over a wide range of polar temperatures given enough time. Acclimation of photosynthesis at −1 °C was probably regulated similarly to high light acclimation.     

High-performance liquid chromatography of the neuroactive steroids alphaxalone and pregnanolone in plasma using dansyl hydrazine as fluorescent label: application to a pharmacokinetic-pharmacodynamic study in rats

This report describes a rapid and sensitive analytical method for the quantification of the neuroactive steroids alphaxalone and pregnanolone in rat plasma using derivatization with dansyl hydrazine as fluorescent label. The method involves protein precipitation, alkaline derivatization and extraction of the compounds and internal standard pregnenolone with dichloromethane, followed by isocratic reversed-phase high-performance liquid chromatography on a 3-μm Microsphere C₁₈ column with fluorescence detection at wavelengths 332 nm and 516 nm for excitation and emission, respectively. The mobile phase consists of a mixture of 25 mM acetate buffer (pH 3.7)–acetonitrile (45:55, v/v for alphaxalone and 40:60, v/v for pregnanolone) with a flow-rate of 1 ml/min. The total run time was 35 min. In the concentration range of 0.010–10 μg ml⁻¹, the intra- and inter-assay coefficients of variation were less than 17% for both methods. In 50 μl plasma samples the corresponding limits of detection were 10 ng ml⁻¹ (signal-to-noise ratio=3). The utility of the analytical method was established by analyzing plasma samples from rats, which had received an intravenous administration of 5 mg kg⁻¹ alphaxalone or pregnanolone. Values for clearance, volume of distribution at steady state and terminal half life were 71.9 ml min⁻¹ kg⁻¹, 814 mg kg⁻¹ and 13.5 min for alphaxalone and 69.2 ml min⁻¹ kg⁻¹, 1638 ml kg⁻¹ and 27.8 min for pregnanolone, respectively. Due to its simplicity and sensitivity this method can be used on a routine basis for pharmacokinetic analysis of neuroactive steroids.     

Inhibition of hepatic lipase by m-aminophenylboronate application of phenylboronate affinity chromatography for purification of human postheparin plasma lipases

Phenylboronates are competitive inhibitors of serine hydrolases including lipases. We studied the effect of m-aminophenylboronate on triglyceride-hydrolyzing activity of hepatic lipase (EC 3.1.1.3). m-Aminophenylbo ronate inhibited hepatic lipase activity with a K₁ value of 55 μM. Furthermore, m-aminophenylboronate protected hepatic lipase activity from inhibition by di-isopropyl fluorophosphate, an irreversible active site inhibitor of serine hydrolases. Inhibition of hepatic lipase activity by m-aminophenylboronate was pH-dependent. The inhibition was maximal at pH 7.5, while at pH 10 it was almost non-existent. These data were used to develop a purification procedure for postheparin plasma hepatic lipase and lipoprotein lipase. The method is a combination of m-aminophenylboronate and heparin-Sepharose affinity chromatographies. Hepatic lipase was purified to homogeneity as analyzed on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The specific activity of purified hepatic lipase was 5.46 mmol free fatty acids h⁻¹ mg⁻¹ protein with a total purification factor of 14 400 and a final recovery of approximately 20%. The recovery of hepatic lipase activity in m-aminophenylboronate affinity chromatography step was 95%. The purified lipoprotein lipase was a homogeneous protein with a specific activity of 8.27 mmol free fatty acids h⁻¹ mg⁻¹ The purification factor was 23 400 and the final recovery approximately 20%. The recovery of lipoprotein lipase activity in the m-aminophenylboronate affinity chromatography step was 87%. The phenylboronate affinity chromatography step can be used for purification of serine hydrolases which interact with boronates.     

<Emphasis Type="Italic">Cannabis sativa</Emphasis> L. growing on heavy metal contaminated soil: growth,cadmium uptake and photosynthesis

The effects of different cadmium concentrations [17 mg(Cd) kg⁻¹(soil) and 72 mg(Cd) kg^{− 1}(soil)] on Cannabis sativa L. growth and photosynthesis were examined. Hemp roots showed a high tolerance to Cd, i.e. more than 800 mg(Cd) kg⁻¹(d.m.) in roots had no major effect on hemp growth, whereas in leaves and stems concentrations of 50 – 100 mg(Cd) kg⁻¹(d.m.) had a strong effect on plant viability and vitality. For control of heavy metal uptake and xylem loading in hemp roots, the soil pH plays a central role. Photosynthetic performance and regulation of light energy consumption were analysed using chlorophyll fluorescence analysis. Seasonal changes in photosynthetic performance were visible in control plants and plants growing on soil with 17 mg(Cd) kg⁻¹(soil). Energy distribution in photosystem 2 is regulated in low and high energy phases that allow optimal use of light and protect photosystem 2 from overexcitation, respectively. Photosynthesis and energy dissipation were negatively influenced by 72 mg(Cd) kg⁻¹(soil). Cd had detrimental effects on chlorophyll synthesis, water splitting apparatus, reaction centre, antenna and energy distribution of PS 2. Under moderate cadmium concentrations, i.e. 17 mg(Cd) kg⁻¹(soil), hemp could preserve growth as well as the photosynthesis apparatus, and long-term acclimation to chronically Cd stress occurred.     

Biochemical characterization of Rhodococcus erythropolis N′4 nitrile hydratase acting on 4-chloro-3-hydroxybutyronitrile

The Rhodococcus erythropolis strain (N′4) possesses the ability to convert 4-chloro-3-hydroxybutyronitrile into the corresponding acid. This conversion was determined to be performed by its nitrile hydratase and amidase. Ammonium sulfate fractionation, DEAE ion exchange chromatography, and phenyl chromatography were used to partially purify nitrile hydratase from cell-free extract. A SDS-PAGE showed that the partially purified enzyme had two subunits and gel filtration chromatography showed that it consisted of four subunits of α₂β₂. The purified enzyme had a high specific activity of 860 U mg⁻¹ toward methacrylonitrile. The enzyme was found to have high activity at low temperature range, with a maximum activity occurring at 25 °C and be stable in the presence of organic acids at higher temperatures. The enzyme exhibited a preference for aliphatic saturated nitrile substrates over aliphatic unsaturated or aromatic ones. It was inhibited by sulfhydryl, oxidizing, and serine protease inhibitors, thus indicating that essential cysteine and serine residues can be found in the active site.The purified nitrile hydratase was able to convert 4-chloro-3-hydroxybutyronitrile into the corresponding amide at 15 °C. GC analysis showed that the initial conversion rate of the reaction was 215 mg substrate consumed min⁻¹ mg⁻¹. This demonstrated that this enzyme could be used in conjunction with a stereoselective amidase to synthesize ethyl (S)-4-chloro-3-hydroxybutyrate, an intermediate for a hypercholesterolemia drug, Atorvastatin.     

High-performance liquid chromatographic method for determination of DDT and its degradation products in rat plasma, liver and brain: validation and application to a pharmacokinetic study

A sensitive and reliable high-performance liquid chromatographic (HPLC) method, using a solid-phase extraction (SPE), was established and validated for determination of p,p′-DDT [1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane] and its metabolite p,p′-DDE [1,1-(2,2-dichloroethanylidene)-bis(4-chlorobenzene)] in rat plasma, liver and brain. After being diluted with water, plasma, liver and brain samples were applied to a solid-phase extraction C₁₈ cartridge. The extraction containing p,p′-DDT and p,p′-DDE from the cartridge were cleaned-up using a Florisil Sep-Pak cartridge. The samples were analyzed by HPLC using UV detection at 238 nm. The limit of detection for p,p′-DDT and p,p′-DDE was 0.1 mg kg⁻¹ liver or brain and 0.1 mg l⁻¹ plasma. For six replicate samples at 40, 4 and 0.2 mg kg⁻¹, intra-day precision values were within 4.9% for plasma, 6.4% for liver, and 9.7% for brain. Inter-day precision values at 4 mg kg⁻¹ were within 8.2% for plasma and tissues. The method performances were shown to be selective for p,p′-DDT and p,p′-DDE, and linear over the range 0.04–12 mg kg⁻¹ (mg l⁻¹ for plasma). The absolute recoveries of p,p′-DDT and p,p′-DDE in rat plasma and tissues were over 92%. The method was proved to be applicable to the pharmacokinetic study of DDT in rats after a single oral administration.