Application of the yeast‐surface‐display system for orally administered salmon calcitonin and safety assessment

High manufacturing costs and oral delivery are the constraints in clinical application of calcitonin. We selected surface‐displayed Saccharomyces cerevisiae as a low‐cost and safe carrier for oral delivery of salmon calcitonin (sCT). The sCT DNA fragment, optimized according to the codon preference of S. cerevisiae, was synthesized and cloned into the plasmid M‐pYD1 to yield recombinant yAGA2‐sCT, which was induced to express sCT by galactose for 0, 12, and 24 h. sCT expression was detected on the cell surface by indirect immunofluorescence and peaked at 12 h. About 65% recombinants expressed sCT on flow cytometry. The in vivo and in vitro activity of recombinant sCT was determined by detecting bioactivity of antiosteoclastic absorption on bone wafers and orally administering yAGA2‐sCT to Wistar rats, respectively. For safety assessment of yAGA2‐sCT, we observed abnormalities, morbidity, and mortality and determined body weight, serum chemistry parameters, hematological parameters, and organ weight. In vitro bioactivity of the recombinant sCT was similar to that of commercial sCT, Miacalcic; oral administration of 5 g/kg yAGA2‐sCT induced a long‐term hypocalcemic effect in Wistar rats and no adverse effects. This study demonstrates that yAGA2‐sCT anchoring sCT protein on a S. cerevisiae surface has potential for low‐cost and safe oral delivery of sCT. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

High-cell-density cultivation for co-production of ergosterol and reduced glutathione by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Two different high-cell-density cultivation processes based on the mutant Saccharomyces cerevisiae GE-2 for simultaneous production of glutathione and ergosterol were investigated. Compared with keeping the ethanol volumetric concentration at a constant low level, feedback control of glucose feeding rate (F) by keeping the descending rate of ethanol volumetric concentration (ΔE/Δt) between −0.1% and 0.15% per hour was much more efficient to achieve a high glutathione and ergosterol productivity. This bioprocess overcomes some disadvantages of traditional S. cerevisiae-based cultivation process, especially shortening cultivation period and making the cultivation process steady-going. A classical on or off controller was used to manipulate F to maintain ΔE/Δt at its set point. The dry cell weight, glutathione yield and ergosterol yield reached 110.0 ± 2.6 g/l, 2,280 ± 76 mg/l, and 1,510 ± 28 mg/l in 32 h, respectively.     

NMR investigations of structural and dynamics features of natively unstructured drug peptide – salmon calcitonin: implication to rational design of potent sCT analogs

Backbone dynamics and conformational properties of drug peptide salmon calcitonin have been studied in aqueous solution using nuclear magnetic resonance (NMR). Although salmon calcitonin (sCT) is largely unfolded in solution (as has been reported in several circular dichroism studies), the secondary H^α chemical shifts and three bond H^N–H^α coupling constants indicated that most of the residues of the peptide are populating the α‐helical region of the Ramachandran (?, ψ) map. Further, the peptide in solution has been found to exhibit multiple conformational states exchanging slowly on the NMR timescale (10²–10³ s^?1), inferred by the multiple chemical shift assignments in the region Leu4–Leu12 and around Pro23 (for residues Gln20–Tyr22 and Arg24). Possibly, these slowly exchanging multiple conformational states might inhibit symmetric self‐association of the peptide and, in part, may account for its reduced aggregation propensity compared with human calcitonin (which lacks this property). The ¹⁵N NMR‐relaxation data revealed (i) the presence of slow (microsecond‐to‐millisecond) timescale dynamics in the N‐terminal region (Cys1–Ser5) and core residues His17 and Asn26 and (ii) the presence of high frequency (nanosecond‐to‐picosecond) motions in the C‐terminal arm. Put together, the various results suggested that (i) the flexible C‐terminal of sCT (from Thr25–Thr31) is involved in identification of specific target receptors, (ii) whereas the N‐terminal of sCT (from Cys1–Gln20) in solution – exhibiting significant amount of conformational plasticity and strong bias towards biologically active α‐helical structure – facilitates favorable conformational adaptations while interacting with the intermembrane domains of these target receptors. Thus, we believe that the structural and dynamics features of sCT presented here will be useful guiding attributes for the rational design of biologically active sCT analogs. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Growth of <Emphasis Type="Italic">Pseudomonas chloritidismutans</Emphasis> AW-1<Superscript>T</Superscript> on <Emphasis Type="Italic">n</Emphasis>-alkanes with chlorate as electron acceptor

Microbial (per)chlorate reduction is a unique process in which molecular oxygen is formed during the dismutation of chlorite. The oxygen thus formed may be used to degrade hydrocarbons by means of oxygenases under seemingly anoxic conditions. Up to now, no bacterium has been described that grows on aliphatic hydrocarbons with chlorate. Here, we report that Pseudomonas chloritidismutans AW-1^T grows on n-alkanes (ranging from C7 until C12) with chlorate as electron acceptor. Strain AW-1^T also grows on the intermediates of the presumed n-alkane degradation pathway. The specific growth rates on n-decane and chlorate and n-decane and oxygen were 0.5 ± 0.1 and 0.4 ± 0.02 day⁻¹, respectively. The key enzymes chlorate reductase and chlorite dismutase were assayed and found to be present. The oxygen-dependent alkane oxidation was demonstrated in whole-cell suspensions. The strain degrades n-alkanes with oxygen and chlorate but not with nitrate, thus suggesting that the strain employs oxygenase-dependent pathways for the breakdown of n-alkanes.     

Enhancement of polysaccharides production in <Emphasis Type="Italic">Ganoderma lucidum</Emphasis> by the addition of ethyl acetate extracts from <Emphasis Type="Italic">Eupolyphaga sinensis</Emphasis> and <Emphasis Type="Italic">Catharsius molossus</Emphasis>

To screen stimulators from Chinese medicinal insects for mycelial growth and polysaccharides production of Ganoderma lucidum, G. lucidum was inoculated into the media with and without supplementation of medicinal insect extracts. The ethyl acetate extract of Eupolyphaga sinensis at 55 mg l⁻¹ lead to significant increase in both biomass and intracellular polysaccharides (IPS) concentration from 8.53 ± 0.41 to 14.16 ± 0.43 and 1.28 ± 0.09 to 2.13 ± 0.11 g l⁻¹, respectively. In addition, the ethyl acetate extract of Catharsius molossus at 55 mg l⁻¹ significantly enhanced extracellular polysaccharides (EPS) production; the EPS yield increased from 350.9 ± 14.1 to 475.1 ± 15.3 mg l⁻¹. There were no new components in the two types of polysaccharides obtained by the addition of the insect extracts.     

Combined effects of zinc and algal food on the competition between planktonic rotifers, <Emphasis Type="Italic">Anuraeopsis fissa</Emphasis> and <Emphasis Type="Italic">Brachionus rubens</Emphasis> (Rotifera)

We evaluated the combined effects of algal (Chlorella vulgaris) food levels (low, 0.5 × 10⁶ (or 2.9 μg C ml⁻¹); and high, 1 × 10⁶ cells ml⁻¹ (or 5.8 μg C ml⁻¹)) and zinc concentrations (0, 0.125, and 0.250 mg l⁻¹ of ZnCl₂) on the competition between two common planktonic rotifers Anuraeopsis fissa and Brachionus rubens using their population growth. Median lethal concentration data (LC₅₀) (mean ± 95% confidence intervals) showed that B. rubens was more resistant to zinc (0.554 ± 0.08 mg l⁻¹) than A. fissa (0.315 ± 0.07 mg l⁻¹). A. fissa when grown alone or with Zn was always numerically more abundant than B. rubens. When grown in the absence of zinc, under low- and high-food levels, the peak abundances of A. fissa varied from 251 ± 24 to 661 ± 77 ind. ml⁻¹, respectively, and the corresponding maxima for B. rubens were 52 ± 3 and 102 ± 18 ind. ml⁻¹. At a given food level, competition for food reduced the peak abundances of both rotifers considerably. Increase in Zn concentration also lowered the rotifer abundances. The impact of zinc on competition between the two-rotifer species was evident at low-food level, mainly for A. fissa. At zinc concentrations of 0 and 0.125 mg l⁻¹, the populations of both rotifers continued to grow for about 10 days, but thereafter B. rubens began to decline. Role of zinc on the competitive outcome of the two species is discussed in relation to the changing algal densities in natural water bodies.     

β-Adrenoreceptor agonists and antagonists modulate the activity of α<Subscript>2</Subscript>-adrenoreceptors in rat cortex cerebral membranes

The influence of isoprenaline- and propranolole-induced activation and inhibition of β-adrenoreceptors on the specific nonselective α₂-antagonist [³H]RX821002 binding was studied on rat cerebral cortex subcellular membrane fractions. It was shown that the ligand-receptor interaction for α₂-adrenoreceptors corresponded to the model that assumed the presence of one receptor pool and binding of two ligand molecules to a receptor dimer. The following parameters were determined for [³H]RX821002 binding to α₂-adrenoreceptors: K _d1 = 1.57 ± 0.27 nM, B _max = 7.24 ± 1.63 fmol/mg of protein, n = 2. In the case of isoprenaline-induced activation of β-adrenoreceptors the binding of radiolabeled ligand to α₂-adrenoreceptors was described by the same model. The affinity of α₂-adrenoreceptors for [³H]RX821002 decreased more than twofold (K _d = 3.55 ± 0.02 nM) and the quantity of active receptors increased by 69% (B _max = 12.24 ± 0.06 fmol/mg of protein). Propranolole changed the model of ligand binding, and two pools of receptors were detected with the following parameters: K _d1 = 0.61 ± 0.02 nM, K _d2 = 3.41 ± 0.13 nM, B _ml = 1.88 ± 0.028 fmol/mg of protein, B _m2 = 9.27 ± 0.08 fmol/mg of protein, n = 2. The data suggest that α₂-adrenoreceptors in subcellular membrane fractions from rat cerebral cortex exist in dimeric form. Isoprenaline and propranolole exhibit modulating effect on the specific antagonist binding to α₂-adrenoreceptors, which results in the inhibition and alteration of [³H]RX821002 binding parameters.     

Optimizing bioavailability of oral administration of small peptides through pharmacokinetic and pharmacodynamic parameters: The effect of water and timing of meal intake on oral delivery of Salmon Calcitonin

Background

To investigate the influence of water intake and dose timing on the pharmacokinetic and pharmacodynamic parameters of an oral formulation of salmon calcitonin (sCT).

Methods

The study was a randomized, partially-blind, placebo-controlled, single dose, exploratory crossover phase I study. 56 healthy postmenopausal women were randomly assigned to receive five treatments. The treatments comprised a combination of study medication (SMC021 (0.8 mg sCT + 200 mg 5-CNAC), SMC021 placebo, or 200 IU Miacalcic^® NS nasal spray), water volume given with the tablet (50 or 200 ml water), and time between dosing and meal (10, 30, or 60 minutes pre-meal). Plasma sCT levels and changes in the bone resorption (C-terminal telopeptide of collagen type I) was investigated. Trial regristration

Results

Oral delivery of 0.8 mg of sCT with 50 ml of water compared to that with 200 ml water resulted in a two-fold increase in maximum concentration (C_max and AUC_0–4) of plasma sCT but comparable time to reach maximum concentration (T_max). The sCT AUC_0–4 with 50 ml of water was 4-fold higher than that obtained with nasal calcitonin. The increased absorption of sCT resulted in increased efficacy demonstrated by AUC of the relative change of serum CTX-I measured in the 6 hours post dosing.

Conclusion

0.8 mg sCT with 50 ml of water taken 30 and 60 minutes prior to meal time resulted in optimal pharmacodynamic and pharmacokinetic parameters. The data suggest that this novel oral formulation may have improved absorption and reduction of bone resorption compared to that of the nasal form.     

Improved hydrogen photoproduction regulated by carbonylcyanide <Emphasis Type="Italic">m</Emphasis>-chlorophenylhrazone from marine green alga <Emphasis Type="Italic">Platymonas subcordiformis</Emphasis> grown in CO<Subscript>2</Subscript>-supplemented air bubble column bioreactor

To develop an integrated process of CO₂-fixation and H₂ photoproduction by marine green microalga Platymonas subcordiformis, the impact of algal cells grown in CO₂-supplemented air bubble column bioreactor was investigated on H₂ photoproduction regulated by carbonylcyanide m-chlorophenylhrazone. Highest cell growth (3.85 × 10⁶ cells ml⁻¹), starch content (0.25 ± 0.08 mg per 10⁶ cells) and hydrogen production (50 ± 3 ml l⁻¹) were achieved at 3% CO₂-supplemented culture, which are respectively 1.4, 2.1, 1.5-fold of the air-supplemented culture. Improved H₂ production correlated well with the increase in starch accumulation. In this process, the algal cells have been recycled for stable H₂ production of 40–50 ml l⁻¹ over five cycles.     

Preparation, characterization, and in vivo evaluation of salmon calcitonin microspheres

Purpose. This study was done to prepare, characterize, and evaluate salmon calcitonin (sCT) microspheres (ms) in vivo using a low molecular weight, hydrophilic 50∶50 poly (D,L-lactide-co-glycolide) polymer (PLGA).Methods. sCT ms were prepared by a dispersion/solvent extraction/evaporation process and characterized for drug content, particle size, surface morphology, and structural integrity of encapsulated peptide. Peptide stability and binding to the polymer was studied in 0.1 M phosphate buffer (PB), pH 7.4, and 0.1 M acetate buffer (AB), pH 4.0. Serum sCT levels were monitored for 2 weeks after subcutaneous injection of sCT ms to rats.Results. sCT ms were essentially free of discernible surface pores with a particle size distribution in the range of 16 to 89 mm and mean particle size of 51 and 53 mm for 2 batches. Fourier Transform Matrix-assisted Laser Desorption mass spectrometry of the extracted peptide showed that the encapsulation process did not alter its chemical structure. The peptide was substantially more stable in AB than in PB. Peptide binding to the polymer was dependent on pH and was markedly higher in PB than in AB. In vivo study proved that elevated serum sCT levels could be sustained for at least 10 days after administration of sCT ms to rats at a dose of 1.0 mg/kg.Conclusions. It was demonstrated that sCT could be incorporated into polymeric ms prepared from a low molecular weight, hydrophilic PLGA using a dispersion technique without altering molecular structure. A 2-week formulation was prepared at a dose of 1.0 mg/kg.     

Assessment of optimal depth and photon irradiance for cultivation of the brown alga, <Emphasis Type="Italic">Sargassum fulvellum</Emphasis> (Turner) C. Agardh

This study was aimed at determining the optimal depth and photon irradiance for growth of Sargassum fulvellum. Sampling and measurement of underwater irradiance were carried out at farms cultivating S. fulvellum at Wando, southwestern coast of Korea, from May 2004 to April 2005. Growth of thalli, underwater irradiance and photosynthetic quantum yield were measured over a range of depths for three culture stages. During their nursery cultivation stage (Stage I), length increase was greatest at 1.5 m depth (2.5 ± 0.2 cm), where the average midday irradiance over 28 days was 488 ± 58 μmol photons m⁻² s⁻¹. During the pre-main cultivation stage (Stage II), the greatest length increase occurred at 1 m depth (10.9 ± 0.1 cm) with an average irradiance of 845 ± 169 μmol photons m⁻² s⁻¹. For the main cultivation stage (Stage III) of the alga, thalli showed maximal length growth in March and early April at depths of 1–2 m and 3 m. These results suggest that growth at each cultivation stage of S. fulvellum could be controlled by depth of cultivation rope. Presented at the 6th Meeting of the Asian Pacific Society of Applied Phycology, Manila, Philippines.     

Improvement of the multiple-stress tolerance of an ethanologenic <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> strain by freeze-thaw treatment

An effective, simple, and convenient method to improve yeast’s multiple-stress tolerance, and ethanol production was developed. After an ethanologenic Saccharomyces cerevisiae strain SC521 was treated by nine cycles of freeze-thaw, a mutant FT9-11 strain with higher multiple-stress tolerance was isolated, whose viabilities under acetic acid, ethanol, freeze-thaw, H₂O₂, and heat-shock stresses were, respectively, 23-, 26-, 10- and 7-fold more than the parent strain at an initial value 2 × 10⁷ c.f.u. per ml. Ethanol production of FT9-11 was similar (91.5 g ethanol l⁻¹) to SC521 at 30°C with 200 g glucose l⁻¹, and was better than the parent strain at 37°C (72.5 g ethanol l⁻¹), with 300 (111 g ethanol l⁻¹) or with 400 (85 g ethanol l⁻¹) g glucose l⁻¹.     

Effects of circadian rhythms of fluctuating temperature on growth and biochemical composition of <Emphasis Type="Italic">Ulva pertusa</Emphasis>

The marcoalga Ulva pertusa was cultured under (20 ± 2)°C, (20 ± 4)°C, (20 ± 6)°C, (20 ± 8)°C and (20 ± 10)°C circadian rhythms of fluctuating temperature conditions, and constant temperature of 20°C was used as the control. The growth rate of macroalga at (20 ± 2)°C, (20 ± 4)°C and (20 ± 6)°C were significantly higher than that at constant temperature of 20°C, while growth rate at (20 ± 8)°C and (20 ± 10)°C were significantly lower than that at constant temperature of 20°C. The growth rate of macroalga was a quadratic function of the thermal amplitude. Such a growth model can be described by G = β ₀ + β ₁(TA) + β ₂(TA)², where G represents the relative growth rate, TA is thermal amplitude in degree Celsius, β ₀ is the intercept on the G axis, and β ₁ and β ₂ are the regression coefficients. The optimal thermal amplitude for the growth of thallus at mean temperature of 20°C was estimated to be ± 3.69°C. Analysis of biochemical composition at the final stages of thaulls growth revealed that diel fluctuating temperature caused various influences (P < 0.05). The content of chlorophyll, protein and total solute carbohydrate at (20 ± 2)°C and (20 ± 4)°C were slightly higher than those at constant temperature of 20°C, however no statistically significant differences were found among them (P > 0.05). While osmolytes (total solute carbohydrate and free proline) at (20 ± 10)°C were significantly higher than that at 20°C (P < 0.05). Therefore, more chlorophyll and carbohydrate production might account for the enhancement in the growth of macroalga at the diel fluctuating temperatures in the present study. Handling editor: S. M. Thomaz     

Contributions of Different Organic Carbon Sources to <Emphasis Type="Italic">Daphnia</Emphasis> in the Pelagic Foodweb of a Small Polyhumic Lake: Results from Mesocosm DI<Superscript>13</Superscript>C-Additions

Abstract Freshwater ecosystems derive organic carbon from both allochthonous and autochthonous sources. We studied the relative contributions of different carbon sources to zooplankton in a small, polyhumic, steeply stratified lake, using six replicate surface-to-sediment enclosures established during summer and autumn 2004. We added ¹³C-enriched bicarbonate to the epilimnion of half the enclosures for three weeks during each season and monitored carbon stable isotope ratios of DIC, DOC, POC and Daphnia, along with physical, chemical and biological variables. During summer, ¹³C-enriched DIC (δ¹³C up to 44 ± 7.2‰) was soon taken up by phytoplankton (δ¹³C up to −5.1 ± 13.6‰) and was transmitted to Daphnia (δ¹³C up to −1.7 ± 7.2‰), demonstrating consumption of phytoplankton. In contrast, during autumn, ¹³C-enriched DIC (δ¹³C up to 56.3 ± 9.8‰) was not transmitted to Daphnia, whose δ¹³C became progressively lower (δ¹³C down to −45.6 ± 3.3‰) concomitant with decreasing methane concentration. Outputs from a model suggested phytoplankton contributed 64–84% of Daphnia diet during summer, whereas a calculated pelagic carbon mass balance indicated only 30–40% could have come from phytoplankton. Although autumn primary production was negligible, zooplankton biomass persisted at the summer level. The model suggested methanotrophic bacteria contributed 64–87% of Daphnia diet during autumn, although the calculated carbon mass balance indicated a contribution of 37–112%. Thus methanotrophic bacteria could supply virtually all the carbon requirement of Daphnia during autumn in this lake. The strongly ¹³C-depleted Daphnia values, together with the outputs from the models and the calculated carbon mass balance showed that methanotrophic bacteria can be a greater carbon source for Daphnia in lakes than previously suspected.     

Carrageenan of <Emphasis Type="Italic">Eucheuma isiforme</Emphasis> (Solieriaceae,Rhodophyta) from Nicaragua

The yield and physicochemical properties of native and alkali treated carrageenan from Eucheuma isiforme harvested from the Nicaraguan coast were investigated. The native carrageenan yield was 57.2% of dry weight and decreased to 43.5% when the alga was alkali treated. Native carrageenan viscosities showed significant differences between native (144.6 ± 3.3 cPs) and treated carrageenan (113.9 ± 2.6 cPs) (p < 0.01). Alkali treatment reduced carrageenan sulphate content by 19.3% and increased 3,6 AG content by 13%. Alkali-treated carrageenan formed very weak gels in 1.5% solutions (<50 g cm⁻²). Chemical analysis and FTIR spectra revealed that Eucheuma isiforme from Nicaragua is a good source of relatively pure iota-carrageenan with sufficient quality to serve as a substitute for traditional iota-carrageenan sources.     

Occurrence and characterization of a nucleopolyhedrovirus from <Emphasis Type="Italic">Maruca vitrata</Emphasis> (Lepidoptera,Pyralidae) isolated in Taiwan

A nucleopolyhedrovirus (MaviMNPV) was isolated from diseased larvae of legume pod borer (LPB), Maruca vitrata, at Tainan in Taiwan. Electron microscopical studies on the ultrastructure of MaviMNPV occlusion bodies (OBs) showed several virions (up to 19) with multiple nucleocapsids (up to 6) packaged within a single viral envelope. The diameter of OBs was 0.9 to 1.3 μm with a mean of 1.152±0.116 μm. The complete sequence of the MaviMNPV polyhedrin (Polh) gene contained 735 nucleotides (GenBank accession number DQ399596). Phylogenetic analyses using the complete sequence of the Polh gene of MaviMNPV indicated that this virus clusters with Group I NPVs. The genome size of MaviMNPV estimated with restriction enzymes viz., HindIII, EcoRI, BglII and PstI was 113.41 ± 1.50 kbp. First instar LPB larvae were the most susceptible stage (LC₅₀ 2.053 × 10² OBs/ml) followed by second, third and fourth instars with the median lethal concentrations (LC₅₀s) 1.410 × 10³, 2.390 × 10³ and 2.636 × 10³ OBs/ml, respectively. This is the first record of this virus from this region. The first and second authors have equal contributions in this paper     

Alcoholic fermentation of xylose and mixed sugars using recombinant <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> engineered for xylose utilization

Previously, a Saccharomyces cerevisiae strain was engineered for xylose assimilation by the constitutive overexpression of the Orpinomyces xylose isomerase, the S. cerevisiae xylulokinase, and the Pichia stipitis SUT1 sugar transporter genes. The recombinant strain exhibited growth on xylose, under aerobic conditions, with a specific growth rate of 0.025 h⁻¹, while ethanol production from xylose was achieved anaerobically. In the present study, the developed recombinant yeast was adapted for enhanced growth on xylose by serial transfer in xylose-containing minimal medium under aerobic conditions. After repeated batch cultivations, a strain was isolated which grew with a specific growth rate of 0.133 h⁻¹. The adapted strain could ferment 20 g l⁻¹ of xylose to ethanol with a yield of 0.37 g g⁻¹ and production rate of 0.026 g l⁻¹ h⁻¹. Raising the fermentation temperature from 30°C to 35°C resulted in a substantial increase in the ethanol yield (0.43 g g⁻¹) and production rate (0.07 g l⁻¹ h⁻¹) as well as a significant reduction in the xylitol yield. By the addition of a sugar complexing agent, such as sodium tetraborate, significant improvement in ethanol production and reduction in xylitol accumulation was achieved. Furthermore, ethanol production from xylose and a mixture of glucose and xylose was also demonstrated in complex medium containing yeast extract, peptone, and borate with a considerably high yield of 0.48 g g⁻¹.     

The impact of different chelating leaving groups on the substitution kinetics of mononuclear PtII(1,2- trans - R , R -diaminocyclohexane)(X–Y) complexes

A set of three oxaliplatin derivatives containing 1,2-trans-R,R-diaminocyclohexane (dach) as a spectator ligand and different chelating leaving groups X–Y, viz., [Pt(dach)(O,O-cyclobutane-1,1-dicarboxylate)], or Pt(dach)(CBDCA), [Pt(dach)(N,O-glycine)]⁺, or Pt(dach)(gly), and [Pt(dach)(N,S-methionine)]⁺, or Pt(dach)(l-Met), where l-Met is l-methionine, were synthesized and the crystal structure of Pt(dach)(gly) was determined by X-ray diffraction. The effect of the leaving group on the reactivity of the resulting Pt(II) complexes was studied for the nucleophiles thiourea, glutathione (GSH) and l-Met under pseudo-first-order conditions as a function of nucleophile concentration and temperature, using UV–vis spectrophotometric techniques. ¹H NMR spectroscopy was used to follow the substitution of the leaving group by guanosine 5′-monophosphate (5′-GMP²⁻) under second-order conditions. The rate constants indicate for all reactions a direct substitution of the X–Y chelate by the selected nucleophiles, thereby showing that the nature of the chelate, viz., O–O (CBDCA²⁻), N–O (glycine) or S–N (l-Met), respectively, plays an important role in the kinetic and mechanistic behavior of the Pt(II) complex. The k ₁ values for the reaction with thiourea, l-Met, GSH and 5′-GMP²⁻ were found to be as follows (10³ k ₁, 37.5 °C, M⁻¹ s⁻¹): Pt(dach)(CBDCA) 61 ± 2, 21.6 ± 0.1, 23 ± 1, 0.352 ± 0.002; Pt(dach)(gly) 82 ± 3, 6.2 ± 0.2, 37 ± 1, 1.77 ± 0.01; Pt(dach)(l-Met) (thiourea, GSH) 62 ± 2, 24 ± 1. The activation parameters for all reactions studied suggest an associative substitution mechanism. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Growth and carrageenan quality of <Emphasis Type="Italic">Kappaphycus striatum</Emphasis> var. <Emphasis Type="Italic">sacol</Emphasis> grown at different stocking densities,duration of culture and depth

Kappaphycus striatum var. sacol was grown in two separate studies: (1) at two stocking densities, and (2) at four different depths, each for three different durations of culture (30, 45 and 60 days) in order to determine the growth rate of the seaweed and evaluate the carrageenan content and its molecular weight. The results demonstrated that stocking density, duration of culture and depth significantly (P < 0.01) affected the growth rate, carrageenan content and molecular weight of K. striatum var. sacol. Decreasing growth rate was observed at both stocking densities and at four depths as duration of culture increased. A lower stocking density (500 g m⁻¹line⁻¹) showed a higher growth rate for the shortest durations, i.e. 30 days, as compared to those grown at a higher density. Likewise, decreasing growth rate was observed as depth increased, except at 50 cm after 60 days of culture. A 45-day culture period produced the highest molecular weight at both stocking densities (500 g m⁻¹line⁻¹ = 1,079.5 ± 31.8 kDa, 1,000 g m⁻¹line⁻¹ = 1,167 ± 270.6 kDa). ‘Sacol’ grown for 30 days at 50 cm (1,178 kDa) to 100 cm (1,200 kDa) depth showed the highest values of molecular weight of carrageenan extracted. The results suggested that K. striatum var. sacol is best grown at a stocking density of 500 g m⁻¹line⁻¹, at a depth of 50–100 cm, and for a duration of 30 days in order to provide the highest growth rate, carrageenan content and molecular weight.     

Continuous cultures of <Emphasis Type="Italic">Pseudomonas putida</Emphasis> mt-2 overcome catabolic function loss under real case operating conditions

The long-term performance and stability of Pseudomonas putida mt-2 cultures, a toluene-sensitive strain harboring the genes responsible for toluene biodegradation in the archetypal plasmid pWW0, was investigated in a chemostat bioreactor functioning under real case operating conditions. The process was operated at a dilution rate of 0.1 h⁻¹ under toluene loading rates of 259 ± 23 and 801 ± 78 g m⁻³ h⁻¹ (inlet toluene concentrations of 3.5 and 10.9 g m⁻³, respectively). Despite the deleterious effects of toluene and its degradation intermediates, the phenotype of this sensitive P. putida culture rapidly recovered from a 95% Tol⁻ population at day 4 to approx. 100% Tol⁺ cells from day 13 onward, sustaining elimination capacities of 232 ± 10 g m⁻³ h⁻¹ at 3.5 g Tol m⁻³ and 377 ± 13 g m⁻³ h⁻¹ at 10.9 g Tol m⁻³, which were comparable to those achieved by highly tolerant strains such as P. putida DOT T1E and P. putida F1 under identical experimental conditions. Only one type of Tol⁻ variant, harboring a TOL-like plasmid with a 38.5 kb deletion (containing the upper and meta operons for toluene biodegradation), was identified.