Scavenging of superoxide anion radical and hydroxyl radical by novel thiazolyl‐thiazolidine‐2,4‐dione compounds

The antioxidant behavior of a series of new synthesized substituted thiazolyl‐thiazolidine‐2,4‐dione compounds (TZDs) was examined using chemiluminescence and electron paramagnetic resonance spin trapping techniques. 5,5‐Dimethyl‐1‐pyrroline‐N‐oxide (DMPO) was used as the spin trap. The reactivity of TZDs with superoxide anion radical (O) and hydroxyl radical (HO^?) was evaluated using potassium superoxide/18‐crown‐6 ether dissolved in dimethylsulfoxide, and the Fenton‐like reaction (Fe²⁺ + H₂O₂), respectively. The results showed that TZDs efficiently inhibited light emission from the O generating system at a concentration of 0.05–1 mmol L^?1 (5–94% reductions were found at 1 mmol L^?1 concentration). The TZD compounds showed inhibition of HO^?‐dependent DMPO–OH spin adduct formation from DMPO (the amplitude decrease ranged from 8 to 82% at 1 mmol L^?1 concentration). The findings showed that examined TZDs had effective activities as radical scavengers. Copyright © 2009 John Wiley & Sons, Ltd.     

Characterization of a biofilm membrane reactor and its prospects for fine chemical synthesis

Biofilms are known to be robust biocatalysts. Conventionally, they have been mainly applied for wastewater treatment, however recent reports about their employment for chemical synthesis are increasingly attracting attention. Engineered Pseudomonas sp. strain VLB120ΔC biofilm growing in a tubular membrane reactor was utilized for the continuous production of (S)‐styrene oxide. A biofilm specific morphotype appeared in the effluent during cultivation, accounting for 60–80% of the total biofilm irrespective of inoculation conditions but with similar specific activities as the original morphotype. Mass transfer of the substrate styrene and the product styrene oxide was found to be dependent on the flow rate but was not limiting the epoxidation rate. Oxygen was identified as one of the main parameters influencing the biotransformation rate. Productivity was linearly dependent on the specific membrane area and on the tube wall thickness. On average volumetric productivities of 24 g L day^?1 with a maximum of 70 g L day^?1 and biomass concentrations of 45 g_BDW L have been achieved over long continuous process periods (≥50 days) without reactor downtimes. Biotechnol. Bioeng. 2010. 105: 705–717. © 2009 Wiley Periodicals, Inc.     

Hydroxyl and superoxide radical scavenging abilities of chromonyl‐thiazolidine‐2,4‐dione compounds

The oxygen free radical scavenging activities of 15 chromonyl‐thiazolidine‐2,4‐dione compounds (CTDs) were examined in chemical systems producing superoxide anion radicals, O (potasium superoxide–18‐crown‐6 ether–DMSO), and hydroxyl radicals, HO^? (a Fenton reaction: Fe(II)–H₂O₂–sodium trifluoroacetate, pH 6.15). Chemiluminescence and electron spin resonance (ESR) spectroscopy using 5,5‐dimethyl‐1‐pyrroline‐1‐oxide (DMPO) as spin trap were applied to evaluate antioxidant behaviour of CTDs towards the oxygen radicals. The results indicated that 11 of the 15 tested compounds showed a significant inhibitory effect on the chemiluminescence generated from the O‐generating system, ranging from 41 to 86%, and 13 CTDs quenched the ESR signal of the DMPO–OH spin adduct by 33–86%, at a concentration of 1 mmol L^?1. Our findings demonstrate that CTDs could be good free radical scavengers. Copyright © 2008 John Wiley & Sons, Ltd.     

High‐pressure systems for gas‐phase free continuous incubation of enriched marine microbial communities performing anaerobic oxidation of methane

Novel high‐pressure biotechnical systems that were developed and applied for the study of anaerobic oxidation of methane (AOM) are described. The systems, referred to as high‐pressure continuous incubation system (HP‐CI system) and high‐pressure manifold‐incubation system (HP‐MI system), allow for batch, fed‐batch, and continuous gas‐phase free incubation at high concentrations of dissolved methane and were designed to meet specific demands for studying environmental regulation and kinetics as well as for enriching microbial biomass in long‐term incubation. Anoxic medium is saturated with methane in the first technical stage, and the saturated medium is supplied for biomass incubation in the second stage. Methane can be provided in continuous operation up to 20 MPa and the incubation systems can be operated during constant supply of gas‐enriched medium at a hydrostatic pressure up to 45 MPa. To validate the suitability of the high‐pressure systems, we present data from continuous and fed‐batch incubation of highly active samples prepared from microbial mats from the Black Sea collected at a water depth of 213 m. In continuous operation in the HP‐CI system initial methane‐dependent sulfide production was enhanced 10‐ to 15‐fold after increasing the methane partial pressure from near ambient pressure of 0.2 to 10.0 MPa at a hydrostatic pressure of 16.0 MPa in the incubation stage. With a hydraulic retention time of 14 h a stable effluent sulfide concentration was reached within less than 3 days and a continuing increase of the volumetric AOM rate from 1.2 to 1.7 mmol L^?1 day^?1 was observed over 14 days. In fed‐batch incubation the AOM rate increased from 1.5 to 2.7 and 3.6 mmol L^?1 day^?1 when the concentration of aqueous methane was stepwise increased from 5 to 15 mmol L^?1 and 45 mmol L^?1. A methane partial pressure of 6 MPa and a hydrostatic pressure of 12 MPa in manifold fed‐batch incubation in the HP‐MI system yielded a sixfold increase in the volumetric AOM rate. Over subsequent incubation periods AOM rates increased from 0.6 to 1.2 mmol L^?1 day^?1 within 26 days of incubation. No inhibition of biomass activity was observed in all continuous and fed‐batch incubation experiments. The organisms were able to tolerate high sulfide concentrations and extended starvation periods. Biotechnol. Bioeng. 2010; 105: 524–533. © 2009 Wiley Periodicals, Inc.     

Scavenging capacities of some thiazolyl thiazolidine‐2,4‐dione compounds on superoxide radical,hydroxyl radical,and DPPH radical

The scavenging effects of eighteen thiazolyl thiazolidine‐2,4‐dione compounds (TTCs) on superoxide radical , hydroxyl radical HO^?, and 1,1‐diphenyl‐2‐picrylhydrazyl (DPPH^?) radical were evaluated by the chemiluminescence technique, electron spin resonance spectrometry (ESR) and visible spectrophotometry, respectively. The examined compounds were shown to have 27–59% scavenging ability, 19–69% HO^? scavenging activity and 2–32% DPPH^? scavenging ability. This property of the tested compound seems to be important in the prevention of various diseases of free radicals etiology. Copyright © 2009 John Wiley & Sons, Ltd.     

Effects of hydrophobicity and anions on self‐assembly of the peptide EMK16‐II

Effects of hydrophobic and electrostatic interactions on the self‐assembling process of the ionic‐complementary peptide EMK16‐II are investigated by atomic force microscopy imaging, circular dichroism spectra, light scattering, and chromatography. It is found that the hydrophobicity of the peptide promotes the aggregation in pure water even at a very low concentration, resulting in a much lower critical aggregation concentration than that of another peptide, EAK16‐II. The effect of anions in solution with different valences on electrostatic interactions is also important. Monovalent anions (Cl^? and Ac^?) with a proper concentration can facilitate the formation of peptide fibrils, with Cl^? of smaller size being more effective than Ac^? of larger size. However, only small amounts of fibrils, but plenty of large amorphous aggregates, are found when the peptide solution is incubated with multivalent anions, such as SO, C₆H₅O, and HPO. More importantly, by gel filtration chromatography, the citrate anion, which induces a similar effect on the self‐assembling process of EMK16‐II as that of SO and HPO, can interact with two or more positively charged residues of the peptide and reside in the amorphous aggregates. This implies a “salt bridge” effect of multivalent anions on the peptide self‐assembling process, which can interpret a previous puzzle why divalent cations inhibit the formation of ordered nanofibrils of the ionic‐complementary peptides. Thus, our results clarify the important effects of hydrophobic and electrostatic interactions on the self‐assembling process of the ionic‐complementary peptides. These are greatly helpful for us to understand the mechanism of peptides' self‐assembling process and protein folding and aggregation. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 318–329, 2010. This article was originally published online as an acceptedpreprint. The “Published Online” date corresponds to the preprintversion. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Removal of nitrate and hexavalent uranium from groundwater by sequential treatment in bioreactors packed with elemental sulfur and zero‐valent iron

The bioreduction of soluble hexavalent uranium (U^VI) to insoluble tetravalent uranium (U^IV) is an attractive bioremediation strategy for the clean‐up of contaminated groundwater. High levels of the common occurring co‐contaminant, nitrate ( ), can potentially interfere with uranium bioremediation. In this study, treatment of a synthetic groundwater containing a mixture of and U^VI was investigated in a sulfur–limestone autotrophic denitrifying (SLAD) bioreactor that was coupled in series with a bioreactor packed with zero‐valent iron (Fe⁰, ZVI) and sand. An additional aim of the study was to explore the possible role of biological activity in enhancing the reduction of U^VI by Fe⁰. The SLAD reactor removed efficiently (99.8%) at loadings of up to 20 mmol L d^?1, with near stoichiometric conversion to benign dinitrogen gas (N₂). The ZVI bioreactor subsequently removed uranium (99.8%) at high (0.22 mM) and low (0.02 mM) influent concentrations of the radionuclide. Aqueous uranium was reliably eliminated to below the maximum contaminant level of 30 µg L^?1 (0.13 µM) when the ZVI reactor was operated at average empty bed hydraulic retention times as low as 2.3 h, demonstrating the feasibility of the sequential treatment strategy in packed bed bioreactors. Sequential extraction of the ZVI reactor packing confirmed that uranium was immobilized as U^IV. Uranium removal was enhanced by microbial activity as confirmed by the increased rate of uranium removal in batch assays inoculated with effluent from the ZVI bioreactor and spiked with Fe⁰ compared to abiotic controls. Biotechnol. Bioeng. 2010;107: 933–942. © 2010 Wiley Periodicals, Inc.     

Reaction engineering analysis of hydrogenotrophic production of acetic acid by Acetobacterium woodii

Great interest has emerged in biological CO₂‐fixing processes in the context of current climate change discussions. One example for such a process is the hydrogenotrophic production of acetic acid by anaerobic microorganisms. Acetogenic microorganisms make use of carbon dioxide in the presence of hydrogen to produce acetic acid and biomass. In order to establish a process for the hydrogenotrophic production of acetic acid, the formation of acetate by Acetobacterium woodii was studied in a batch‐operated stirred‐tank bioreactor at different hydrogen partial pressures (pH₂) in the gas phase. The volumetric productivity of the batch processes increased with increasing hydrogen partial pressure. A maximum of the volumetric productivity of 7.4 g_acetate L⁻¹ day⁻¹ was measured at a pH₂ of 1,700 mbar. At this pH₂ a final acetate concentration of 44 g L⁻¹ was measured after a process time of 11 days, if the pH was controlled at pH 7.0 (average cell density of 1.1 g L⁻¹ cell dry weight). The maximum cell specific actetate productivity was 6.9 g_acetate g day⁻¹ under hydrogenotrophic conditions. Biotechnol. Bioeng. 2011;108: 470–474. © 2010 Wiley Periodicals, Inc.     

Anoxic oxidation of arsenite linked to chemolithotrophic denitrification in continuous bioreactors

In this study, the anoxic oxidation of arsenite (As(III)) linked to chemolithotrophic denitrification was shown to be feasible in continuous bioreactors. Biological oxidation of As(III) was stable over prolonged periods of operation ranging up to 3 years in continuous denitrifying bioreactors with granular biofilms. As(III) was removed with a high conversion efficiency (>92%) to arsenate (As(V)) in periods with high volumetric loadings (e.g., 3.5–5.1 mmol As L day^?1). The maximum specific activity of sampled granular sludge from the bioreactors was 0.98 ± 0.04 mmol As(V) formed g^?1 VSS day^?1 when determined at an initial concentration of 0.5 mM As(III). The microbial population adapted to high influent concentrations of As(III) up to 5.2 mM. However, the As(III) oxidation process was severely inhibited when 7.6–8.1 mM As(III) was fed. Activity was restored upon lowering the As(III) concentration to 3.8 mM. Several experimental strategies were utilized to demonstrate a dependence of the nitrate removal on As(III) oxidation as well as a dependence of the As(III) removal on nitrate reduction. The molar stoichiometric ratio of As(V) formed to nitrate removed (corrected for endogenous denitrification) in the bioreactors approximated 2.5, indicating complete denitrification was occurring. As(III) oxidation was also shown to be linked to the complete denitrification of NO to N₂ gas by demonstrating a significantly enhanced production of N₂ beyond the background endogenous production in a batch bioassay spiked with 3.5 mM As(III). The N₂ production also corresponded closely to the expected stoichiometry of 2.5 mol As(III) mol^?1 N₂–N for complete denitrification. Biotechnol. Bioeng. 2010;105: 909–917. © 2009 Wiley Periodicals, Inc.     

Engineered catalytic biofilms for continuous large scale production of n‐octanol and (S)‐styrene oxide

This study evaluates the technical feasibility of biofilm‐based biotransformations at an industrial scale by theoretically designing a process employing membrane fiber modules as being used in the chemical industry and compares the respective process parameters to classical stirred‐tank studies. To our knowledge, catalytic biofilm processes for fine chemicals production have so far not been reported on a technical scale. As model reactions, we applied the previously studied asymmetric styrene epoxidation employing Pseudomonas sp. strain VLB120ΔC biofilms and the here‐described selective alkane hydroxylation. Using the non‐heme iron containing alkane hydroxylase system (AlkBGT) from P. putida Gpo1 in the recombinant P. putida PpS81 pBT10 biofilm, we were able to continuously produce 1‐octanol from octane with a maximal productivity of 1.3 g L day^?1 in a single tube micro reactor. For a possible industrial application, a cylindrical membrane fiber module packed with 84,000 polypropylene fibers is proposed. Based on the here presented calculations, 59 membrane fiber modules (of 0.9 m diameter and 2 m length) would be feasible to realize a production process of 1,000 tons/year for styrene oxide. Moreover, the product yield on carbon can at least be doubled and over 400‐fold less biomass waste would be generated compared to classical stirred‐tank reactor processes. For the octanol process, instead, further intensification in biological activity and/or surface membrane enlargement is required to reach production scale. By taking into consideration challenges such as biomass growth control and maintaining a constant biological activity, this study shows that a biofilm process at an industrial scale for the production of fine chemicals is a sustainable alternative in terms of product yield and biomass waste production. Biotechnol. Bioeng. 2013; 110: 424–436. © 2012 Wiley Periodicals, Inc.     

Microbial diversity and community structure of a highly active anaerobic methane‐oxidizing sulfate‐reducing enrichment

Anaerobic oxidation of methane (AOM) is an important methane sink in the ocean but the microbes responsible for AOM are as yet resilient to cultivation. Here we describe the microbial analysis of an enrichment obtained in a novel submerged‐membrane bioreactor system and capable of high‐rate AOM (286 μmol g_{dry weight}^?1 day^?1) coupled to sulfate reduction. By constructing a clone library with subsequent sequencing and fluorescent in situ hybridization, we showed that the responsible methanotrophs belong to the ANME‐2a subgroup of anaerobic methanotrophic archaea, and that sulfate reduction is most likely performed by sulfate‐reducing bacteria commonly found in association with other ANME‐related archaea in marine sediments. Another relevant portion of the bacterial sequences can be clustered within the order of Flavobacteriales but their role remains to be elucidated. Fluorescent in situ hybridization analyses showed that the ANME‐2a cells occur as single cells without close contact to the bacterial syntrophic partner. Incubation with ¹³C‐labelled methane showed substantial incorporation of ¹³C label in the bacterial C₁₆ fatty acids (bacterial; 20%, 44% and 49%) and in archaeal lipids, archaeol and hydroxyl‐archaeol (21% and 20% respectively). The obtained data confirm that both archaea and bacteria are responsible for the anaerobic methane oxidation in a bioreactor enrichment inoculated with Eckernförde bay sediment.     

High capacity Na+/H+ exchange activity in mineralizing osteoblasts

Osteoblasts synthesize bone in polarized groups of cells sealed by tight junctions. Large amounts of acid are produced as bone mineral is precipitated. We addressed the mechanism by which cells manage this acid load by measuring intracellular pH (pHi) in non‐transformed osteoblasts in response to weak acid or bicarbonate loading. Basal pHi in mineralizing osteoblasts was ～7.3 and decreased by ～1.4 units upon replacing extracellular Na⁺ with N‐methyl‐D ‐glucamine. Loading with 40 mM acetic or propionic acids, in normal extracellular Na⁺, caused only mild cytosolic acidification. In contrast, in Na⁺‐free solutions, weak acids reduced pHi dramatically. After Na⁺ reintroduction, pHi recovered rapidly, in keeping with Na⁺/H⁺ exchanger (NHE) activity. Sodium‐dependent pHi recovery from weak acid loading was inhibited by amiloride with the Ki consistent with NHEs. NHE1 and NHE6 were expressed strongly, and expression was upregulated highly, by mineralization, in human osteoblasts. Antibody labeling of mouse bone showed NHE1 on basolateral surfaces of all osteoblasts. NHE6 occurred on basolateral surfaces of osteoblasts mainly in areas of mineralization. Conversely, elevated HCO alkalinized osteoblasts, and pH recovered in medium containing Cl^?, with or without Na⁺, in keeping with Na⁺‐independent Cl^?/HCO exchange. The exchanger AE2 also occurred on the basolateral surface of osteoblasts, consistent with Cl^?/HCO exchange for elimination of metabolic carbonate. Overexpression of NHE6 or knockdown of NHE1 in MG63 human osteosarcoma cells confirmed roles of NHE1 and NHE6 in maintaining pHi. We conclude that in mineralizing osteoblasts, slightly basic basal pHi is maintained, and external acid load is dissipated, by high‐capacity Na⁺/H⁺ exchange via NHE1 and NHE6. J. Cell. Physiol. 226: 1702–1712, 2011. © 2010 Wiley‐Liss, Inc.     

Free‐energy profiles for ions in the influenza M2‐TMD channel

M₂ transmembrane domain channel (M₂‐TMD) permeation properties are studied using molecular dynamics simulations of M₂‐TMD (1NYJ) embedded in a lipid bilayer (DMPC) with 1 mol/kg NaCl or KCl saline solution. This study allows examination of spontaneous cation and anion entry into the selectivity filter. Three titration states of the M₂‐TMD tetramer are modeled for which the four His³⁷ residues, forming the selectivity filter, are net uncharged, +2 charged, or +3 charged. M₂‐TMD structural properties from our simulations are compared with the properties of other models extracted from NMR and X‐ray studies. During 10 ns simulations, chloride ions occasionally occupy the positively‐charged selectivity filter region, and from umbrella sampling simulations, Cl^? has a lower free‐energy barrier in the selectivity‐filter region than either Na⁺ or NH, and NH has a lower free‐energy barrier than Na⁺. For Na⁺ and Cl^?, the free‐energy barriers are less than 5 kcal/mol, suggesting that the 1NYJ conformation would probably not be exquisitely proton selective. We also point out a rotameric configuration of Trp⁴¹ that could fully occlude the channel. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Characterization of the transition state of Lysozyme unfolding. I. Effect of protein-solvent interactions on the transition state

The influence of proline cis-trans isomerization on the kinetics of lysozyme unfolding was examined carefully according to the theory of Hagerman and Baldwin [(1976) Biochemistry 15, 1462–1473]. As a result, the kinetics of lysozyme unfolding was found to follow the two-state transition model well. The temperature dependencies of k_uf and k_f over a wide temperature range showed that ΔC = 0 and ΔC = ?6.7 kJ K^?1 mol^?1 in solutions of different concentrations of GuHCl. The data observed in solutions containing other denaturants also supported the conclusion that ΔC is nearly equal to zero. The activation enthalpies of unfolding (ΔH) were observed at various concentrations of several kinds of denaturants. They were independent of species and concentrations of denaturants ΔH = 200 kJ mol^?1). These facts indicate that the aspect of interaction between protein and different kinds of solvent molecules varies only slightly during the unfolding to the transition state, that is, the transition state is at compact as the native one. Therefore, it is also suggested that ΔH of 200 kJ mol^?1 is primarily required for the disruption of long-range interactions among different structural domains through a subtle conformational change. We compared the effects of several kinds of denaturants on the unfolding rate. The addition of PrOH more remarkably increases the unfolding rate than do other hydrophilic denaturants. This is probably because PrOH molecules can penetrate into the hydrophobic core of lysozyme, but hydrophilic reagents cannot because of the compactness of the transition state.     

Study of laccase production by Pleurotus ostreatus in a 5 l bioreactor and application of the enzyme to determine the antioxidant concentration of human plasma

Aims: To achieve high laccase production from Pleurotus ostreatus in a bench top bioreactor and to utilize the enzyme for determination of the total antioxidant concentration (TAC) of human plasma. Methods and Results: Laccase production by P. ostreatus studied in a benchtop bioreactor was as high as, 874·0 U ml^?1 in presence of copper sulfate. The enzyme was used to replace metmyoglobin and hydrogen peroxide for the estimation of TAC in human plasma. The trolox equivalent antioxidant concentrations determined by the laccase‐based method and metmyoglobin method ranged from 1·63 ± 0·011 to 1·80 ± 0·006 mmol l^?1 and from 1·41 ± 0·004 to 1·51 ± 0·008 mmol l^?1 plasma, respectively. Conclusions: Pleurotus ostreatus produced high amount of extracellular laccase in a benchtop bioreactor. The enzyme can be used to assay TAC of blood plasma without the interference encountered with the hydrogen peroxide and metmyoglobin mediated assay method. Significance and Impact of the Study: Laccase production by P. ostreatus obtained in this study was the highest among all reported laccase producing white‐rot fungi. Moreover, an accurate laccase‐based assay method was developed for detection of TAC in human plasma.     

Diversity decoupled from sulfur isotope fractionation in a sulfate‐reducing microbial community

The extent of fractionation of sulfur isotopes by sulfate‐reducing microbes is dictated by genomic and environmental factors. A greater understanding of species‐specific fractionations may better inform interpretation of sulfur isotopes preserved in the rock record. To examine whether gene diversity influences net isotopic fractionation in situ, we assessed environmental chemistry, sulfate reduction rates, diversity of putative sulfur‐metabolizing organisms by 16S rRNA and dissimilatory sulfite reductase (dsrB) gene amplicon sequencing, and net fractionation of sulfur isotopes along a sediment transect of a hypersaline Arctic spring. In situ sulfate reduction rates yielded minimum cell‐specific sulfate reduction rates < 0.3 × 10^?15 moles cell^?1 day^?1. Neither 16S rRNA nor dsrB diversity indices correlated with relatively constant (38‰–45‰) net isotope fractionation (ε³⁴S_{sulfide‐sulfate}). Measured ε³⁴S values could be reproduced in a mechanistic fractionation model if 1%–2% of the microbial community (10%–60% of Deltaproteobacteria) were engaged in sulfate respiration, indicating heterogeneous respiratory activity within sulfate‐reducing populations. This model indicated enzymatic kinetic diversity of Apr was more likely to correlate with sulfur fractionation than DsrB. We propose that, above a threshold Shannon diversity value of 0.8 for dsrB, the influence of the specific composition of the microbial community responsible for generating an isotope signal is overprinted by the control exerted by environmental variables on microbial physiology.     

Evaluation of growth rate and semi‐refined carrageenan properties of tissue‐cultured Kappaphycus alvarezii (Rhodophyta,Gigartinales)

This study aimed to evaluate and compare the quality of κ‐carrageenan obtained from tissue‐cultured and field‐cultured Kappaphycus alvarezii. Carrageenan properties including yield, viscosity, gel strength and sulfate content were studied. After 60 days of cultivation, tissue‐cultured K. alvarezii showed a higher growth rate (6.3 ± 0.01% day^?1) than field‐cultured seedlings (3.4 ± 0.3% day^?1). The obtained carrageenan yield from tissue‐cultured (67.3 ± 16.4%) was higher than field‐cultured K. alvarezii (51.5 ± 21.0%). Gel viscosity of carrageenans from tissue‐cultured K. alvarezii (1280.0 ± 25.0 cP) was found significantly higher than field‐cultured samples (87.8 ± 20.9 cP). The 1.5% gel solution of tissue‐cultured and field‐cultured K. alvarezii exhibited gel strengths of 703.5 ± 14.1 and 288.3 ± 19.3 g cm^?2, respectively. The average sulfate content of carrageenans was found to be significantly different between tissue‐cultured and field‐cultured K. alvarezii with 34.2 ± 10.9 and 7.5 ± 6.7%, respectively. Tissue culture is recommended to produce high quality seedlings by providing optimized culture conditions to the seaweed. This approach can serve as an alternative way to solve the seedling shortage problems currently faced by the seaweed industry.     

Hetero-α-helical,two-chain coiled-coils. Clam–worm hybrid paramyosins

The specificity of the interaction between the α-helices in two-chain coiled-coils is investigated by studying the formation of hybrid molecules in which one α-helix is a clam paramyosin chain and the other a worm paramyosin chain. Hybrids are formed by mixing, denaturation, and subsequent renaturation. Comparison is made with a blank solution in which renaturation precedes mixing, thus precluding hybridization. Hybrids are detected by a ruse based on the presence of free sulfhydryl functions on calm chains. This allows molecules comprising two clam chains to be covalently crosslinked by oxidation with 5,5′-dithiobis(2-nitrobenzoate). Worm paramyosin chains have no sulfhydryl, so molecules comprising two worm chains or hybrid molecules comprising one chain of each type cannot crosslink. When run on sodium dodecyl sulfate polyacrylamide gel electrophoresis, therefore, the protein separates into two well-resolved regions, one containing one-chain species and the other two-chain species. When the gels are scanned and quantitated, the hybrids show up as an increase in the fraction of material in the one-chain band compared with the fraction in the blank solution. When renaturation is direct, we find that the fraction of renaturated molecules that are hybrids varies from ～10% at 5°C to ～5% at 25°C. These are judged to be nonequilibrium (quenched) values. When renaturation is by slow annealing, the equilibrium fraction hybrids are ～4% and show a modest, but measurable, increase with increasing temperature. These data allow calculation of the equilibrium constant K_h and standard free energy for the hybridization reaction: (1/2)CC + (½)WW = CW, in which C(W) stands for an α-helical clam (worm) polypeptide chain. The temperature dependence gives the standard enthalpy and entropy of the reaction. We find ΔH ? 1800 cal mol^?1 and ΔH ? 1.4 cal mol^?1 K^?1, using molarity concentration units and the infinitely dilute solution in NaCl/phosphate buffer as reference state. The possible molecular significance of these values is discussed, and it is concluded that the observed standard entropy arises essentially entirely from the rotational dissymmetry of the hybrids.