Temperature-induced changes of the packing of double-stranded linear DNA molecules in particles of liquid-crystalline dispersions

The circular dichroism spectra of liquid-crystalline dispersions obtained by phase exclusion of linear double-stranded DNA molecules from aqueous saline solutions of polyethylene glycol (120 ≤ C_PEG ≤ 300 mg/mL) have been investigated. The formation of liquid-crystalline dispersions at polyethylene glycol concentrations ranging from 120 to 200 mg/mL was accompanied by the emergence of an abnormal negative band in the spectrum of circular dichroism; this is indicative of cholesteric packing of the double stranded DNA molecules in the particles of the dispersion. Liquid-crystalline dispersions formed at PEG concentrations higher than 220 mg/mL and room temperature did not show any abnormal bands in the circular dichroism spectra; this is indicative of hexagonal packing of double-stranded DNA molecules in the particles of the dispersions. Heating of optically inactive liquid crystal dispersions induced a transition of the dispersions into a different state accompanied by the emergence of an abnormal negative band in the spectrum of circular dichroism. This transition is considered within the concept of the transformation of a hexagonal packing of DNA molecules into a cholesteric packing. A qualitative mechanism of such a transition is proposed that is formulated in the terms of the “quasinematic” layers of double-stranded DNA molecules that change their spatial orientation under the competing influences of the osmotic pressure of the solvent, orientational elasticity of the cholesteric packing, and thermal fluctuations.     

Electron microscopy of liquid crystalline DNA: direct evidence for cholesteric-like organization of DNA in dinoflagellate chromosomes

Freeze-fracture-etch replicas of concentrated DNA solutions which appeared, by polarized light microscopy, to be in a cholesteric-like liquid crystalline state were examined by high resolution transmission electron microscopy (TEM). Individual DNA molecules were resolvable, and the microscopic morphologies observed for such replicas confirmed the cholesteric organization of DNA molecules in this liquid crystalline state. Furthermore, replica morphologies were strikingly similar to TEM images of dinoflagellate chromosomes in both thin section and freeze-etch replicas, providing strong support for the cholesteric DNA packing model proposed for the organization of DNA in these chromosomes by Bouligand and Livolant.     

Linear clusters of gold nanoparticles in quasinematic layers of DNA liquid-crystalline dispersion particles

The effects of small size (～2 nm) gold nanoparticles on the properties of particles of cholesteric liquid-crystalline dispersions formed by double-stranded DNA molecules were analyzed. It has been shown that gold nanoparticles induce two different processes. First, they facilitate reorganization of the spatial cholesteric structure of dispersion particles to nematic one. This process is accompanied by the fast decrease in the amplitude of abnormal band in the CD spectrum. Second, they can form ensembles consisting of gold nanoparticles. This process is accompanied by the development and displacement of surface plasmon resonance band in the visible region of the absorption spectrum. The appearance of this band is analyzed by considering two different models of the formation of ensembles consisting of gold nanoparticles. By small-angle X-ray scattering we performed structural analysis of phases formed by DNA cholesteric liquid-crystalline dispersion particles treated with gold nanoparticles. As a result of this study it was possible to prove the formation of linear clusters of gold nanoparticles in the “free space” between the adjacent DNA molecules fixed in the quasinematic layers of liquid-crystalline particles. It has been hypothesized that the formation of linear clusters of gold nanoparticles is most likely related to DNA molecules, ordered in the spatial structure of quasinematic layers, and the toxicity of these nanoparticles in biological systems hypothesized.     

Retention of gold nanoparticles in the structure of quasinematic layers formed by DNA molecules

Gold nanoparticles are shown to get incorporated into double-stranded DNA molecules forming quasinematic layers in the cholesteric liquid-crystalline dispersion particles. The process of nanoparticle incorporation results in distortion in an ordered arrangement of the neighboring dsDNA molecules in a layer and in global spatial structure of particles of the dispersion, which may be one of the possible causes of the genotoxicity of gold nanoparticles.     

Isolation and characterization of novel ginsenoside-hydrolyzing glycosidase from Microbacterium esteraromaticum that transforms ginsenoside Rb2 to rare ginsenoside 20(S)-Rg3

Ginsenoside Rb2 was transformed by recombinant glycosidase (Bgp2) into ginsenosides Rd and 20(S)-Rg3. The bgp2 gene consists of 2,430 bp that encode 809 amino acids, and this gene has homology to the glycosyl hydrolase family 2 protein domain. SDS-PAGE was used to determine that the molecular mass of purified Bgp2 was 87 kDa. Using 0.1 mg ml^?1 of enzyme in 20 mM sodium phosphate buffer at 40 °C and pH 7.0, 1.0 mg ml^?1 ginsenoside Rb2 was transformed into 0.47 mg ml^?1 ginsenoside 20(S)-Rg3 within 120 min, with a corresponding molar conversion yield of 65 %. Bgp2 hydrolyzed the ginsenoside Rb2 along the following pathway: Rb2 → Rd → 20(S)-Rg3. This is the first report of the biotransformation of ginsenoside Rb2 to ginsenoside 20(S)-Rg3 using the recombinant glycosidase.     

Production of cyclodextrin glycosyltransferase by immobilized <Emphasis Type="Italic">Bacillus</Emphasis> sp. on chitosan matrix

The whole-cell immobilization on chitosan matrix was evaluated. Bacillus sp., as producer of CGTase, was grown in solid-state and batch cultivation using three types of starches (cassava, potato and cornstarch). Biomass growth and substrate consumption were assessed by flow cytometry and modified phenol–sulfuric acid assays, respectively. Qualitative analysis of CGTase production was determined by colorless area formation on solid culture containing phenolphthalein. Scanning electron microscopy (SEM) analysis demonstrated that bacterial cells were immobilized on chitosan matrix efficiently. Free cells reached very high numbers during batch culture while immobilized cells maintained initial inoculum concentration. The maximum enzyme activity achieved by free cells was 58.15 U ml^?1 (36 h), 47.50 U ml^?1 (36 h) and 68.36 U ml^?1 (36 h) on cassava, potato and cornstarch, respectively. CGTase activities for immobilized cells were 82.15 U ml^?1 (18 h) on cassava, 79.17 U ml^?1 (12 h) on potato and 55.37 U ml^?1 (in 6 h and max 77.75 U ml^?1 in 36 h) on cornstarch. Application of immobilization technique increased CGTase activity significantly. The immobilized cells produced CGTase with higher activity in a shorter fermentation time comparing to free cells.     

Endophytic actinomycetes isolated from Aquilaria crassna Pierre ex Lec and screening of plant growth promoters production

A total of 10 endophytic actinomycete strains were successfully isolated from healthy shoots and roots of Aquilaria crassna Pierre ex Lec (eaglewood). Analysis of 16S rDNA sequencing of those isolates showed that they belong to members of the genera Streptomyces (2 isolates), Nonomuraea (1 isolate), Actinomadura (1 isolate), Pseudonocardia (1 isolate) and Nocardia (3 isolates). The remaining 2 isolates were unidentified. All of isolates produced the amount of indole-3-acetic acid (IAA) and ammonia ranging between 9.85 ± 0.31 to 15.14 ± 0.22 μg ml^?1 and 2 to 60 mg ml^?1, respectively. Among 10 isolates tested, the amount of hydroxamate-type siderophore produced by 2 isolates was undetectable. While the remaining 8 isolates produced the amount of hydroxamate-type ranging between 3.21 ± 0.12 and 39.30 ± 0.40 μg ml^?1. Also, catechols-type siderophore produced by 9 isolates was undetectable. Actinomadura glauciflava is only one isolate that produced catechols-type 4.12 ± 0.90 μg ml^?1. In addition, 10 endophytic actinomycetes showed protease activity ranging from undetectable to 8.16 ± 0.15 unit ml^?1. Genetic relatedness amongst these isolates was determined base on Random amplified polymorphic DNA (RAPD) and Enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC PCR). Both methodologies generated specific patterns corresponding to particular genotypes. RAPD fingerprinting proved to be slightly more discriminatory than ERIC PCR. This study is the first published report that actinomycetes can be isolated as endophytes within this plant. It is also the first published report that endophytic actinomycetes are capable of producing IAA and siderophores.     

Production of aglycone protopanaxatriol from ginseng root extract using Dictyoglomus turgidum β-glycosidase that specifically hydrolyzes the xylose at the C-6 position and the glucose in protopanaxatriol-type ginsenosides

The hydrolytic activity of a recombinant β-glycosidase from Dictyoglomus turgidum that specifically hydrolyzed the xylose at the C-6 position and the glucose in protopanaxatriol (PPT)-type ginsenosides followed the order Rf > Rg₁ > Re > R₁ > Rh₁ > R₂. The production of aglycone protopanaxatriol (APPT) from ginsenoside Rf was optimal at pH 6.0, 80 °C, 1 mg ml^?1 Rf, and 10.6 U ml^?1 enzyme. Under these conditions, D. turgidum β-glycosidase converted ginsenoside R₁ to APPT with a molar conversion yield of 75.6 % and a productivity of 15 mg l^?1 h^?1 after 24 h by the transformation pathway of R₁ → R₂ → Rh₁ → APPT, whereas the complete conversion of ginsenosides Rf and Rg₁ to APPT was achieved with a productivity of 1,515 mg l^?1 h^?1 after 6.6 h by the pathways of Rf → Rh₁ → APPT and Rg₁ → Rh₁ → APPT, respectively. In addition, D. turgidum β-glycosidase produced 0.54 mg ml^?1 APPT from 2.29 mg ml^?1 PPT-type ginsenosides of Panax ginseng root extract after 24 h, with a molar conversion yield of 43.2 % and a productivity of 23 mg l^?1 h^?1, and 0.62 mg ml^?1 APPT from 1.35 mg ml^?1 PPT-type ginsenosides of Panax notoginseng root extract after 20 h, with a molar conversion yield of 81.2 % and a productivity of 31 mg l^?1 h^?1. This is the first report on the APPT production from ginseng root extract. Moreover, the concentrations, yields, and productivities of APPT achieved in the present study are the highest reported to date.     

Kocuria SM1 controls vibriosis in rainbow trout (Oncorhynchus mykiss,Walbaum)

Aims: To develop probiotics for the control of vibriosis caused by Vibrio anguillarum and Vibrio ordalii in finfish. Methods and Results: Kocuria SM1, isolated from the digestive tract of rainbow trout, was administered orally to rainbow trout (Oncorhynchus mykiss) for 2 weeks at a dose equivalent to c. 10⁸ cells per g of feed and then challenged intraperitoneally with V. anguillarum and V. ordalii. Use of SM1 led to a reduction in mortalities to 15–20% compared to 74–80% mortalities in the controls. SM1 stimulated both cellular and humoral immune responses in rainbow trout, by elevation of leucocytes (5·5 ± 0·8 × 10⁶ ml^?1 from 3·7 ± 0·8 × 10⁶ ml^?1), erythrocytes (1·2 ± 0·1 × 10⁸ ml^?1 from 0·8 ± 0·1 × 10⁸ ml^?1), protein (23 ± 4·4 mg ml^?1 from 16 ± 1·3 mg ml^?1), globulin (15·7 ± 0·2 mg ml^?1 from 9·9 ± 0·1 mg ml^?1) and albumin (7·3 ± 0·2 mg ml^?1 from 6·1 ± 0·1 mg ml^?1) levels, upregulation of respiratory burst (0·05 ± 0·01 from 0·02 ± 0·01), complement (56 ± 7·2 units ml^?1 from 40 ± 8·0 units ml^?1), lysozyme (920 ± 128·8 units ml^?1 from 760 ± 115·3 units ml^?1) and bacterial killing activities. Conclusions: Kocuria SM1 successfully controlled vibriosis in rainbow trout, and the mode of action reflected stimulation of the host innate immune system. Significance and Impact of the Study: Probiotics can contribute a significant role in fish disease control strategies, and their use may replace some of the inhibitory chemicals currently used in fish farms.     

Non-toxic plant metabolites regulate Staphylococcus viability and biofilm formation: a natural therapeutic strategy useful in the treatment and prevention of skin infections

In the present study, the efficacy of generally recognised as safe (GRAS) antimicrobial plant metabolites in regulating the growth of Staphylococcus aureus and S. epidermidis was investigated. Thymol, carvacrol and eugenol showed the strongest antibacterial action against these microorganisms, at a subinhibitory concentration (SIC) of ≤ 50 μg ml^?1. Genistein, hydroquinone and resveratrol showed antimicrobial effects but with a wide concentration range (SIC = 50–1,000 μg ml^?1), while catechin, gallic acid, protocatechuic acid, p-hydroxybenzoic acid and cranberry extract were the most biologically compatible molecules (SIC ≥ 1000 μg ml^?1). Genistein, protocatechuic acid, cranberry extract, p-hydroxybenzoic acid and resveratrol also showed anti-biofilm activity against S. aureus, but not against S. epidermidis in which, surprisingly, these metabolites stimulated biofilm formation (between 35% and 1,200%). Binary combinations of cranberry extract and resveratrol with genistein, protocatechuic or p-hydroxibenzoic acid enhanced the stimulatory effect on S. epidermidis biofilm formation and maintained or even increased S. aureus anti-biofilm activity.     

Structural nucleic acid nanotechnology: Liquid-crystalline approach

The properties of the particles of cholesteric liquid-crystalline dispersions formed by double-stranded DNA molecules obtained as a result of phase exclusion of these molecules from water-salt polymer-containing solutions are briefly described. Physicochemical properties of quasinematic layers of dispersion particles and double-stranded DNA molecules in their content are taken into account in the course of developing fundamental background of the liquid-crystalline approach to the DNA structural nanotechnology. According to different versions of this approach, which is based on intraparticle gelation of cholesteric liquid-crystalline dispersions, spatial structures (DNA nanoconstructions, “rigid” DNA particles) with unique properties, are created. By means of atomic force microscopy images of “rigid” DNA particles of different type are registered. Specific properties of metallic nanoparticles (in particular, gold nanoparticles) are considered while developing the other approach to DNA structural nanotechnology, which provides the basis for “metallized” DNA nanoconstructions.     

Structural polymorphism of liquid-crystalline dispersions of double-stranded DNA complexed with synthetic polycations

Linear double-stranded DNA molecules interact with positively charged polyconidine molecules in aqueous salt solutions to yield liquid-crystalline dispersions (LCDs) with a mean particle diameter of ～6000 Å. The packing density of (DNA-polycation) complexes differs among LCD particles formed at different ionic strengths. X-ray data on the liquid-crystalline phases of (DNA-polyconidine) complexes formed under different conditions were compared with a phase diagram, reflecting polymorphism of liquid crystals of linear double-stranded DNA. It was shown that LCD was hexagonal at 0.15 M ≤ C _NaCl < 0.4 M and cholesteric at 0.4 M ≤ C _NaCl < 0.55 M. Cholesteric LCD displayed abnormal optical activity in the circular dichroism spectrum. A similar situation was observed with poly(2,5-ionene), another polycation differing in chemical structure from polyconidine. The results demonstrated structural polymorphism of (DNA-polycation) LCDs. It was assumed that the packing mode of (DNA-polycation) complexes in LCD particles can be regulated by changing NaCl concentration. The mechanism generating the cholesteric liquid-crystalline state of DNA in a narrow range of NaCl concentrations is discussed.     

The Usefulness of Non-Toxic Plant Metabolites in the Control of Bacterial Proliferation

The effect of generally recognised as safe (GRAS) plant metabolites in regulating the growth of human pathogenic and probiotic bacteria and in the formation of biofilm was investigated. Thymol, carvacrol and eugenol showed the strongest antibacterial action against both pathogenic and probiotic microorganisms, at a subinhibitory concentration (SIC) of ≤50 μg ml^?1. Genistein, hydroquinone, p-hydroxybenzoic acid and resveratrol also showed antibacterial effects but at a wide concentration range (SIC = 50–1000 μg ml^?1). Catechin, gallic acid, protocatechuic acid and cranberry extracts were the most biologically compatible molecules (SIC ≥ 1000 μg ml^?1). Regarding the effect on biofilm, it was observed that thymol, carvacrol and eugenol showed antibiofilm activity against all potential pathogenic bacteria tested whilst specifically enhancing probiotic aggregation. Catechin, genistein and cranberry extracts did not inhibit the pathogenic aggregation but they stimulated probiotic biofilm formation, whilst gallic acid, protocateuchic acid, hydroquinone, p-hydroxybenzoic acid and resveratrol did not show opposite effect on biofilm formation between pathogenic and probiotic microorganisms. These results indicate that an appropriate combination of GRAS plant metabolites, which have traditionally been used as dietary constituents due to their health-promoting characteristics, can also be extremely useful in the regulation of bacterial proliferation in the intestinal microbiota. Hence, it is suggested to apply these natural GRAS molecules as dietary supplements in the food industry in order to promote probiotic viability and to prevent or reduce colonisation or proliferation of intestinal pathogens.     

A rapid knockdown effect of Penicillium citrinum for control of the mosquito Culex quinquefasciatus in Thailand

Twenty local isolates of entomopathogenic fungi were determined for control of the larvae and adults of Culex quinquefasciatus. In a laboratory experiment, a Penicillium sp. CM-010 caused 100 % mortality of third-instar larvae within 2 h using a conidial suspension of 1 × 10⁶ conidia ml^?1. Its LC₅₀ was 3 × 10⁵ conidia ml^?1, and the lethal time (LT₅₀) was 1.06 h. Cloning and sequencing of its internal transcribed spacer region indicated that this Penicillium species is Penicillium citrinum (100 % identity in 434 bp). Mortality of the adult was highest with Aspergillus flavus CM-011 followed with Metarhizium anisopliae CKM-048 from 1 × 10⁹ conidia ml^?1. P. citrinum CM-010 at 1 × 10⁶ conidia ml^?1 killed 100 % larvae within 2 h while Bacillus thuringiensis var. israelensis at 5 ITU ml^?1 required 24 h. This P. citrinum CM-010 also greatly reduced survival of C. quinquefasciatus larvae in an unreplicated field test. Light and transmission electron micrographs showed that the fungal conidia were ingested by the larvae and deposited in the gut. The metabolite patulin was produced by P. citrinum CM-010 instead of citrinin.     

Nisin A and Polymyxin B as Synergistic Inhibitors of Gram-positive and Gram-negative Bacteria

Nisin A and polymyxin B were tested alone and in combination in order to test their antagonism against Listeria innocua HPB13 and Escherichia coli RR1, respectively. While the combination of both antibacterial substances was synergistically active against both target bacteria, nisin A alone did not show any inhibition of E. coli RR1. The nisin A/polymyxin B combination at 1.56/2.5 μg ml^?1 caused lysis of about 35.86 ± 0.35 and 73.36 ± 0.14% of L. innocua HPB13 cells after 3 and 18 h, respectively. Polymyxin B at 0.12 μg ml^?1 and nisin A/polymyxin B at 4.64/0.12 μg ml^?1 decreased the numbers of viable E. coli RR1 cells by about 0.23 and 0.65 log₁₀ CFU ml^?1, respectively, compared to the control. Our data suggest that the concentration of nisin A required for the effective control of pathogenic strains Listeria spp. could be lowered considerably by combination with polymyxin B. The use of lower concentrations of nisin A or polymyxin B should slow the emergence of bacterial populations resistant to these agents.     

Optimizing modes of inoculation of <Emphasis Type="Italic">Rhipicephalus</Emphasis> ticks (Acari: Ixodidae) with a mitosporic entomopathogenic fungus in the laboratory

The process of strain selection is an important step in the development of insect pathogens for biological control. Bioassays were conducted in the laboratory to evaluate the efficacy of different methods of inoculation using Rhipicephalus pulchellus Gerstäcker (Acari: Ixodidae) as a model. Initially, an oil-based formulation of Metarhizium anisopliae (Metsch.) Sorok. (Ascomycota: Hypocreales) titred at 10⁹ conidia ml^?1 was applied to R. pulchellus adults using a Burgerjon spray tower or a microapplicator. Inoculation by microapplicator yielded poor results (25.0% tick mortality) compared to Burgerjon’s spray tower (52.3% tick mortality), although the mean number of fungal conidia on R. pulchellus adults was lower (1.5 × 10⁴ ± 1.1 × 10³ conidia ml^?1) after spraying by Burgerjon’s spray tower compared to 1 × 10⁶ conidia ml^?1 obtained with the microapplicator. Thus, inoculation by Burgerjon’s spray tower was selected for further investigations. Different modes of inoculation were tested and included direct spray of inoculum on the tick and substrate (SS), direct spray on the substrate and tick followed by transfer of the tick to clean uncontaminated Petri dish (SP) or indirect inoculation of ticks through substrate (SW). The LC₅₀ values following contamination of nymphs (LC₅₀ = 1.4 × 10⁷ conidia ml^?1) and adults (LC₅₀ = 6.7 × 10⁷ conidia ml^?1) in SS were significantly lower compared to SP; nymphs (LC₅₀ = 5.7 × 10⁸ conidia ml^?1) and adults (LC₅₀ = 5.3 × 10⁹ conidia ml^?1) and SW; nymphs (LC₅₀ = 5 × 10⁸ conidia ml^?1). Although the LC₅₀ value in SS was the lowest, it recorded the highest tick mortality among control ticks (24.2% at 2 weeks post-treatment) and (23.3% at 3 weeks post-treatment) in nymphs and adults respectively compared to SP (2.5 and 5.8%, respectively) and SW (0.0 and 0.0). Results show that among the modes of inoculation tested, SP was the most appropriate for inoculating R. pulchellus adults. SW and SP were identified as appropriate techniques for infecting the R. pulchellus nymphs with conidia formulated in oil.     

Grazing impact on the cyanobacterium Microcystis aeruginosa by the heterotrophic flagellate Collodictyon triciliatum in an experimental pond

We estimated the grazing impact of the heterotrophic flagellate Collodictyon triciliatum on the harmful, bloom-forming cyanobacterium Microcystis aeruginosa in an experimental pond during a Microcystis bloom from summer to winter in 2010. For these experiments, we calculated the grazing rates from the digestion rate of C. triciliatum and its food vacuole contents. During the study period, M. aeruginosa exhibited one bloom event with a maximum density of 1.1 × 10⁵ cells ml^?1. The cell density of C. triciliatum fluctuated from below the detection limit to 291 cells ml^?1. The number of M. aeruginosa cells ingested by C. triciliatum food vacuoles ranged between 0.4 and 10.8 cells flagellate^?1, and the digestion rate of C. triciliatum at 25 °C was 0.73 % cell contents min^?1. The grazing rate of C. triciliatum on the M. aeruginosa prey was 0.2–6.9 cells flagellate^?1 h^?1, and its grazing impact was 0.0–25.3 % standing stock day^?1. The functional response of C. triciliatum to the M. aeruginosa prey followed the Michaelis–Menten model of significance (r ² = 0.873, p < 0.001) in our experimental systems, in which the prey concentration varied from 1.0 × 10⁴ to 2.1 × 10⁶ cells ml^?1. The maximum grazing rate was 6.2 prey cells grazer^?1 h^?1, and the half-saturation constant was 1.2 × 10⁵ cells ml^?1. We present evidence that C. triciliatum grazing explained the remarkable decrease in M. aeruginosa cell density in the pond. The present study is the first demonstration of the high potential of protistan grazing on M. aeruginosa to reduce cyanobacterial blooms.     

Iodine from bacterial iodide oxidization by Roseovarius spp. inhibits the growth of other bacteria

Microbial activities in brine, seawater, or estuarine mud are involved in iodine cycle. To investigate the effects of the microbiologically induced iodine on other bacteria in the environment, a total of 13 bacteria that potentially participated in the iodide-oxidizing process were isolated from water or biofilm at a location containing 131 μg ml^?1 iodide. Three distinct strains were further identified as Roseovarius spp. based on 16 S rRNA gene sequences after being distinguished by restriction fragment length polymorphism analysis. Morphological characteristics of these three Roseovarius spp. varied considerably across and within strains. Iodine production increased with Roseovarius spp. growth when cultured in Marine Broth with 200 μg ml^?1 iodide (I^?). When 10⁶ CFU/ml Escherichia coli, Pseudomonas aeruginosa, and Bacillus pumilus were exposed to various concentrations of molecular iodine (I₂), the minimum inhibitory concentrations (MICs) were 0.5, 1.0, and 1.0 μg ml^?1, respectively. However, fivefold increases in the MICs for Roseovarius spp. were obtained. In co-cultured Roseovarius sp. IOB-7 and E. coli in Marine Broth containing iodide (I^?), the molecular iodine concentration was estimated to be 0.76 μg ml^?1 after 24 h and less than 50 % of E. coli was viable compared to that co-cultured without iodide. The growth inhibition of E. coli was also observed in co-cultures with the two other Roseovarius spp. strains when the molecular iodine concentration was assumed to be 0.52 μg ml^?1.     

Renal, metabolic and hematological effects of trans-retinoic acid during critical developmental windows in the embryonic chicken

All-trans-retinoic acid (tRA), an active metabolite of vitamin A, directly influences the developing kidney, and is a major regulatory signal during vertebrate organogenesis. The aim of the current study was to specifically target potential critical windows in renal development, and assess altered renal function through disruptions in embryonic fluid compartments. In addition, the effect of exogenous tRA administration on embryonic growth and metabolism was determined. Embryos were exposed to 0.1 or 0.3 mg tRA on embryonic day 8. Morphological and physiological measurements were made on days 12, 14, 16 and 18. Embryo wet mass on day 18 was reduced by 23 % (0.1 mg tRA) and 44 % (0.3 mg tRA). tRA exposure elevated mass-specific oxygen consumption in embryos exposed to 0.1 mg (21.2 ± 0.3 μL^?1 g^?1 min^?1) and 0.3 mg (23.4 ± 0.4 μL^?1 g^?1 min^?1) when compared to sham (18.9 ± 0.6 μL^?1 g^?1 min^?1) on day 14, but not subsequent incubation days. Osmolality of blood plasma was transiently lowered in embryos exposed to 0.3 mg tRA between days 14 and 16. Allantoic fluid osmolality was significantly elevated by tRA to ~220 mmol L^?1 from days 16 to 18 compared to controls. Blood plasma [Na⁺] was reduced by ~17 % over the same period, while allantoic fluid [Na⁺] was elevated in tRA-treated embryos compared to control embryos. Collectively, our data indicates that exogenous administration of tRA produces significant alterations to the developmental trajectory of the developing embryonic chicken.     

Enhanced production of ATP-binding cassette protein exporter-dependent lipase by modifying the growth medium components of Pseudomonas fluorescens

The industrially-important thermostable lipase, TliA, was extracellularly produced in the recombinant Pseudomonas fluorescens by the homologous expression of TliA and its cognate ABC protein exporter, TliDEF. To increase the secretory production of TliA, we optimized the growth temperature and the culture medium of P. fluorescens. The total amount and the specific productivity of lipase was highest at 25 °C of cell growth temperature, although maximal cell growth was observed at 30 °C. Using the culture medium composed of 20 g dextrin l^?1, 40 g Tween 80 l^?1 and 30 g peptone l^?1, TliA was produced at a level of 2,200 U ml^?1 in a flask culture. The TliA production increased about 3.8-fold (8,450 U ml^?1) in batch fermentation using a 2.5 l fermentor, which was about 7.7-fold higher than that of previously reported TliA production.