Oxygen transfer as a tool for fine-tuning recombinant protein production by <Emphasis Type="Italic">Pichia pastoris</Emphasis> under glyceraldehyde-3-phosphate dehydrogenase promoter

Effects of oxygen transfer on recombinant protein production by Pichia pastoris under glyceraldehyde-3-phosphate dehydrogenase promoter were investigated. Recombinant glucose isomerase was chosen as the model protein. Two groups of oxygen transfer strategies were applied, one of which was based on constant oxygen transfer rate where aeration rate was Q _O/V = 3 and 10 vvm, and agitation rate was N = 900 min^?1; while the other one was based on constant dissolved oxygen concentrations, C _DO = 5, 10, 15, 20 and 40 % in the fermentation broth, by using predetermined exponential glucose feeding with μ _o = 0.15 h^?1. The highest cell concentration was obtained as 44 g L^?1 at t = 9 h of the glucose fed-batch phase at C _DO = 20 % operation while the highest volumetric and specific enzyme activities were obtained as 4440 U L^?1 and 126 U g^?1 cell, respectively at C _DO = 15 % operation. Investigation of specific enzyme activities revealed that keeping C _DO at 15 % was more advantageous with an expense of relatively higher by-product formation and lower specific cell growth rate. For this strategy, the highest oxygen transfer coefficient and oxygen uptake rate were K _L a = 0.045 s^?1 and OUR = 8.91 mmol m^?3 s^?1, respectively.     

Structural dynamics of proximal heme pocket in HemAT-Bs associated with oxygen dissociation

HemAT from Bacillus subtilis (HemAT-Bs) is a heme-containing O₂ sensor protein that acts as a chemotactic signal transducer. Binding of O₂ to the heme in the sensor domain of HemAT-Bs induces a conformational change in the protein matrix, and this is transmitted to a signaling domain. To characterize the specific mechanism of O₂-dependent conformational changes in HemAT-Bs, we investigated time-resolved resonance Raman spectra of the truncated sensor domain and the full-length HemAT-Bs upon O₂ and CO dissociation. A comparison between the O₂ and CO complexes provides insights on O₂/CO discrimination in HemAT-Bs. While no spectral changes upon CO dissociation were observed in our experimental time window between 10 ns and 100 μs, the band position of the stretching mode between the heme iron and the proximal histidine, ν(Fe–His), for the O₂-dissociated HemAT-Bs was lower than that for the deoxy form on time-resolved resonance Raman spectra. This spectral change specific to O₂ dissociation would be associated with the O₂/CO discrimination in HemAT-Bs. We also compared the results obtained for the truncated sensor domain and the full-length HemAT-Bs, which showed that the structural dynamics related to O₂ dissociation for the full-length HemAT-Bs are faster than those for the sensor domain HemAT-Bs. This indicates that the heme proximal structural dynamics upon O₂ dissociation are coupled with signal transduction in HemAT-Bs.     

枯草菌HemAT蛋白质结合配基O2的构象变化——紫外共振拉曼光谱研究

枯草菌HemAT蛋白质是新近发现的一种基于血红素的趋氧性同型二聚体蛋白质。作者对此蛋白质进行了表达和提纯。用紫外共振拉曼光谱研究了全分子和传感域HemAT在与配基O2结合时的构象变化。发现O2配基与HemAT蛋白质的结合使传感域中Trp和Tyr的环境发生变化,而对连接域中Tyr的环境影响可忽略不计。信号发送域对O2配基引起的Trp和Tyr的环境变化不产生影响。O2配基与HemAT蛋白质的结合使得G-螺旋发生位移,传感域与信号发送域通过某种互感方式把O2结合信号从传感域传递到信号发送域。     

Effects of dietary fibre and protein on urea transport across the cecal mucosa of piglets

In ruminants, gastrointestinal recycling of urea is acutely enhanced by fibre-rich diets that lead to high ruminal concentration of short chain fatty acids (SCFA), while high ammonia has inhibitory effects. This study attempted to clarify if urea flux to the porcine cecum is similarly regulated. Thirty-two weaned piglets were fed diets containing protein (P) of poor prececal digestibility and fibre (F) at high (H) or low levels (L) in a 2 × 2 factorial design. After slaughter, cecal content was analyzed and the cecal mucosa incubated in Ussing chambers to measure the effect of pH, SCFA and NH₄ ⁺ on the flux rates of urea, short-circuit current (I _sc) and tissue conductance (G _t). NH₄ ⁺ significantly enhanced I _sc (from 0.5 ± 0.2 to 1.2 ± 0.1 μEq cm^?2 h^?1). No acute effects of SCFA or ammonia on urea flux were observed. Tissue conductance was significantly lower in the high dietary fibre groups irrespective of the protein content. Only the HP-LF group emerged as different from all others in terms of urea flux (74 ± 6 versus 53 ± 3 nmol cm^?2 h^?1), associated with higher cecal ammonia concentration and reduced fecal consistency. The data suggest that as in the rumen, uptake of ammonia by the cecum may involve electrogenic transport of the ionic form (NH₄ ⁺). In contrast to findings in the rumen, neither a high fibre diet nor acute addition of SCFA enhanced urea transport across the pig cecum. Instead, a HP-LF diet had stimulatory effects. A potential role for urea recycling in stabilizing luminal pH is discussed.     

Pressure-tuning FT-IR spectroscopic study on the helix–coil transition of Ala-rich oligopeptide in aqueous solution

We investigated the effect of pressure on the helix–coil transition of an Ala-rich peptide (AK16: YGAAKAAAAKAAAAKA-NH₂) in aqueous solution by FT-IR spectroscopy. The spectra of the amide I' region of AK16 in aqueous solution was decomposed into some component bands using a curve fitting method. The peak at around 1635 cm ^?1 corresponding to the solvent exposed α-helix conformer increases with increasing pressures, while the peak at around 1655 cm ^?1 corresponding to the random coil conformer decreases. From the pressure dependence of the band intensities, we determined the volume change from the α-helix to random coil conformers of AK16 to be + 10.5 ± 0.3 cm³/mol. The positive volume change is different from the negative volume change generally observed in the pressure denaturation of proteins.     

Theoretical description of the magnetic properties of μ3-hydroxo bridged trinuclear copper(II) complexes

A theoretical study of the magnetic properties, using density functional theory, of a family of trinuclear μ₃-OH copper(II) complexes reported in the literature is presented. The reported X-ray crystal structures of [Cu₃(μ₃-OH)(aat)₃(H₂O)₃](NO₃)₂·H₂O (HUKDUM), where aat: 3-acetylamine-1,2,4-triazole; [Cu₃(μ₃-OH)(aaat)₃(H₂SO₄)(HSO₄)(H₂O)] (HUKDOG), where aaat: 3-acetylamine-5-amine-1,2,4-triazole; [Cu₃(μ₃-OH)(PhPyCNO)₃(tchlphac)₂] (HOHQUR), where PhPyCNO: phenyl 2-pyridyl-ketoxime and tchlphac: acid 2,4,5-trichlorophenoxyacetic; [Cu₃(μ₃-OH)(PhPyCNO)₃(NO₃)₂(CH₃OH)] (ILEGEM); [Cu₃(μ₃-OH)(pz)₃(Hpz)₃(ClO₄)₂] (QOPJIP), where Hpz?=?pyrazole; [Cu₃(μ₃-OH)(pz)₃(Hpz)(Me₃CCOO)₂]?2Me₃CCOOH (DEFSEN) and [Cu₃(μ₃-OH)(8-amino-4-methyl-5-azaoct-3-en-2-one)₃][CuI₃] (RITXUO), were used in the calculations. The magnetic exchange constants were calculated using the broken-symmetry approach. The calculated J values are for HUKDUM J₁?=??68.6 cm^?1, J₂?=??69.9 cm^?1, J₃?=??70.4 cm^?1; for HUKDOG, J₁?=??73.5 cm^?1, J₂?=??58.9 cm^?1, J₃?=??62.1 cm^?1; for HOHQUR J₁?=??128.3 cm^?1, J₂?=??134.1 cm^?1, J₃?=??120.4 cm^?1; for ILEGEM J₁?=??151.6 cm^?1, J₂?=??173.9 cm^?1, J₃?=??186.9 cm^?1; for QOPJIP J₁?=??118.3 cm^?1, J₂?=??106.0 cm^?1, J₃?=??120.6 cm^?1; for DEFSEN J₁?=??74.9 cm^?1, J₂?=??64.0 cm^?1, J₃?=??57.7 cm^?1 and for RITXUO J₁?=??10.9 cm^?1, J₂?=?+14.3 cm^?1, J₃?=??35.4 cm^?1. The Kahn-Briat model was used to correlate the calculated magnetic properties with the overlap of the magnetic orbitals. Spin density surfaces show that the delocalization mechanism is predominant in all the studied compounds.

Identification and characterization of a carboxysomal γ-carbonic anhydrase from the cyanobacterium Nostoc sp. PCC 7120

Carboxysomes are proteinaceous microcompartments that encapsulate carbonic anhydrase (CA) and ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco); carboxysomes, therefore, catalyze reversible HCO₃ ^? dehydration and the subsequent fixation of CO₂. The N- and C-terminal domains of the β-carboxysome scaffold protein CcmM participate in a network of protein–protein interactions that are essential for carboxysome biogenesis, organization, and function. The N-terminal domain of CcmM in the thermophile Thermosynechococcus elongatus BP-1 is also a catalytically active, redox regulated γ-CA. To experimentally determine if CcmM from a mesophilic cyanobacterium is active, we cloned, expressed and purified recombinant, full-length CcmM from Nostoc sp. PCC 7120 as well as the N-terminal 209 amino acid γ-CA-like domain. Both recombinant proteins displayed ethoxyzolamide-sensitive CA activity in mass spectrometric assays, as did the carboxysome-enriched TP fraction. NstCcmM209 was characterized as a moderately active and efficient γ-CA with a k _cat of 2.0 × 10⁴ s^?1 and k _cat/K _m of 4.1 × 10⁶ M^?1 s^?1 at 25 °C and pH 8, a pH optimum between 8 and 9.5 and a temperature optimum spanning 25–35 °C. NstCcmM209 also catalyzed the hydrolysis of the CO₂ analog carbonyl sulfide. Circular dichroism and intrinsic tryptophan fluorescence analysis demonstrated that NstCcmM209 was progressively and irreversibly denatured above 50 °C. NstCcmM209 activity was inhibited by the reducing agent tris(hydroxymethyl)phosphine, an effect that was fully reversed by a molar excess of diamide, a thiol oxidizing agent, consistent with oxidative activation being a universal regulatory mechanism of CcmM orthologs. Immunogold electron microscopy and Western blot analysis of TP pellets indicated that Rubisco and CcmM co-localize and are concentrated in Nostoc sp. PCC 7120 carboxysomes.     

Can uranium follow the iron-acquisition pathway? Interaction of uranyl-loaded transferrin with receptor 1

Transferrin receptor 1 (R_D) binds iron-loaded transferrin and allows its internalization in the cytoplasm. Human serum transferrin also forms complexes with metals other than iron, including uranium in the uranyl form (UO₂ ²⁺). Can the uranyl-saturated transferrin (TUr₂) follow the receptor-mediated iron-acquisition pathway? In cell-free assays, TUr₂ interacts with R_D in two different steps. The first is fast, direct rate constant, k ₁ = (5.2 ± 0.8) × 10⁶ M^?1 s^?1; reverse rate constant, k _?1 = 95 ± 5 s^?1; and dissociation constant K ₁ = 18 ± 6 μM. The second occurs in the 100-s range and leads to an increase in the stability of the protein–protein adduct, with an average overall dissociation constant K _d = 6 ± 2 μM. This kinetic analysis implies in the proposed in vitro model possible but weak competition between TUr₂ and the C-lobe of iron-loaded transferrin toward the interaction with R _D.     

Direct discrimination of different plant populations and study on temperature effects by Fourier transform infrared spectroscopy

Fourier transform infrared spectroscopy was used to characterise highland and lowland populations of Polygonum minus Huds. grown in different controlled environments. A thermal perturbation technique of two-dimensional correlation infrared spectroscopy (2D-IR) correlation spectra was applied to establish differences between the populations. The absorption peaks at 3,480 cm^?1 (hydroxyl group), 2,927 cm^?1 (methyl group), 1,623 cm^?1 (carbonyl group), and 1,068 cm^?1 (C–O group) were particularly powerful in separating the populations. These peaks, which indicate the presence of carbohydrate, terpenes, amide and flavonoids were more intense for the highland populations than lowland populations, and increased in environments with a higher temperature. Wavenumbers (1,634, 669 cm^?1) and (1,634, 1,555 cm^?1) in the 2D-IR correlation spectra provided fingerprint signals to differentiate plants grown at different temperatures. This study demonstrates that IR fingerprinting, which combines mid-IR spectra and 2D-IR correlation spectra, can directly discriminate different populations of P. minus and the effects of temperature.     

Subcellular Localization and Kinetic Characterization of a Gill (Na+, K+)-ATPase from the Giant Freshwater Prawn Macrobrachium rosenbergii

The stimulation by Mg²⁺, Na⁺, K⁺, NH₄ ⁺, and ATP of (Na⁺, K⁺)-ATPase activity in a gill microsomal fraction from the freshwater prawn Macrobrachium rosenbergii was examined. Immunofluorescence labeling revealed that the (Na⁺, K⁺)-ATPase α-subunit is distributed predominantly within the intralamellar septum, while Western blotting revealed a single α-subunit isoform of about 108 kDa M _r. Under saturating Mg²⁺, Na⁺, and K⁺ concentrations, the enzyme hydrolyzed ATP, obeying cooperative kinetics with V _M = 115.0 ± 2.3 U mg^?1, K _0.5 = 0.10 ± 0.01 mmol L^?1. Stimulation by Na⁺ (V _M = 110.0 ± 3.3 U mg^?1, K _0.5 = 1.30 ± 0.03 mmol L^?1), Mg²⁺ (V _M = 115.0 ± 4.6 U mg^?1, K _0.5 = 0.96 ± 0.03 mmol L^?1), NH₄ ⁺ (V _M = 141.0 ± 5.6 U mg^?1, K _0.5 = 1.90 ± 0.04 mmol L^?1), and K⁺ (V _M = 120.0 ± 2.4 U mg^?1, K _M = 2.74 ± 0.08 mmol L^?1) followed single saturation curves and, except for K⁺, exhibited site–site interaction kinetics. Ouabain inhibited ATPase activity by around 73 % with K _I = 12.4 ± 1.3 mol L^?1. Complementary inhibition studies suggest the presence of F₀F₁–, Na⁺-, or K⁺-ATPases, but not V(H⁺)- or Ca²⁺-ATPases, in the gill microsomal preparation. K⁺ and NH₄ ⁺ synergistically stimulated enzyme activity (≈25 %), suggesting that these ions bind to different sites on the molecule. We propose a mechanism for the stimulation by both NH₄ ⁺, and K⁺ of the gill enzyme.     

Kinetics study of pyridine biodegradation by a novel bacterial strain,Rhizobium sp. NJUST18

Biodegradation of pyridine by a novel bacterial strain, Rhizobium sp. NJUST18, was studied in batch experiments over a wide concentration range (from 100 to 1,000 mg l^?1). Pyridine inhibited both growth of Rhizobium sp. NJUST18 and biodegradation of pyridine. The Haldane model could be fitted to the growth kinetics data well with the kinetic constants μ* = 0.1473 h^?1, K _s = 793.97 mg l^?1, K _i = 268.60 mg l^?1 and S _m = 461.80 mg l^?1. The true μ _max, calculated from μ*, was found to be 0.0332 h^?1. Yield coefficient Y _X/S depended on S _i and reached a maximum of 0.51 g g^?1 at S _i of 600 mg l^?1. V _max was calculated by fitting the pyridine consumption data with the Gompertz model. V _max increased with initial pyridine concentration up to 14.809 mg l^?1 h^?1. The q _S values, calculated from $V_{ \hbox{max} }$ , were fitted with the Haldane equation, yielding q _Smax = 0.1212 g g^?1 h^?1 and q* = 0.3874 g g^?1 h^?1 at S _m′ = 507.83 mg l^?1, K _s′ = 558.03 mg l^?1, and K _i′ = 462.15 mg l^?1. Inhibition constants for growth and degradation rate value were in the same range. Compared with other pyridine degraders, μ _max and S _m obtained for Rhizobium sp. NJUST18 were relatively high. High K _i and K _i′ values and extremely high K _s and K _s′ values indicated that NJUST18 was able to grow on pyridine within a wide concentration range, especially at relatively high concentrations.     

The carbohydrate-binding module of Fragaria × ananassa expansin 2 (CBM-FaExp2) binds to cell wall polysaccharides and decreases cell wall enzyme activities “in vitro”

A putative carbohydrate binding module (CBM) from strawberry (Fragaria × ananassa Duch.) expansin 2 (CBM-FaExp2) was cloned and the encoding protein was over-expressed in Escherichia coli and purified in order to evaluate its capacity to bind different cell wall polysaccharides “in vitro”. The protein CBM-FaExp2 bound to microcrystalline cellulose, xylan and pectin with different affinities (K_ad = 33.6 ± 0.44 mL g^?1, K_ad = 11.37 ± 0.87 mL g^?1, K_ad = 10.4 ± 0.19 mL g^?1, respectively). According to “in vitro” enzyme assays, this CBM is able to decrease the activity of cell wall degrading enzymes such as polygalacturonase, endo-glucanase, pectinase and xylanase, probably because the binding of CBM-FaExp2 to the different substrates interferes with enzyme activity. The results suggest that expansins would bind not only cellulose but also a wide range of cell wall polymers.     

Spectroscopic evidence for an all-ferrous [4Fe–4S]<Superscript>0</Superscript> cluster in the superreduced activator of 2-hydroxyglutaryl-CoA dehydratase from <Emphasis Type="Italic">Acidaminococcus </Emphasis><Emphasis Type="Italic">fermentans</Emphasis>

The key enzyme of the fermentation of glutamate by Acidaminococcus fermentans, 2-hydroxyglutarylcoenzyme A dehydratase, catalyzes the reversible syn-elimination of water from (R)-2-hydroxyglutaryl-coenzyme A, resulting in (E)-glutaconylcoenzyme A. The dehydratase system consists of two oxygen-sensitive protein components, the activator (HgdC) and the actual dehydratase (HgdAB). Previous biochemical and spectroscopic studies revealed that the reduced [4Fe–4S]⁺ cluster containing activator transfers one electron to the dehydratase driven by ATP hydrolysis, which activates the enzyme. With a tenfold excess of titanium(III) citrate at pH 8.0 the activator can be further reduced, yielding about 50% of a superreduced [4Fe–4S]⁰ cluster in the all-ferrous state. This is inferred from the appearance of a new Mössbauer spectrum with parameters δ = 0.65 mm/s and ΔE _Q = 1.51–2.19 mm/s at 140 K, which are typical of Fe(II)S₄ sites. Parallel-mode electron paramagnetic resonance (EPR) spectroscopy performed at temperatures between 3 and 20 K showed two sharp signals at g = 16 and 12, indicating an integer-spin system. The X-band EPR spectra and magnetic Mössbauer spectra could be consistently simulated by adopting a total spin S _t = 4 for the all-ferrous cluster with weak zero-field splitting parameters D = ?0.66 cm^?1 and E/D = 0.17. The superreduced cluster has apparent spectroscopic similarities with the corresponding [4Fe–4S]⁰ cluster described for the nitrogenase Fe-protein, but in detail their properties differ. While the all-ferrous Fe-protein is capable of transferring electrons to the MoFe-protein for dinitrogen reduction, a similar physiological role is elusive for the superreduced activator. This finding supports our model that only one-electron transfer steps are involved in dehydratase catalysis. Nevertheless we discuss a common basic mechanism of the two diverse systems, which are so far the only described examples of the all-ferrous [4Fe–4S]⁰ cluster found in biology.     

Toxicity of an Annonin-Based Commercial Bioinsecticide Against Three Primary Pest Species of Stored Products

The effects of a bioinsecticide formulation based on extract of Annona squamosa L. (Annonaceae) containing 10,000 mg L^?1 of acetogenin annonin as the main active ingredient were investigated against three primary pest species of stored grains in Brazil [maize weevil Sitophilus zeamais Motschulsky (Coleoptera: Curculionidae), Mexican bean weevil Zabrotes subfasciatus (Boheman) (Coleoptera: Chrysomelidae: Bruchinae), and cowpea weevil Callosobruchus maculatus (Fabricius) (Coleoptera: Chrysomelidae: Bruchinae)] by means of residual contact bioassays. In a concentration-dependent manner, the annonin-based commercial bioinsecticide caused significant adult mortality of C. maculatus (LC₅₀ = 6890 μL kg^?1), S. zeamais (LC₅₀ = 2781 μL kg^?1), and Z. subfasciatus (LC₅₀ = 2120 μL kg^?1) after 120 h of residual contact exposure. In addition to acute toxicity, the tested bioinsecticide also promoted a significant reduction of the number of eggs laid by females of C. maculatus (EC₅₀ = 5949.7 μL kg^?1) and Z. subfasciatus (EC₅₀ = 552.7 μL kg^?1). Moreover, the bioinsecticide significantly reduced the number of emerged insects (F₁ generation) of C. maculatus (EC₅₀ = 2763.0 μL kg^?1), S. zeamais (EC₅₀ = 1380.8 μL kg^?1), and Z. subfasciatus (EC₅₀ = 561.5 μL kg^?1). The bioinsecticide also reduced the percentage of damaged grains for the three pest species studied, and its grain-protectant properties are comparable to or superior in efficacy in relation to a diatomaceous earth-based insecticide (Insecto® at 1000 mg kg^?1) used as a positive control. Thus, this standardized formulation has promising bioactivity against stored insect species and can be a useful component for IPM of stored grains in Brazil and elsewhere.     

Fine Root Morphology,Biochemistry and Litter Quality Indices of Fast- and Slow-growing Woody Species in Ethiopian Highland Forest

Fine root turnover of trees is a major C input to soil. However, the quality of litter input is influenced by root morphological traits and tissue chemical composition. In this study, fine roots of ten tropical woody species were collected from an Afromontane forest in the northern highlands of Ethiopia. The fine roots were analysed for root morphological traits and tissue chemistry measured as proxy carbon fractionations. Based on stem increment, the 10 species were divided into faster- and slower-growing species. Faster-growing species exhibited higher specific root length (1362 cm g^?1) than slower-growing species (923 cm g^?1). Similarly specific root area was higher in faster-growing species (223 cm² g^?1) than in slower-growing species (167 cm² g^?1). Among the carbon fractions, the acid-insoluble fraction (AIF) was the highest (44–51%). The carbon content, AIF, and the lignocellulose index were higher for slower-growing species. Root tissue density was lower in faster-growing species (0.33 g cm^?3) than slower-growing species (0.40 g cm^?3) and showed a strong positive correlation with carbon content (r ² = 0.84) and the AIF (r _pearson = 0.93). The morphological traits of fine roots between faster- and slower-growing species reflect the ecological strategy they employ. Slower-growing species have a higher tissue density which may reflect a greater longevity.     

DNA Methylation Detection Using Resonance and Nanobowtie-Antenna-Enhanced Raman Spectroscopy

We show that DNA carrying 5-methylcytosine modifications or methylated DNA (m-DNA) can be distinguished from DNA with unmodified cytosine by Raman spectroscopy enhanced by both a bowtie nanoantenna and excitation resonance. In particular, m-DNA can be identified by a peak near 1000 cm^?1 and changes in the Raman peaks in the 1200–1700 cm^?1 band that are enhanced by the ring-absorption resonance. The identification is robust to the use of resonance Raman and nanoantenna excitation used to obtain significant signal improvement. The primary differences are three additional Raman peaks with methylation at 1014, 1239, and 1639 cm^?1 and spectral intensity inversion at 1324 (C₅=C₆) and 1473 cm^?1 (C₄=N₃) in m-DNA compared to that of DNA with unmodified cytosine. We attribute this to the proximity of the methyl group to the antenna, which brings the (C₅=C₆) mode closer to experiencing a stronger near-field enhancement. We also show distinct Raman spectral features attributed to the transition of DNA from a hydrated state, when dissolved, to a dried/denatured state. We observe a general broadening of the larger lines and a transfer of spectral weight from the ～1470 cm^?1 vibration to the two higher-energy lines of the dried m-DNA solution. We attribute the new spectral characteristics to DNA softening under high salt conditions and find that the m-DNA is still distinguishable via the ～1000 cm^?1 peak and distribution of the signal in the 1200–1700 cm^?1 band. The nanoantenna gain exceeds 20,000, whereas the real signal ratio is much less because of a low average enhanced region occupancy even with these relatively high DNA concentrations. It is improved when fixed DNA in a salt crystal lies near the nanoantenna. The Raman resonance gain profile is consistent with A-term expectations, and the resonance is found at ～259 nm excitation wavelength.     

Age validation and seasonal growth patterns of a subtropical marsh fish: The Gulf Killifish, <Emphasis Type="Italic">Fundulus grandis</Emphasis>

Fundulus grandis (Baird and Girard), the Gulf Killifish, is an abundant species throughout the marshes of the northern Gulf of Mexico. Its wide distribution and high site fidelity makes it an ideal indicator species for brackish and salt marshes, which experience a variety of anthropogenic disturbances. Despite the ecological, commercial, and scientific importance of F. grandis, age determination methods have not been validated and little is known of its growth pattern. By combining a tag-recapture study with a chemical marker to stain otoliths, we validated an ageing method for F. grandis adults (49–128 mm TL) using whole sagittal otoliths and determined growth rates of recaptured individuals in winter (n = 58) and summer (n = 36) in Louisiana. Mean somatic growth in length was significantly greater during the winter (0.085 mm d^?1) than summer (0.054 mm d^?1). In contrast, mean otolith growth was significantly greater in summer (1.37 μm d^?1) than winter (0.826 μm d^?1). The uncoupling of somatic and otolith growth may be primarily attributed to warm summer temperatures, which led to enhanced otolith growth while simultaneously reducing somatic growth. Fundulus grandis was aged to a maximum of 2.25 years. The parameters of the von Bertalanffy growth model were estimated as: L _∞  = 87.27 mm, k = 2.43 year^?1, and t ₀ = ?0.022. These findings reveal essential age and growth information for F. grandis and provide a benchmark to evaluate responses to environmental disturbances.     

Molecular Structure Evaluation of Wheat Gluten during Frozen Storage

The effects of frozen storage (0–120 day) on the secondary structure and molecular chain conformation of hydrated gluten were investigated using Fourier transform infrared spectroscopy (FTIR) and atomic force microscopy (AFM). After frozen storage, no changes were observed in the secondary structure of the 60% hydrated gluten; spectra were consistent with a tight ordered structure with many interchain hydrogen bond interactions. For the dehydrated gluten, more complex changes took place: during frozen storage for up to 60 days, there were distinctive changes in the low-frequency region of the amide I band (1618–1633 cm^?1) which were attributed to changes in the β-sheet structure. However, with the increase of frozen storage from 60 to 120 days, a band near 1614 cm^?1 replaced that at 1659 cm^?1 illustrate that the formation of protein aggregates during the long-time frozen storage, which along with the establishment of new intermolecular non-covalent bonds within the protein molecule or between two neighboring molecules. AFM images showed that the gluten chain formed a fibril-like branched network, and this network was weakened with increasing frozen storage time.     

Spectral Characteristics of Near-Infrared Surface Plasmon Resonance

Intrinsic properties of surface plasmons (SPs) excited with Kretschmann configuration were analyzed as a function of wavelength, including the propagation length, the penetration depth, the Goos–Hänchen (GH) shift, and the field enhancement. The calculated results indicate that there exists a critical thickness (t _cr) of the gold layer and that the maximum GH shift occurring exactly at the SP resonance wavelength (λ _R) rapidly varies from positive to negative with changing of the gold layer thickness from t?<?t _cr to t?>?t _cr. The maximum field enhancement happens not at λ _R but at a wavelength smaller than λ _R due to the phase retardation between the transmitted and reflected light. Simulations also reveal that a broadband collimated near-infrared beam can simultaneously excite two SPs with different responses to a refractive index (RI) change: the shorter-wavelength SP able to make a small redshift and the longer-wavelength SP capable of yielding a large blueshift. Only the shorter-wavelength SP was experimentally observed and its RI sensitivity was measured to increase from 3,539 nm/RIU at λ _R?=?707.6 nm to 57,143 nm/RIU at λ _R?=?1,398 nm. The SP at λ _R?=?1,013 nm moved to λ _R?=?1,029 nm in response to the saturation adsorption of bovine serum albumin, and the corresponding surface coverage was determined to be Γ?=?1.565 ng/mm² based on a quasilinear dependence of Γ on the resonance wavelength shift (?λ _R) deduced theoretically. Butyrylcholinesterase adsorption from a dilute solution of 10 nM protein in phosphate buffer solution leads to a redshift of ?λ _R?=?10 nm, corresponding to Γ?≈?0.97 ng/mm².