Quaternary structure and functional properties of Penaeus monodon hemocyanin

The hemocyanin of the tiger shrimp, Penaeus monodon, was investigated with respect to stability and oxygen binding. While hexamers occur as a major component, dodecamers and traces of higher aggregates are also found. Both the hexamers and dodecamers were found to be extremely stable against dissociation at high pH, independently of the presence of calcium ions, in contrast to the known crustacean hemocyanins. This could be caused by only a few additional noncovalent interactions between amino acids located at the subunit-subunit interfaces. Based on X-ray structures and sequence alignments of related hemocyanins, the particular amino acids are identified. At all pH values, the p50 and Bohr coefficients of the hexamers are twice as high as those of dodecamers. While the oxygen binding of hexamers from crustaceans can normally be described by a simple two-state model, an additional conformational state is needed to describe the oxygen-binding behaviour of Penaeus monodon hemocyanin within the pH range of 7.0 to 8.5. The dodecamers bind oxygen according to the nested Monod-Whyman-Changeaux (MWC) model, as observed for the same aggregation states of other hemocyanins. The oxygen-binding properties of both the hexameric and dodecameric hemocyanins guarantee an efficient supply of the animal with oxygen, with respect to the ratio between their concentrations. It seems that under normoxic conditions, hexamers play the major role. Under hypoxic conditions, the hexamers are expected not to be completely loaded with oxygen. Here, the dodecamers are supposed to be responsible for the oxygen supply.     

Attenuation of tick-borne encephalitis virus by structure-based site-specific mutagenesis of a putative flavivirus receptor binding site

The impact of a specific region of the envelope protein E of tick-borne encephalitis (TBE) virus on the biology of this virus was investigated by a site-directed mutagenesis approach. The four amino acid residues that were analyzed in detail (E308 to E311) are located on the upper-lateral surface of domain III according to the X-ray structure of the TBE virus protein E and are part of an area that is considered to be a potential receptor binding determinant of flaviviruses. Mutants containing single amino acid substitutions, as well as combinations of mutations, were constructed and analyzed for their virulence in mice, growth properties in cultured cells, and genetic stability. The most significant attenuation in mice was achieved by mutagenesis of threonine 310. Combining this mutation with deletion mutations in the 3'-noncoding region yielded mutants that were highly attenuated. The biological effects of mutation Thr 310 to Lys, however, could be reversed to a large degree by a mutation at a neighboring position (Lys 311 to Glu) that arose spontaneously during infection of a mouse. Mutagenesis of the other positions provided evidence for the functional importance of residue 308 (Asp) and its charge interaction with residue 311 (Lys), whereas residue 309 could be altered or even deleted without any notable consequences. Deletion of residue 309 was accompanied by a spontaneous second-site mutation (Phe to Tyr) at position 332, which in the three-dimensional structure of protein E is spatially close to residue 309. The information obtained in this study is relevant for the development of specific attenuated flavivirus strains that may serve as future live vaccines.     

The effect of electrical gradients on current fluctuations and impedance recorded fromNecturus gallbladder

Summary Transepithelial current fluctuations were recorded inNecturus gallbladder, clamped at negative as well as positive potentials up to 64 mV. With NaCl-Ringer's (+10mm TAP) on both sides a mucosa-negative potential enhanced the relaxation noise component, present at zero potential, and produced peaking in the power spectrum at potentials above –36mV. Concomitantly at these potentials an inductive as well as a capacitive low-frequency feature appeared in the impedance locus. Clamping at positive potentials of 18 mV suppressed the relaxation noise component. At potentials above 51mV the spectral values increased predominantly at low frequencies. In this case the power spectrum showed only a 1/f noise component. The experiments confirm the previous finding that a K⁺ efflux through fluctuating apical K⁺ channels exists under normal conditions. With serosal KCl-Ringer's the initial Lorentzian component was enhanced at negative but suppressed at positive potentials. The increase at negative potentials was less pronounced than in experiments with NaCl-Ringer's on both sides, indicating saturation of the fluctuating K⁺ current component. With mucosal KCl-Ringer's a negative potential depressed the initial relaxation noise component, whereas it was enhanced at +18 mV clamp potential. In the latter case an additional Lorentzian component became apparent at higher frequencies. At potentials of 36 mV and above the low-frequency Lorentzian disappeared whereas the corner frequency of the high-frequency component increased. The latter experiments demonstrate that the relaxation noise component inNecturus gallbladder consists of two superimposed Lorentzians. As the relaxation times of these two components behave differently under an electrical field, there may exist two different types of K⁺ channels. It is demonstrated that peaking in the plateau of power spectra can be explained by frequency-dependent attenuation effects, caused by a polarization impedance.     

Retarded Charge–Carrier Recombination in Photoelectrochemical Cells from Plasmon‐Induced Resonance Energy Transfer

N‐type metal oxides such as hematite (α‐Fe₂O₃) and bismuth vanadate (BiVO₄) are promising candidate materials for efficient photoelectrochemical water splitting; however, their short minority carrier diffusion length and restricted carrier lifetime result in undesired rapid charge recombination. Herein, a 2D arranged globular Au nanosphere (NS) monolayer array with a highly ordered hexagonal hole pattern (hereafter, Au array) is introduced onto the surface of photoanodes comprised of metal oxide films via a facile drying and transfer‐printing process. Through plasmon‐induced resonance energy transfer, the Au array provides a strong electromagnetic field in the near‐surface area of the metal oxide film. The near‐field coupling interaction and amplification of the electromagnetic field suppress the charge recombination with long‐lived photogenerated holes and simultaneously enhance the light harvesting and charge transfer efficiencies. Consequently, an over 3.3‐fold higher photocurrent density at 1.23 V versus reversible hydrogen electrode (RHE) is achieved for the Au array/α‐Fe₂O₃. Furthermore, the high versatility of this transfer printing of Au arrays is demonstrated by introducing it on the molybdenum‐doped BiVO₄ film, resulting in 1.5‐fold higher photocurrent density at 1.23 V versus RHE. The tailored metal film design can provide a potential strategy for the versatile application in various light‐mediated energy conversion and optoelectronic devices.     

HNCCH-TOCSY,a triple resonance experiment for the correlation of backbone 13Cα and 15N resonances with aliphatic side-chain proton resonances and for measuring vicinal 13CO, 1Hβ coupling constants in proteins

Summary A 3D triple resonance experiment has been designed to provide intraresidual and sequential correlations between amide nitrogens and -carbons in uniformly ¹³C¹⁵N-labeled proteins. In-phase ¹³C magnetization is transferred to the aliphatic side-chain protons via the side-chain carbons using a CC-TOCSY mixing sequence. Thus, the experiment alleviates the resonance assignment process by providing information about the amino acid type as well as establishing sequential connectivities. Leaving the carbonyl spins untouched throughout the transfer from ¹³C to ¹H leads to E.COSY-type cross peaks, from which the ³J_{H co} coupling constants can be evaluated. The pulse sequence is applied to oxidized Desulfovibrio vulgaris flavodoxin.     

T1R3 is expressed in brush cells and ghrelin-producing cells of murine stomach

Various digestive and enteroendocrine signaling processes are constantly being adapted to the chemical composition and quantity of the chyme contained in the diverse compartments of the gastrointestinal tract. The chemosensory monitoring that underlies the adaptive capacity of the gut is thought to be performed by so-called brush cells that share morphological and molecular features with gustatory sensory cells. A substantial population of brush cells is localized in the gastric mucosa. However, no chemosensory receptors have been found to be expressed in these cells so far, challenging the concept that they serve a chemosensory function. The canonical chemoreceptors for the detection of macronutrients are taste receptors belonging to the T1R family; these have been identified in several tissues in addition to the gustatory system including the small intestine. We demonstrate the expression of the T1R subtype T1R3, which is essential for the detection of both sugars and amino acids in the gustatory system, in two distinct cell populations of the gastric mucosa. One population corresponds to open-type brush cells, emphasizing the notion that they are a chemosensory cell type; T1R3 immunoreactivity in these cells is restricted to the apical cell pole, which might provide the basis for the detection of luminal macronutrient compounds. The second gastric T1R3-positive population consists of closed-type endocrine cells that produce ghrelin. This finding suggests that ghrelin-releasing cells, which lack access to the stomach lumen, might receive chemosensory input from macronutrients in the circulation via T1R3.     

Transcriptional Repressor HIC1 Contributes to Suppressive Function of Human Induced Regulatory T Cells

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Preferential use of root litter compared to leaf litter by beech seedlings and soil microorganisms

Background and aims

Litter decomposition is regulated by e.g. substrate quality and environmental factors, particularly water availability. The partitioning of nutrients released from litter between vegetation and soil microorganisms may, therefore, be affected by changing climate. This study aimed to elucidate the impact of litter type and drought on the fate of litter-derived N in beech seedlings and soil microbes.

Methods

We quantified ¹⁵N recovery rates in plant and soil N pools by adding ¹⁵N-labelled leaf and/or root litter under controlled conditions.

Results

Root litter was favoured over leaf litter for N acquisition by beech seedlings and soil microorganisms. Drought reduced ¹⁵N recovery from litter in seedlings thereby affecting root N nutrition. ¹⁵N accumulated in seedlings in different sinks depending on litter type.

Conclusions

Root turnover appears to influence (a) N availability in the soil for plants and soil microbes and (b) N acquisition and retention despite a presumably extremely dynamic turnover of microbial biomass. Compared to soil microorganisms, beech seedlings represent a very minor short-term N sink, despite a potentially high N residence time. Furthermore, soil microbes constitute a significant N pool that can be released in the long term and, thus, may become available for N nutrition of plants.     

High-level expression of the mycobacterial porin MspA in Escherichia coli and purification of the recombinant protein

MspA is the prototype of a new family of tetrameric porins and provides the main general diffusion pathway for hydrophilic compounds through the outer membrane of Mycobacterium smegmatis. Structural analysis was hampered by the scarce amount of pure protein. After replacement of the GC-rich codons of the mspA gene by codons optimal for high-level expression in Escherichia coli, the mature MspA protein was overproduced in E. coli. The recombinant MspA (rMspA) monomer (M(r) 20000) was purified by anion exchange and hydrophobic interaction chromatography yielding 2.6 mg pure protein per liter of culture. This exceeded the yield of the native protein 10-fold. Circular dichroism revealed that rMspA is folded in a native-like structure. rMspA assembled partially to the channel-forming tetramer both during expression in E. coli and after purification in vitro. Thus, overexpression in E. coli and chromatographic purification are key steps towards a high resolution structure of MspA.