The Voltage-Dependent Anion Channel (VDAC) of Pacific Oysters <Emphasis Type="Italic">Crassostrea gigas</Emphasis> Is Upaccumulated During Infection by the Ostreid Herpesvirus-1 (OsHV-1): an Indicator of the Warburg Effect

Voltage-dependent anion channel (VDAC) is a key mitochondrial protein. VDAC drives cellular energy metabolism by controlling the influx and efflux of metabolites and ions through the mitochondrial membrane, playing a role in its permeabilization. This protein exerts a pivotal role during the white spot syndrome virus (WSSV) infection in shrimp, through its involvement in a particular metabolism that plays in favor of the virus, the Warburg effect. The Warburg effect corresponds to an atypical metabolic shift toward an aerobic glycolysis that provides energy for rapid cell division and resistance to apoptosis. In the Pacific oyster Crassostrea gigas, the Warburg effect occurs during infection by Ostreid herpesvirus (OsHV-1). At present, the role of VDAC in the Warburg effect, OsHV-1 infection and apoptosis is unknown. Here, we developed a specific antibody directed against C. gigas VDAC. This tool allowed us to quantify the tissue-specific expression of VDAC, to detect VDAC oligomers, and to follow the amount of VDAC in oysters deployed in the field. We showed that oysters sensitive to a mortality event in the field presented an accumulation of VDAC. Finally, we propose to use VDAC quantification as a tool to measure the oyster susceptibility to OsHV-1 depending on its environment.     

TALEN-Induced Double-Strand Break Repair of CTG Trinucleotide Repeats

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Phosphorus speciation and micro-scale spatial distribution in North-American temperate agricultural soils from micro X-ray fluorescence and X-ray absorption near-edge spectroscopy

Background and Aims

Phosphorus (P) is an essential nutrient for plants but its low availability often necessitates amendments for agronomical issues. Objectives were to determine P spatial distribution and speciation that remain poorly understood in cultivated soils.

Methods

Aquic Argiudoll soil samples developed on a calcareous loam glacial till were collected from experimental plots submitted to contrasting crop rotations and amendments. Micro-X-ray fluorescence (μ-XRF) maps were collected on undisturbed samples. X-ray absorption near edge structure (XANES) spectra were collected on bulk samples and on fractions thereof, and on points of interests selected from μ-XRF maps. Results were compared with chemical analyses and extraction techniques results.

Results

Chemical analyses show variations in total and exchangeable P contents depending on the samples but no significant difference is observed in terms of P distribution and speciation. P distribution is dominated by a low-concentration diffuse background with a minor contribution from minute hot spots. P speciation is dominated by phosphate groups bound to clay-humic complexes. No modification of P distribution and speciation is observed close to roots.

Conclusions

This study evidenced minor effect of cropping and fertilizing practices on P speciation in cultivated soils. Despite analytical challenges, the combined use of μ-XRF and XANES provides relevant information on P speciation in heterogeneous soil media.

     

In vivo phosphorylation of a peptide tag for protein purification

Objectives

To design a new system for the in vivo phosphorylation of proteins in Escherichia coli using the co-expression of the α-subunit of casein kinase II (CKIIα) and a target protein, (Nanofitin) fused with a phosphorylatable tag.

Results

The level of the co-expressed CKIIα was controlled by the arabinose promoter and optimal phosphorylation was obtained with 2 % (w/v) arabinose as inductor. The effectiveness of the phosphorylation system was demonstrated by electrophoretic mobility shift assay (NUT-PAGE) and staining with a specific phosphoprotein-staining gel. The resulting phosphorylated tag was also used to purify the phosphoprotein by immobilized metal affinity chromatography, which relies on the specific interaction of phosphate moieties with Fe(III).

Conclusion

The use of a single tag for both the purification and protein array anchoring provides a simple and straightforward system for protein analysis.

     

鱼类卵子发育能力的进一步研究

作者中的二人[Tung,and Tung.(童第周与叶毓芬)1944;Tung,Chang,andTung.(童第周、张致一与叶毓芬)1945]曾以分割的方法,研究金鱼(Carassiusauratus)卵子的发育能力。他们将2至8细胞时期的金鱼卵子,在去膜后,用玻璃针,按第一次或第二次的分裂平面,将整个卵子分割为二半。每半含有半数分裂球,一半卵黄,和一半未流入分裂球的细胞质。分割后,每半都能继续发育,但产生下列6种不同的情形:(1)二半都发育为完整的,一半大小的胚胎;(2)一个为完整的胚胎,另一个为不完整的胚胎;(3)一个为完整的胚胎,另一人为囊胚;     

Dissecting structural basis of the unique substrate selectivity of human enteropeptidase catalytic subunit

Enteropeptidase is a key enzyme in the digestion system of higher animals. It initiates enzymatic cascade cleaving trypsinogen activation peptide after a unique sequence DDDDK. Recently, we have found specific activity of human enteropeptidase catalytic subunit (L-HEP) being significantly higher than that of its bovine ortholog (L-BEP). Moreover, we have discovered that L-HEP hydrolyzed several nonspecific peptidic substrates. In this work, we aimed to further characterize species-specific enteropeptidase activities and to reveal their structural basis. First, we compared hydrolysis of peptides and proteins lacking DDDDK sequence by L-HEP and L-BEP. In each case human enzyme was more efficient, with the highest hydrolysis rate observed for substrates with a large hydrophobic residue in P2-position. Computer modeling suggested enzyme exosite residues 96 (Arg in L-HEP, Lys in L-BEP) and 219 (Lys in L-HEP, Gln in L-BEP) to be responsible for these differences in enteropeptidase catalytic activity. Indeed, human-to-bovine mutations Arg96Lys, Lys219Gln shifted catalytic properties of L-HEP toward those of L-BEP. This effect was amplified in case of the double mutation Arg96Lys/Lys219Gln, but still did not cover the full difference in catalytic activities of human and bovine enzymes. To find a missing link, we studied monopeptide benzyl-arginine-β-naphthylamide hydrolysis. L-HEP catalyzed it with an order lower K (m) than L-BEP, suggesting the monopeptide-binding S1 site input into catalytic distinction between two enteropeptidase species. Together, our findings suggest structural basis of the unique catalytic properties of human enteropeptidase and instigate further studies of its tentative physiological and pathological roles.