Modulation of TNF-α mRNA stability by human antigen R and miR181s in sepsis-induced immunoparalysis.

Immunoparalysis is an important pathological mechanism in sepsis. However, an effective small molecule therapy is lacking. Here, we show that ouabain, a Na(+),K(+)-ATPase ligand, can reverse immunoparalysis in vitro, in vivo, and in clinical samples. Notably, the effect of ouabain was critically dependent on TNF-α expression. However, ouabain had opposing effects on the stability of TNF-α mRNA: Ouabain triggered miR-181 transcription, which promoted TNF-α mRNA degradation and induced immunoparalysis, and ouabain triggered the nuclear export of human antigen R (HuR), which stabilized TNF-α mRNA and suppressed immuno-paralysis. Interestingly, because the miR-181 binding site is located within the HuR binding site in the 3'-untranslated region of TNF-α, in ouabain-treated cells, HuR competed with miR-181 for binding to TNF-α mRNA and recruited TNF-α mRNA to stress granules, thereby stabilizing TNF-α mRNA and reversing immunoparalysis. Ouabain also induced GM-CSF and interferon-γ expression in a HuR-dependent manner. Hence, the fine-tuning of TNF-α mRNA stability by HuR and miR181 plays a crucial role in immunoparalysis, and Na(+),K(+)-ATPase ligands are promising agents for immunoparalysis therapy.

A Novel Biocatalyst and Its Optimized Process for Preparing L-theanine / 中国生物工程杂志

L-theanine (γ-glutamylethylamide) is the main free amino acid component of tea and its favorable physiological effects on mammals have been reported. An enzymatic method for optically pure L-theanine production with a new L-aminoacylases-production fungi Cunnighamella echinulata 9980 was developed. The optimum conditions for enzymatic catalysis were at pH 6.5 with 50 mmol/L N-Acyl-DLtheanine and 40 mmol/L CoCl2. After 12-h incubation at 50℃,22.5 mmol/L L-theanine was obtained, the conversion rate against N-Acyl-L-theanine being 90%. Cunnighamella echinulata and the aminoacylase were applied in preparation of L-theanine.

A Novel Biocatalyst and Its Optimized Process for Preparing L-theanine / 中国生物工程杂志

L-theanine (γ-glutamylethylamide) is the main free amino acid component of tea and its favorable physiological effects on mammals have been reported. An enzymatic method for optically pure L-theanine production with a new L-aminoacylases-production fungi Cunnighamella echinulata 9980 was developed. The optimum conditions for enzymatic catalysis were at pH 6.5 with 50 mmol/L N-Acyl-DLtheanine and 40 mmol/L CoCl2. After 12-h incubation at 50℃,22.5 mmol/L L-theanine was obtained, the conversion rate against N-Acyl-L-theanine being 90%. Cunnighamella echinulata and the aminoacylase were applied in preparation of L-theanine.

The Research of Enzymology Characterization about Arginine Deiminase from Enterococcus faecalis / 微生物学通报

Arginine Deiminase(ADI) was purified to homogeneity using ammonium sulfate precipitation,Q-Sepharose Fast Flow anion exchange chromatography and SephadexG-75 gel filtration chromatography. This purification protocol resulted in a 34.5-fold purification of ADI with 31.4% final yield. A molecular weight of about 190 kD determined by native gradient polyacrylamide gel electrophoresis. The enzyme has only one kind of 46 kD subunit determined by SDS-PAGE. Combining the results from the two kinds of electrophoresis,the authors deduce that the enzyme may be a tetramer. The optimum pH and temperature for lipolytic activity of ADI was pH 6.5 and 50℃,respectively. It was extremely stable at 45℃ and retained 97.9% of its original activity for 30 min. The stability declined rapidly as soon as the temperature rose over 50℃. ADI was highly stable in the pH range from pH 5-8. ADI acted on L-arginine but not on D-arginine. ADI catabolism was dependent on metal ions. At their adequate concentration,Mn2+,Mg2+ and Co2+ were the effective promoter,while superfluous Zn2+and Co2+ inhibited ADI activity. L-citrulline did not act on ADI,but L-ornithine inhibited ADI activity. The degradation of L-arginine with ADI catalysis was according to simple Michaelis-Menten equation. The Michaelis constant was 3.2686 mmol/L and the maxi-mum velocity was 2.44 ?mol/min.

Characterization of Aminoacylase in the Mycelial Cell of Cunnighamella echinulata / 微生物学通报

The strain of Cunninghamella echinulata 9980 was first selected with high aminoacylase activity . In three submerged cultures, the aminoacylase activity in the mycelial cell was compared . A number of factors have effects on the resolution reaction. The results showed that, peptone culture gave the highest aminoacylase activity with 680U/g.The optium temperature,pH,and substrate concentration were 55℃, 7.0,and 0.2mol/L,respectively. The ions in the buffer lowered the activity,but the Co~(2+) in 10~(-4)~10~(-3 )mol/L was necessary for its activity.