Improved l-Leucine Production in Corynebacterium glutamicum by Optimizing the Aminotransferases.

The production of branched-chain amino acids (BCAAs) is still challenging, therefore we rationally engineered Corynebacterium glutamicum FA-1 to increase the l-leucine production by optimizing the aminotransferases. Based on this, we investigated the effects of the native aminotransferases, i.e., branched-chain amino acid aminotransferase (BCAT; encoded by ilvE) and aspartate aminotransferase (AspB; encoded by aspB) on l-leucine production in C. glutamicum. The strain FA-1△ilvE still exhibited significant growth without leucine addition, while FA-1△ilvE△aspB couldn't, which indicated that AspB also contributes to L-leucine synthesis in vivo and the yield of leucine reached 20.81 ± 0.02 g/L. It is the first time that AspB has been characterized for l-leucine synthesis activity. Subsequently, the aromatic aminotransferase TyrB and the putative aspartate aminotransferases, the aspC, yhdR, ywfG gene products, were cloned, expressed and characterized for leucine synthesis activity in FA-1△ilvE△aspB. Only TyrB was able to synthesize l-leucine and the l-leucine production was 18.55 ± 0.42 g/L. The two putative branched-chain aminotransferase genes, ybgE and CaIlvE, were also cloned and expressed. Both genes products function efficiently in BCAAs biosynthesis. This is the first report of a rational modification of aminotransferase activity that improves the l-leucine production through optimizing the aminotransferases.

[Activity and role of tryptase after entrance of amniotic fluid into blood in rats].

OBJECTIVE: To investigate the activity and role of tryptase after entrance of amniotic fluid into blood in rats. METHODS: Thirty female Wistar rats (20 day pregnancy) were divided into the control group (10, injected with normal saline), amniotic group (10, injected with amniotic fluid), meconium group (10, injected with 1% meconium). After injection, pulmonary tissue was taken out. Tryptase activity was measured by special substrate. The histology of pulmonary tissue was determined by immunohistochemistry (HE). RESULTS: (1) Dropsy, hemorrhage, and infiltration of neutrophil (PMN), macrophage, leukomonocyte were observed in two experimental groups, but no such changes were found in control group. (2) After injection, tryptase activity in meconium group 176.4 +/- 8.6 and amniotic fluid group 165.4 +/- 7.4 was significantly higher than preexperimental groups 146.8 +/- 8.9 and 147.8 +/- 9.5, respectively (t = 7.58 and t = 4.64, P < 0.01); tryptase activity in control group was 145.3 +/- 10.6 before injection and 146.9 +/- 9.4 after injection, respectively, there was no difference (t = 0.37, P > 0.05). After injection, tryptase activity in meconium and amniotic fluid groups was significantly increased than that in control group (F = 30.66, P < 0.05). CONCLUSION: The activity of tryptase was significantly increased after entrance of amniotic fluid into blood in rats. Degranulation of mast cells to release tryptase may be the important cause of the pathophysiologic change after entrance of amniotic fluid into blood. These results suggest a role for mast cell activation in the mechanism of amniotic fluid embolism. This method is sensitive and effective for diagnosis of amniotic fluid embolism in clinic.

Inhibition of topoisomerase II by 8-chloro-adenosine triphosphate induces DNA double-stranded breaks in 8-chloro-adenosine-exposed human myelocytic leukemia K562 cells.

8-Chloro-cAMP and 8-chloro-adenosine (8-Cl-Ado) are known to inhibit proliferation of cancer cells by converting 8-Cl-Ado into an ATP analog, 8-chloro-ATP (8-Cl-ATP). Because type II topoisomerases (Topo II) are ATP-dependent, we infer that 8-Cl-Ado exposure might interfere with Topo II activities and DNA metabolism in cells. We found that 8-Cl-Ado exposure inhibited Topo II-catalytic activities in K562 cells, as revealed by decreased relaxation of the supercoiled pUC19 DNA and inhibited decatenation of the kinetoplast DNA (kDNA). In vitro assays showed that 8-Cl-ATP, but not 8-Cl-Ado, could directly inhibit Topo IIalpha-catalyzed relaxation and decatenation of substrate DNA. Furthermore, 8-Cl-ATP inhibited Topo II-catalyzed ATP hydrolysis and increased salt-stabilized closed clamp. In addition, 8-Cl-Ado exposure decreased bromo-deoxyuridine (BrdU) incorporation into DNA and led to enhanced DNA double-stranded breaks (DSBs) and to increased formation of gamma-H2AX nuclear foci in exposed K562 cells. Together, 8-Cl-Ado/8-Cl-ATP can inhibit Topo II activities in cells, thereby inhibiting DNA synthesis and inducing DNA DSBs, which may contribute to 8-Cl-Ado-inhibited proliferation of cancers.