Effect of lon Protease Overexpression on Endotoxin Production and Stress Resistance in Bacillus thuringiensis.

Lon protease, an intracellular protease, plays a key role in cell homeostasis in bacteria and is involved in numerous physiological processes. In this work, we aimed to study the impact of Lon on the production of endotoxins and stress response in Bacillus thuringiensis, which is an important bioinsecticide alternative for toxic chemicals. For this purpose, lon gene was cloned into a multi-copy vector with its original promoter and transcriptional terminator and expressed in B. thuringiensis serovar israelensis ATCC 35,646. Our results showed that the recombinant lon gene transcribed and translated efficiently and the resulting protein was active. Although the sporulation efficiency of the recombinant strain was found to be reduced and its mobility impaired, overexpression of the lon gene triggered the production of endotoxin. Together with increased biofilm formation, recombinant strain exhibited significantly better adaptation to osmotic and heat shock stresses and UV exposure compared to wild type and the control strain with empty plasmid. This study suggested a possible link between Lon protease and the production of insecticide and stress response in B. thuringiensis and provides a platform for future studies focusing on enhancing bio-insecticidal production using this bacterium.

One Extra Copy of lon Gene Causes a Dramatic Increase in Actinorhodin Production by Streptomyces coelicolor A3(2).

ATP-dependent Lon protease plays important roles in different physiological processes, including cellular differentiation of the bacteria and is a part of an important stress response regulon (HspR/HAIR). In Streptomyces, biosynthesis of secondary metabolites starts with cellular differentiation and stress is one of the factor that affect metabolite production. To clarify the effect of Lon protease on secondary metabolite production, we constructed a recombinant strain of Streptomyces coelicolor A3(2) that has one extra copy of lon gene with its own promoter and transcriptional terminator in its genome. Expression of lon gene in the recombinant strain was determined by quantitative real time (RT-qPCR). Actinorhodin and undecylprodigiosin production of the recombinant cell was measured in liquid R2YE and it was found to produce about 34 times more actinorhodin and 9 times more undecylprodigiosin than the wild-type at 168 h of growth. Development of stable Streptomyces strains capable of producing high amounts of secondary metabolites is valuable for biotechnology industry. One extra copy of lon gene is enough to boost antibiotic production by S. coelicolor A3(2) and this change do not cause any metabolic burden in the cell.

Sevoflurane exerts brain-protective effects against sepsis-associated encephalopathy and memory impairment through caspase 3/9 and Bax/Bcl signaling pathway in a rat model of sepsis.

Objective We compared the effects of sevoflurane and isoflurane on systemic inflammation, sepsis-associated encephalopathy, and memory impairment in a rat sepsis model of cecal ligation and puncture (CLP)-induced polymicrobial peritonitis. Methods Twenty-four rats were assigned to sham, CLP, CLP + sevoflurane, and CLP + isoflurane groups. At 72 hours after CLP, the rats underwent behavior tests. Serum cytokines were evaluated. Brain tissue samples were collected for determination of glutathione peroxidase (GPX), superoxide dismutase (SOD), and catalase; the wet/dry weight ratio; myeloperoxidase (MPO) and malondialdehyde (MDA); apoptotic gene release; and histologic examinations. Results The MPO level, wet/dry weight ratio, and histopathology scores were lower and the Bcl2a1 and Bcl2l2 expressions were upregulated in both the CLP + sevoflurane and CLP + isoflurane groups compared with the CLP group. The interleukin-6, interleukin-1ß, MDA, and caspase 3, 8, and 9 levels were lower; the GPX, SOD, Bax, Bcl2, and Bclx levels were higher; and non-associative and aversive memory were improved in the CLP + sevoflurane group compared with the CLP + isoflurane group. Conclusion Sevoflurane decreased apoptosis and oxidative injury and improved memory in this experimental rat model of CLP. Sevoflurane sedation may protect against brain injury and memory impairment in septic patients.

Thoracic epidural bupivacaine attenuates inflammatory response, intestinal lipid peroxidation, oxidative injury, and mucosal apoptosis induced by mesenteric ischemia/reperfusion.

BACKGROUND: We conducted this study to evaluate the effects of thoracic epidural anesthesia (TEA) on inflammatory response, lipid peroxidation, and oxidative stress in a rat model of mesenteric ischemia/reperfusion (I/R). METHOD: Rats were divided into 4 groups: sham group (n=6; sham laparotomy), control group (n=6; I/R), bupivacaine group (n=6; mesenteric I/R and 20 µL/h 0.5% bupivacaine), and saline group (n=6, mesenteric I/R and 20 µL/h 0.9% saline). I/R injury was established by occluding the superior mesenteric artery for 1 hour followed by 12 hours reperfusion. Blood gas, tumor necrosis factor-α, interleukin-6, interleukin-1ß, glutathione peroxidise, superoxide dismutase, catalese, myeloperoxidase concentrations, immunohistochemical examinations (intracellular adhesion molecule-1), apoptosis determination, and wet/dry ratio of intestinal edema were determined. RESULTS: Bupivacaine significantly decreased the cytokine, malondialdehyde, and myeloperoxidase levels and increased the antioxidant enzyme levels. Wet/dry ratio comparison showed a significant decrease in the bupivacaine (2.88±0.17) group in comparison with control (5.45±0.67) and saline (5.87±0.17) groups. The intestinal injury score was significantly decreased in rats in the epidural bupivacaine (2 [1-2]) infusion group in comparison with rats in the control (3 [2-3]) and saline (3 [2-4]) groups. Bupivacaine (63%) caused a significant decrease in the percentage of apoptotic cells in comparison with control (85%) only. ICAM-1 levels in the bupivacaine (27.4±7.1) group decreased in comparison with control (12.3±7.4) and saline (24.9±3.2) groups. CONCLUSION: This study demonstrated that epidural bupivacaine attenuates the mesenteric I/R-related inflammatory response and intestinal damage.