Vibrio parahaemolyticus is a recognized enteropathogen causing diarrhea in humans and is one of the major causes of seafoodborne gastroenteritis. An important virulence factor is thermostable direct hemolysin (TDH), a pore-forming toxin, which is able to lyse eukaryotic cells. The active toxin is a tetramer of four identical protein subunits, which is secreted by the pathogen after cleavage of a signal peptide. To establish diagnostic detection systems for TDH we expressed the hemolysin with and without the signal peptide in a prokaryotic cell-free system to obtain pure toxin. In order to purify and to facilitate the isolation from cell lysates we synthesized TDH variants with different tags. Important regulatory sequences for cell-free protein synthesis as well as sequences for N-terminal Strep-tag and C-terminal 6xHis-tag were added by a two-step PCR. For the expression in the cell-free system these linear tdh templates were subjected directly to prokaryotic cell extracts. Protein yields were in the range of 500-600 μg/ml for the preproteins and approx. 300-400 μg/ml for the mature proteins. The identities of expressed proteins were further confirmed by SDS-PAGE, immunological and MALDI-TOF mass spectrometric analyses. The functionality of newly synthesized toxin variants was tested by performing qualitative and semiquantitative hemolysis assays. Cell-free produced mature TDH and its variants were active while the preprotein and its derivatives lacked hemolytic activity. A C-terminal 6xHis-tag showed less influence on functionality compared to the N-terminal Strep-tag.

Objective— Perivascular adipose tissue (PVAT) wraps blood vessels and modulates vasoreactivity by secretion of vasoactive molecules. Mammalian target of rapamycin complex 2 (mTORC2) has been shown to control inflammation and is expressed in adipose tissue. In this study, we investigated whether adipose-specific deletion of rictor and thereby inactivation of mTORC2 in PVAT may modulate vascular function by increasing inflammation in PVAT. Approach and Results— Rictor, an essential mTORC2 component, was deleted specifically in mouse adipose tissue (rictor ad−/− ). Phosphorylation of mTORC2 downstream target Akt at Serine 473 was reduced in PVAT from rictor ad−/− mice but unaffected in aortic tissue. Ex vivo functional analysis of thoracic aortae revealed increased contractions and impaired dilation in rings with PVAT from rictor ad−/− mice. Adipose rictor knockout increased gene expression and protein release of interleukin-6, macrophage inflammatory protein-1α, and tumor necrosis factor-α in PVAT as shown by quantitative real-time polymerase chain reaction and Bioplex analysis for the cytokines in the conditioned media, respectively. Moreover, gene and protein expression of inducible nitric oxide synthase was upregulated without affecting macrophage infiltration in PVAT from rictor ad−/− mice. Inhibition of inducible nitric oxide synthase normalized vascular reactivity in aortic rings from rictor ad−/− mice with no effect in rictor fl/fl mice. Interestingly, in perivascular and epididymal adipose depots, high-fat diet feeding induced downregulation of rictor gene expression. Conclusions— Here, we identify mTORC2 as a critical regulator of PVAT-directed protection of normal vascular tone. Modulation of mTORC2 activity in adipose tissue may be a potential therapeutic approach for inflammation-related vascular damage.

Obesity involves hypoxic adipose tissue and low-grade chronic inflammation. We investigated the impact of hypoxia on inflammatory response to TNF-in white and brown adipocytes. In response to TNF-, the expression of the inducible enzymes iNOS and COX-2 was prominently and selectively potentiated during hypoxia while only moderately under normoxia. Levels of their products, nitrite and prostaglandinE2 were elevated accordingly. NS398, a selective COX-2 inhibitor, reduced nitrite levels. The expression of PGC-1, a transcriptional co-activator involved in mitochondrial biogenesis, and PPAR, a transcription factor involved in adipocyte homeostasis, was reduced by TNF-during hypoxia. These results suggest that hypoxia potentiates the inflammatory response by TNF-in both white and brown adipocytes and downregulates the transcription factors involved in adipocyte function. AbstractObesity involves hypoxic adipose tissue and low-grade chronic inflammation. We investigated the impact of hypoxia on inflammatory response to TNF-α in white and brown adipocytes. In response to TNF-α, the expression of the inducible enzymes iNOS and COX-2 was prominently and selectively potentiated during hypoxia while only moderately under normoxia. Levels of their products, nitrite and prostaglandinE 2 were elevated accordingly. NS398, a selective COX-2 inhibitor, reduced nitrite levels.The expression of PGC-1α, a transcriptional co-activator involved in mitochondrial biogenesis, and PPARγ, a transcription factor involved in adipocyte homeostasis, was reduced by TNF-α during hypoxia. These results suggest that hypoxia potentiates the inflammatory response by TNF-α in both white and brown adipocytes and downregulates the transcription factors involved in adipocyte function.

Abstract-The mammalian target of rapamycin complex 2 (mTORC2) contains the essential protein RICTOR and is activated by growth factors. mTORC2 in adipose tissue contributes to the regulation of glucose and lipid metabolism. In the perivascular adipose tissue, mTORC2 ensures normal vascular reactivity by controlling expression of inflammatory molecules. To assess whether RICTOR/mTORC2 contributes to blood pressure regulation, we applied a radiotelemetry approach in control and Rictor knockout (Rictor aP2KO ) mice generated using adipocyte protein-2 gene promoter-driven CRE recombinase expression to delete Rictor. The 24-hour mean arterial pressure was increased in Rictor aP2KO mice, and the physiological decline in mean arterial pressure during the dark period was impaired. In parallel, heart rate and locomotor activity were elevated during the dark period with a pattern similar to blood pressure changes. This phenotype was associated with mild cardiomyocyte hypertrophy, decreased cardiac natriuretic peptides, and their receptor expression in adipocytes. Moreover, clock gene expression was reduced or phase-shifted in perivascular adipose tissue. No differences in clock gene expression were observed in the master clock suprachiasmatic nucleus, although Rictor gene expression was also lower in brain of Rictor aP2KO mice. Thus, this study highlights the importance of RICTOR/mTORC2 for interactions between vasculature, adipocytes, and brain to tune physiological outcomes, such as blood pressure and locomotor activity. (Hypertension. 2015;66:332-339.