Generation of Synthetic Shuttle Vectors Enabling Modular Genetic Engineering of Cyanobacteria.

Cyanobacteria have raised great interest in biotechnology due to their potential for a sustainable, photosynthesis-driven production of fuels and value-added chemicals. This has led to a concomitant development of molecular tools to engineer the metabolism of those organisms. In this regard, however, even cyanobacterial model strains lag behind compared to their heterotrophic counterparts. For instance, replicative shuttle vectors that allow gene transfer independent of recombination into host DNA are still scarce. Here, we introduce the pSOMA shuttle vector series comprising 10 synthetic plasmids for comprehensive genetic engineering of Synechocystis sp. PCC 6803. The series is based on the small endogenous plasmids pCA2.4 and pCB2.4, each combined with a replicon from Escherichia coli, different selection markers as well as features facilitating molecular cloning and the insulated introduction of gene expression cassettes. We made use of genes encoding green fluorescent protein (GFP) and a Baeyer-Villiger monooxygenase (BVMO) to demonstrate functional gene expression from the pSOMA plasmids in vivo. Moreover, we demonstrate the expression of distinct heterologous genes from individual plasmids maintained in the same strain and thereby confirmed compatibility between the two pSOMA subseries as well as with derivatives of the broad-host-range plasmid RSF1010. We also show that gene transfer into the filamentous model strain Anabaena sp. PCC 7120 is generally possible, which is encouraging to further explore the range of cyanobacterial host species that could be engineered via pSOMA plasmids. Altogether, the pSOMA shuttle vector series displays an attractive alternative to existing plasmid series and thus meets the current demand for the introduction of complex genetic setups and to perform extensive metabolic engineering of cyanobacteria.

Splenic pooling and loss of VCAM-1 causes an engraftment defect in patients with myelofibrosis after allogeneic hematopoietic stem cell transplantation.

Myelofibrosis is a myeloproliferative neoplasm that results in cytopenia, bone marrow fibrosis and extramedullary hematopoiesis. Allogeneic hematopoietic stem cell transplantation is the only curative treatment but is associated with a risk of delayed engraftment and graft failure. In this study, patients with myelofibrosis (n=31) and acute myeloid leukemia (n=31) were analyzed for time to engraftment, graft failure and engraftment-related factors. Early and late neutrophil engraftment and late thrombocyte engraftment were significantly delayed in patients with myelofibrosis as compared to acute myeloid leukemia, and graft failure only occurred in myelofibrosis (6%). Only spleen size had a significant influence on engraftment efficiency in myelofibrosis patients. To analyze the cause for the engraftment defect, clearance of hematopoietic stem cells from peripheral blood was measured and immunohistological staining of bone marrow sections was performed. Numbers of circulating CD34+ were significantly reduced at early time points in myelofibrosis patients, whereas CD34+CD38- and colony-forming cells showed no significant difference in clearance. Staining of bone marrow sections for homing proteins revealed a loss of VCAM-1 in myelofibrosis with a corresponding significant increase in the level of soluble VCAM-1 within the peripheral blood. In conclusion, our data suggest that reduced engraftment and graft failure in myelofibrosis patients is caused by an early pooling of CD34+ hematopoietic stem cells in the spleen and a bone marrow homing defect caused by the loss of VCAM-1. Improved engraftment in myelofibrosis might be achieved by approaches that reduce spleen size and cleavage of VCAM-1 in these patients prior to hematopoietic stem cell transplantation.

The influence of oral antidiabetic drugs on cellular drug uptake mediated by hepatic OATP family members.

As patients with type 2 diabetes receiving oral antidiabetic drugs are often concomitantly treated with other drugs, they are of increased risk for drug interactions. Drugs have to be taken up into hepatocytes before their intracellular drug action or before they are metabolized, and therefore, uptake transporters are important modulators of drug pharmacokinetics and drug effects. To gain more insights into the role of uptake transporters for drug interactions, we investigated whether frequently prescribed oral antidiabetic drugs interact with the transport of drugs, mediated by the hepatic uptake transporters OATP1B1 (gene symbol SLCO1B1), OATP1B3 (gene symbol SLCO1B3) and OATP2B1 (gene symbol SLCO2B1). Using HEK293 cells recombinantly over-expressing these uptake transporters, we analysed whether glibenclamide, glimepiride, nateglinide and pioglitazone influence the transport of the model transport substrate bromosulfophthalein. Furthermore, we investigated the influence of the same oral antidiabetic drugs and of repaglinide and rosiglitazone on the uptake of the HMG-CoA-reductase inhibitor atorvastatin. The oral antidiabetic drugs glibenclamide, glimepiride and nateglinide inhibited the transport of the model substrate bromosulfophthalein, particularly the OATP2B1-mediated uptake. The OATP-mediated atorvastatin uptake was inhibited in a similar manner. For glibenclamide, inhibitory constants (Ki values) of 13.6 µM, 8.1 µM and 0.5 µM for OATP1B1-, OATP1B3- and OATP2B1-mediated BSP uptake were determined. In conclusion, these in vitro results demonstrate that several oral antidiabetic drugs may influence hepatic OATP-mediated drug uptake. The in vivo consequences of these results have to be analysed in further studies.

Characterization of ursodeoxycholic and norursodeoxycholic acid as substrates of the hepatic uptake transporters OATP1B1, OATP1B3, OATP2B1 and NTCP.

Ursodeoxycholic acid (UDCA) is the only approved treatment for primary biliary cirrhosis, and norursodeoxycholic acid (norUDCA) is currently tested in clinical trials for future treatment of primary sclerosing cholangitis because of beneficial effects in cholestatic Mdr2 knock-out mice. Uptake of UDCA and norUDCA into hepatocytes is believed to be a prerequisite for subsequent metabolism and therapeutic action. However, the molecular determinants of hepatocellular uptake of UDCA and norUDCA are poorly understood. We therefore investigated whether UDCA and norUDCA are substrates of the hepatic uptake transporters OATP1B1, OATP1B3, OATP2B1 and Na(+) -taurocholate co-transporting polypeptide (NTCP), which are localized in the basolateral membrane of hepatocytes. Uptake of [(3) H]UDCA and [(14) C]norUDCA into Human embryonic kidney (HEK) cells stably expressing OATP1B1, OATP1B3, OATP2B1 or NTCP was investigated and compared with uptake into vector control cells. Uptake ratios were calculated by dividing uptake into transporter-transfected cells by uptake into respective control cells. Uptake ratios of OATP1B1-, OATP1B3- and OATP2B1-mediated UDCA and norUDCA uptake were at maximum 1.23 and 1.49, respectively. Uptake of UDCA was significantly higher into HEK-NTCP cells only at the lowest tested concentration (1 µM, p < 0.001) compared with the control cells with an uptake ratio of 1.34-fold. NorUDCA was not significantly transported by NTCP. The low uptake rates suggest that OATP1B1, OATP1B3, OATP2B1 and NTCP are not relevant for hepatocellular uptake and effects of UDCA and norUDCA in human beings.

Transporter-mediated drug-drug interactions with oral antidiabetic drugs.

Uptake transporters (e.g., members of the SLC superfamily of solute carriers) and export proteins (e.g., members of the ABC transporter superfamily) are important determinants for the pharmacokinetics of drugs. Alterations of drug transport due to concomitantly administered drugs that interfere with drug transport may alter the kinetics of drug substrates. In vitro and in vivo studies indicate that many drugs used for the treatment of metabolic disorders and cardiovascular diseases (e.g., oral antidiabetic drugs, statins) are substrates for uptake transporters and export proteins expressed in the intestine, the liver and the kidney. Since most patients with type 2 diabetes receive more than one drug, transporter-mediated drug-drug interactions are important molecular mechanisms leading to alterations in oral antidiabetic drug pharmacokinetics with the risk of adverse drug reactions. This review focuses on uptake transporters of the SLCO/SLC21 (OATP) and SLC22 (OCT/OAT) family of solute carriers and export pumps of the ABC (ATP-binding cassette) transporter superfamily (especially P-glycoprotein) as well as the export proteins of the SLC47 (MATE) family and their role for transporter-mediated drug-drug interactions with oral antidiabetic drugs.

Effects of inducible nitric oxide synthase inhibition or norepinephrine on the neurovascular coupling in an endotoxic rat shock model.

INTRODUCTION: The inducible nitric oxide synthase (iNOS) plays a crucial role in early sepsis-related microcirculatory dysfunction. Compared to a catecholamine therapy we tested effects of a specific iNOS-inhibitor (1400W) on the microcirculatory function in the brain. METHODS: Seventy SD-rats (280-310 g) were divided into 1 control and 6 sepsis groups. Sepsis groups received 1 or 5 mg/kg lipopolysaccharide (LPS) intravenously to induce a moderate or severe sepsis syndrome. Thirty minutes later rats were further randomized into subgroups receiving moderate volume therapy alone or additionally continuous norepinephrine (NE) or 1400W infusion. Separately, effects of 1400W on neurofunctional parameters were investigated in 3 rats without sepsis induction. Performing electric forepaw-stimulation evoked potentials (N2-P1 amplitude, P1-latency) and local hemodynamic responses were recorded with surface electrodes and laser Doppler over the somatosensory cortex at baseline and repeatedly after LPS administration. Cytokine levels (tumor necrosis factor-alpha (TNFalpha), interleukin-6 (IL6), interferon-gamma (IFNgamma)) and cell destruction markers (neuron-specific enolase (NSE), S-100 calcium binding protein B (S100B)) were obtained at the end of experiments. RESULTS: During sepsis progression resting cerebral blood flow increased and functionally activated hemodynamic responses decreased in a dose-dependent manner. Whereas 1400W and NE improved blood pressure, only 1400W stabilized resting flow levels. However, both regimens were ineffective on the functionally coupled flow responses and destruction markers were similar between groups. CONCLUSIONS: NE and 1400W appeared to be ineffective in mitigating the effects of sepsis on the neurovascular coupling. Other regimens are needed to protect the cerebral microcirculation under septic conditions.

Endotoxin-induced gene expression differences in the brain and effects of iNOS inhibition and norepinephrine.

PURPOSE: We studied gene expression differences in brain homogenate, hippocampus, somatosensory cortex and cerebellum of rats suffering from sepsis-associated delirium and analyzed the effects of norepinephrine and 1,400 W (specific inhibitor of the inducible nitric-oxide synthase). METHODS: We applied microarray screenings to rat brain homogenate 1, 3 and 4.5 h after lipopolysaccharide (LPS, 5 mg/kg) or 0.9% NaCl treatment. Therapy groups were analyzed after 4.5 h. Validations and compartment specific investigations were carried out by real-time PCR. RESULTS: Most striking gene expression differences were seen 4.5 h after LPS administration, especially within the hippocampus (chemokines and endothelial cell-specific molecule 1). Norepinephrine resulted in a discrete chemokine up-regulation, while 1,400 W had hardly any effect. CONCLUSION: Strongest gene regulations were found within the hippocampus. Norepinephrine showed a tendency of having a proinflammatory influence, while 1,400 W had no clear-cut effect onto the gene expression level.