The chloride dependence of the human organic anion transporter 1 (hOAT1) is blunted by mutation of a single amino acid

Organic anion transporter 1 (OAT1) is key for the secretion of organic anions in renal proximal tubules. These organic anions comprise endogenous as well as exogenous compounds including frequently used drugs of various chemical structures. The molecular basis for the polyspecificity of OAT1 is not known. Here we mutated a conserved positively charged arginine residue (Arg(466)) in the 11(th) transmembrane helix of human OAT1. The replacement by the positively charged lysine (R466K) did not impair expression of hOAT1 at the plasma membrane of Xenopus laevis oocytes but decreased the transport of p-aminohippurate (PAH) considerably. Extracellular glutarate inhibited and intracellular glutarate trans-stimulated wild type and mutated OAT1, suggesting for the mutant R466K an unimpaired interaction with dicarboxylates. However, when Arg(466) was replaced by the negatively charged aspartate (R466D), glutarate no longer interacted with the mutant. PAH uptake by wild type hOAT1 was stimulated in the presence of chloride, whereas the R466K mutant was chloride-insensitive. Likewise, the uptake of labeled glutarate or ochratoxin A was chloride-dependent in the wild type but not in R466K. Kinetic experiments revealed that chloride did not alter the apparent K(m) for PAH but influenced V(max) in wild type OAT1-expressing oocytes. In R466K mutants the apparent K(m) for PAH was similar to that of the wild type, but V(max) was not changed by chloride removal. We conclude that Arg(466) influences the binding of glutarate, but not interaction with PAH, and interacts with chloride, which is a major determinant in substrate translocation.     

Rheological and flow properties of blood investigated by ultrasound

Ultrasonic waves of 1-15 MHz frequencies easily propagate through soft biological tissues, thus providing qualitative and quantitative information on mechanical and flow properties of blood and red blood cell (RBC) suspensions. Two types of techniques allow to investigate blood behaviors: echographic devices via amplitude detection and Doppler effect based devices via frequency detection of the ultrasonic signal. When ever B mode serves to construct images of tissue slabs from the ultrasonic backscattering coefficient and can give qualitative information on the mechanical properties of blood, A-mode allows to quantify the ultrasonic backscattering coefficient. Ultrasonic Doppler modes also provide both qualitative and quantitative information on blood flow velocity: continuous and pulsed Doppler modes provide curves of blood flow versus time when color Doppler and power Doppler imaging visualize blood flowing in human vessels. Association of echographic and Doppler modes to investigate simultaneously structure and velocity of blood is commercially available. Some examples of results given by such ultrasonic techniques that contribute to characterize, both in vitro and in vivo, structure and flow properties of blood or red blood cell (RBC) suspensions are presented.     

beta1-Adrenoreceptor activation contributes to ischemia-reperfusion damage as well as playing a role in ischemic preconditioning

Protein kinase A (PKA) activation has been implicated in early-phase ischemic preconditioning. We recently found that during ischemia PKA activation causes inactivation of cytochrome-c oxidase (CcO) and contributes to myocardial damage due to ischemia-reperfusion. It may be that beta-adrenergic stimulation during ischemia via endogenous catecholamine release activates PKA. Thus beta-adrenergic stimulation may mediate both myocardial protection and damage during ischemia. The present studies were designed to determine the role of the beta(1)-adrenergic receptor (beta(1)-AR) in myocardial ischemic damage and ischemic preconditioning. Langendorff-perfused rabbit hearts underwent 30-min ischemia by anterior coronary artery ligation followed by 2-h reperfusion. Occlusion-reperfusion damage was evaluated by delineating the nonperfused volume of myocardium at risk and volume of myocardial necrosis after 2-h reperfusion. In some hearts ischemic preconditioning was accomplished by two 5-min episodes of global low-flow ischemia separated by 10 min before coronary occlusion-reperfusion. Orthogonal electrocardiograms were recorded, and coronary flow was monitored by a drip count. Three hearts from each experimental group were used to determine mitochondrial CcO and aconitase activities. Two-hour reperfusion after occlusion caused an additional decrease in CcO activity vs. that after 30-min occlusion alone. Blocking the beta(1)-AR during occlusion-reperfusion reversed CcO activity depression and preserved myocardium at risk for necrosis. Similarly, mitochondrial aconitase activity exhibited a parallel response after occlusion-reperfusion as well as for the other interventions. Furthermore, classic ischemic preconditioning had no effect on CcO depression. However, blocking the beta(1)-AR during preconditioning eliminated the cardioprotection. If the beta(1)-AR was blocked after preconditioning, the myocardium was preserved. Interestingly, in both of the latter cases the depression in CcO activity was reversed. Thus the beta(1)-AR plays a dual role in myocardial ischemic damage. Our findings may lead to therapeutic strategies for preserving myocardium at risk for infarction, especially in coronary reperfusion intervention.     

Nonviral vector-based gene transfection of primary human skeletal myoblasts

Low-level transgene efficiency is one of the main obstacles in ex vivo nonviral vector-mediated gene transfer into primary human skeletal myoblasts (hSkMs). We optimized the cholesterol:N-[1-(2, 3-dioleoyloxy)propyl]-N, N, N-trimethylammonium methylsulfate liposome (CD liposome) and 22-kDa polyethylenimine (PEI22)- and 25-kDa polyethylenimine (PEI25)-mediated transfection of primary hSkMs for angiogenic gene delivery. We found that transfection efficiency and cell viability of three nonviral vectors were cell passage dependent: early cell passages of hSkMs had higher transfection efficiencies with poor cell viabilities, whereas later cell passages of hSkMs had lower transfection efficiencies with better cell viabilities. Trypsinization improved the transfection efficiency by 20% to 60% compared with adherent hSkMs. Optimum gene transfection efficiency was found with passage 6 trypsinized hSkMs: transfection efficiency with CD lipoplexes was 6.99 +/- 0.13%, PEI22 polyplexes was 18.58 +/- 1.57%, and PEI25 polyplexes was 13.32 +/- 0.88%. When pEGFP (a plasmid encoding the enhanced green fluorescent protein) was replaced with a vector containing human vascular endothelial growth factor 165 (phVEGF(165)), the optimized gene transfection conditions resulted in hVEGF(165) expression up to Day 18 with a peak level at Day 2 after transfection. This study demonstrated that therapeutic angiogenic gene transfer through CD or PEI is feasible and safe after optimization. It could be a potential strategy for treatment of ischemic disease for angiomyogenesis.     

Role of Organic Amendments to Mitigate Cd Toxicity and Its Assimilation in <i>Triticum aestivum</i> L.

In soil biota, higher and enduring concentration of heavy metals like cadmium (Cd) is hazardous and associated with great loss in growth, yield, and quality parameters of most of the crop plants. Recently, in-situ applications of eco-friendly stabilizing agents in the form of organic modifications have been utilized to mitigate the adverse effects of Cd-toxicity. This controlled experiment was laid down to appraise the imprints of various applied organic amendments namely poultry manure (PM), farmyard manure (FYM), and sugarcane press mud (PS) to immobilize Cd in polluted soil. Moreover, phytoavailability of Cd in wheat was also accessed under an alkaline environment. Results revealed that the addition of FYM (5–10 ton ha^-1 ) in Cd-contaminated soil significantly increased germination rate, leaf chlorophyll content, plant height, spike length, biological and grain yield amongst all applied organic amendments. Moreover, the addition of FYM (5–10 ton ha^-1 ) also reduced the phytoavailability of Cd by 73–85% in the roots, 57–83% in the shoots, and 81–90% in grains of wheat crop. Thus, it is affirmed that incorporation of FYM (5–10 ton ha^-1 ) performed better to enhance wheat growth and yield by remediating Cd. Thus, the application of FYM (5–10 ton ha^-1 ) reduced the toxicity induced by Cd to plants by declining its uptake and translocation as compared to all other applied organic amendments to immobilize Cd under sandy alkaline polluted soil.     

Role of the Cytosolic Heat Shock Protein 70 Ssa5 in the Ciliate Protozoan Tetrahymena thermophila

Heat shock protein 70 (Hsp70) is a member of a family of conserved chaperone proteins whose function is well investigated in many model organisms. Here we focus on an Hsp70 called Ssa5 in the ciliate protozoan Tetrahymena thermophila, and reveal that its translation is heat inducible as for general Hsps. Moreover, the protein is abundantly expressed in the cytoplasm during sexual reproduction (conjugation) as well as in response to heat‐stress. Knocking out of SSA5 (ΔSSA5) does not affect the survival of the cell under heat‐stress, likely due to other Hsp70 paralogs compensating for the defect. During conjugation, ΔSSA5 leads to a fertilization defect in which the two pronuclei are in close proximity but never fuse. The unfertilized pronuclei differentiate, resulting in a heterokaryon with developed haploid germline and somatic nuclei. In addition, degeneration of the parental somatic nucleus is not affected. These results suggest a specific involvement of Ssa5 in pronuclear fusion and fertilization.     

Simultaneous removal of inorganic nutrients and organic carbon by symbiotic co-culture of <Emphasis Type="Italic">Chlorella vulgaris</Emphasis> and <Emphasis Type="Italic">Pseudomonas putida</Emphasis>

The co-culture system of photosynthetic microalgae Chlorella vulgaris and aerobic heterotrophic bacteria Pseudomonas putida was investigated as a possible combination of symbiotic mixed culture for the simultaneous removal of nutrients (ammonium and phosphate) and organic contaminants. Using synthetic municipal wastewater, the co-culture system exhibited symbiotic enhancement in the removal of nutrients and organic carbon compared to each of axenic cultures. The co-culture system performed successfully in removing both of ammonium and chemical oxygen demand (COD), showing around 80% removal for 4 days. Strategies of nitrogen and phosphorous starvation in C. vulgaris for two days prior to main treatment did not increase the performance of nutrients removal, indicating that the nutrient starvation as a pretreatment is unnecessary. Without alkalinity (as bicarbonate), nutrients and COD were not removed significantly, implying that the existence of alkalinity is essential for symbiotic treatment of both nutrients and organics. Results demonstrated that coculture system composed of C. vulgaris and P. putida can be a potential candidate of mixed culture system for the simultaneous removal of nutrients and organic carbon in wastewater treatment using a single reactor.