Development of the gateway recycling cloning system for multiple linking of expression cassettes in a defined order, and direction on gateway compatible binary vectors.

We developed the Gateway recycling cloning system, which allows multiple linking of expression cassettes by multiple rounds of the Gateway LR reaction. Employing this system, the recycling donor vector pRED419 was subjected to the first LR reaction with an attR1-attR2 type destination vector. Then conversion vector pCON was subjected to an LR reaction to restore the attR1-attR2 site on the destination vector for the next cloning cycle. By repetition of these two simple steps, we linked four expression cassettes of a reporter gene in Gateway binary vector pGWB1, introduced the constructs into tobacco BY-2 cells, and observed the expression of transgenes.

Attenuation of folic acid-induced renal inflammatory injury in platelet-activating factor receptor-deficient mice.

Platelet-activating factor (PAF), a potent lipid mediator with various biological activities, plays an important role in inflammation by recruiting leukocytes. In this study we used platelet-activating factor receptor (PAFR)-deficient mice to elucidate the role of PAF in inflammatory renal injury induced by folic acid administration. PAFR-deficient mice showed significant amelioration of renal dysfunction and pathological findings such as acute tubular damage with neutrophil infiltration, lipid peroxidation observed with antibody to 4-hydroxy-2-hexenal (day 2), and interstitial fibrosis with macrophage infiltration associated with expression of monocyte chemoattractant protein-1 and tumor necrosis factor-alpha in the kidney (day 14). Acute tubular damage was attenuated by neutrophil depletion using a monoclonal antibody (RB6-8C5), demonstrating the contribution of neutrophils to acute phase injury. Macrophage infiltration was also decreased when treatment with a PAF antagonist (WEB2086) was started after acute phase. In vitro chemotaxis assay using a Boyden chamber demonstrated that PAF exhibits a strong chemotactic activity for macrophages. These results indicate that PAF is involved in pathogenesis of folic acid-induced renal injury by activating neutrophils in acute phase and macrophages in chronic interstitial fibrosis. Inhibiting the PAF pathway might be therapeutic to kidney injury from inflammatory cells.

Pulse total-hemoglobinometer provides accurate noninvasive monitoring.

OBJECTIVE: Rapid noninvasive measurement of total hemoglobin would be extremely useful for various clinical situations. This study determined the clinical accuracy and utility for a pulse total-hemoglobinometer using four wavelengths: 660 nm (reduced hemoglobin), 805 nm (isosbestic point), 940 nm (oxygenated hemoglobin), and 1300 nm (water density). DESIGN: Clinical trial. SETTING: University school of medicine. PATIENTS: Patients were 122 individuals (age, 18-82 yrs; 49.4 +/- 16.0 yrs [mean +/- SD]), including 71 healthy volunteers, 24 patients undergoing surgery, and 27 patients undergoing hemodialysis. INTERVENTIONS: The hemoglobinometer probe, which simultaneously indicated peripheral oxygen saturation, pulse rate, and hemoglobin, was placed on the fingertip similarly to a regular pulse oximeter. The hemoglobin values were compared with those obtained by the co-oximeter or the sodium lauryl sulfate-methemoglobin method. Those hemoglobin values were assigned to either the training set or the validation set for statistical evaluation. MEASUREMENTS AND MAIN RESULTS: Multiple regression analysis including the ratio of the pulsatile optical density (phi(ij)) derived from the four wavelengths and other factors demonstrated that the mean value of the normalized pulse wave obtained from the photodiode at 805 nm (DC805) and the ratios of DC940 and DC1300 (DC940/DC1300) were the pivotal factors in the hemoglobinometer's increased accuracy in the clinically useful range. The coefficient of determination between both methods was r2 = .81 (p < .0001) in the training set and r2 = .75 (p < .0001) in the validation set. When the cutoff value of anemia was set at 10 g/dL, and anemia was defined as <10 g/dL, the respective sensitivity and specificity of hemoglobinometer values to detect anemia in intraoperative patients were 84.3% and 84.6% (n = 20). CONCLUSIONS: The data demonstrated the necessity for consideration of light scattering in red blood cells for pulse-spectrophotometric hemoglobin measurement. This was accomplished with additional factors, such as DC805 and DC940/DC1300. With these improvements, the pulse hemoglobinometer provided noninvasive, clinically acceptable measurement of hemoglobin. The pulse hemoglobinometer is a versatile tool that might be useful for routine health checkups of neonates and young children, intraoperative monitoring of bleeding, and emergency care.