No abstract

Hydroxyeicosatetraenoic acid (20-HETE) is a major cytochrome P-450-arachidonic acid metabolite in the rat kidney, and its synthesis along the nephron is specifically localized to the proximal tubule, where receptor density for epidermal growth factor (EGF) is the highest. EGF stimulated endogenous 20-HETE formation in a concentration and time-dependent manner, i.e., from 1.6 to 2.6 +/- 0.3 and 3.0 +/- 0.6 pmol 20-HETE.mg-1.min-1 at 10(-8) and 10(-7) M EGF, respectively. The effect of 20-HETE on proximal tubular cell proliferation was examined using primary cultures of rat proximal tubular cells and proximal tubular-derived cell lines, LLC-PK1 and opossum kidney OK. In both cell lines, 20-HETE increased thymidine incorporation into DNA with maximal effect at 10(-9) M. Addition of 20-HETE to serum-deprived LLC-PK1 or OK cells for 48 h caused a concentration-dependent increase in cell number with maximal effect at 10(-9) M. This effect was specific, as structurally similar eicosanoids such as 20-COOH-arachidonic acid, 19(R)-HETE, and 19(S)-HETE did not increase cell number. In 4-day primary cultures of proximal tubular cells, EGF (10(-9) M) and 20-HETE (10(-9) M) increased bromodeoxyuridine (BrdU) incorporation by 40 and 28%, respectively. Addition of both resulted in a twofold increase in BrdU incorporation. Although 20-HETE synthesis in cultured cells is greatly diminished with time, significant picomolar concentrations can be obtained in 4-day cultures. Addition of 17-octadecynoic acid (17-ODYA), an inhibitor of 20-HETE synthesis, significantly inhibited EGF-stimulated BrdU incorporation. The demonstrations that EGF stimulates proximal tubular 20-HETE production and that the latter is a potent mitogen to these cells suggests that 20-HETE may act as a mediator of the EGF effect on cellular growth in the proximal tubule.

No abstract

Obesity is involved in several cardiovascular diseases including coronary artery disease and endothelial dysfunction. Endothelial Endothelium vasodilator and vasoconstrictor agonists play a key role in regulation of vascular tone. In this study, we evaluated coronary vascular response in an 8 weeks diet-induced obese C57BL/6 mice model. Coronary perfusion pressure in response to acetylcholine in isolated hearts from obese mice showed increased vasoconstriction and reduced vasodilation responses compared with control mice. Vascular nitric oxide assessed in situ with DAF-2 DA showed diminished levels in coronary arteries from obese mice in both basal and acetylcholine-stimulated conditions. Also, released prostacyclin was decreased in heart perfusates from obese mice, along with plasma tetrahydrobiopterin level and endothelium nitric oxide synthase dimer/monomer ratio. Obesity increased thromboxane A2 synthesis and oxidative stress evaluated by superoxide and peroxynitrite levels, compared with control mice. Obese mice treated with apocynin, a NADPH oxidase inhibitor, reversed all parameters to normal levels. These results suggest that after 8 weeks on a high-fat diet, the increase in oxidative stress lead to imbalance in vasoactive substances and consequently to endothelial dysfunction in coronary arteries.

The polymerase chain reaction (PCR) is a sought-after nucleic acid amplification technique used in the detection of several diseases. However, one of the main limitations of this and other nucleic acid amplification assays is the complexity, size, maintenance, and cost of their operational instrumentation. This limits the use of PCR applications in settings that cannot afford the instruments but that may have access to basic electrical, electronic, and optical components and the expertise to build them. To provide a more accessible platform, we developed a low-cost, palm-size, and portable instrument to perform real-time PCR (qPCR). The thermocycler leverages a copper-sheathed power resistor and a computer fan, in tandem with basic electronic components controlled from a single-board computer. The instrument incorporates a 3D-printed chassis and a custom-made fluorescence optical setup based on a CMOS camera and a blue LED. Results are displayed in real-time on a tablet. We also fabricated simple acrylic microdevices consisting of four wells (2 μL in volume each) where PCR reactions take place. To test our instrument, we performed qPCR on a series of cDNA dilutions spanning 4 orders of magnitude, achieving similar limits of detection as those achieved by a benchtop thermocycler. We envision our instrument being utilized to enable routine monitoring and diagnosis of certain diseases in low-resource areas.