Electroneutral K+/HCO3- cotransport in cells of medullary thick ascending limb of rat kidney.

The renal medullary thick ascending limb (MTAL) of the rat absorbs bicarbonate through luminal H+ secretion and basolateral HCO3- transport into the peritubular space. To characterize HCO3- transport, intracellular pH (pHi) was monitored by use of the pH-sensitive fluorescent probe (2',7')-bis-(carboxyethyl)-(5,6)-carboxyfluorescein in fresh suspensions of rat MTAL tubules. When cells were preincubated in HCO3-/CO2-containing solutions and then abruptly diluted into HCO3-/CO2-free media, the pHi response was an initial alkalinization due to CO2 efflux, followed by an acidification (pHi recovery). The pHi recovery required intracellular HCO3-, was inhibited by 10(-4) M diisothiocyanostilbene-2-2'-disulphonic acid (DIDS), and was not dependent on Cl- or Na+. As assessed by use of the cell membrane potential-sensitive fluorescent probe 3,3'-dipropylthiadicarbocyanine, cell depolarization by abrupt Cl- removal from or addition of 2 mM barium into the external medium did not affect HCO3(-)-dependent pHi recovery, and the latter was not associated per se with any change in potential difference, which indicated that HCO3- transport was electroneutral. The HCO3(-)-dependent pHi recovery was inhibited by raising extracellular potassium concentration and by intracellular potassium depletion. Finally, as measured by use of a K(+)-selective extracellular electrode, a component of K+ efflux out of the cells was HCO3- dependent and DIDS sensitive. The results provide evidence for an electroneutral K+/HCO3- cotransport in rat MTAL cells.     

Chondrogenesis by bone marrow‐derived mesenchymal stem cells grown in chondrocyte‐conditioned medium for auricular reconstruction

Bone marrow‐derived mesenchymal stem cells (BMSCs) can be obtained by minimally invasive means and would be a favourable source for cell‐based cartilage regeneration. However, controlling the differentiation of the BMSCs towards the desired chondrogenic pathway has been a challenge hampering their application. The major aim of the present study was to determine if conditioned medium collected from cultured auricular chondrocytes could promote chondrogenic differentiation of BMSCs. Auricular chondrocytes were isolated and grown in BMSC standard culture medium (SM) that was collected and used as chondrocyte‐conditioned medium (CCM). The BMSCs were expanded in either CCM or SM for three passages. Cells were seeded onto fibrous collagen scaffolds and precultured for 2 weeks with or without transforming growth factor‐beta 3 (TGF‐β3). After preculture, constructs were implanted subcutaneously in nude mice for 6 and 12 weeks and evaluated with real‐time polymerase chain reaction, histology, immunohistochemistry and biochemistry. Real‐time polymerase chain reaction results showed upregulation of COL2A1 in the constructs cultured in CCM compared with those in SM. After 12 weeks in vivo, abundant neocartilage formation was observed in the implants that had been cultured in CCM, with or without TGF‐β3. In contrast, very little cartilage matrix formation was observed within the SM groups, regardless of the presence of TGF‐β3. Osteogenesis was only observed in the SM group with TGF‐β3. In conclusion, CCM even had a stronger influence on chondrogenesis than the supplementation of the standard culture medium with TGF‐β3, without signs of endochondral ossification. Efficient chondrogenic differentiation of BMSCs could provide a promising alternative cell population for auricular regeneration. Copyright © 2016 John Wiley & Sons, Ltd.     

Acute metabolic acidosis enhances circulating parathyroid hormone, which contributes to the renal response against acidosis in the rat.

Acute PTH administration enhances final urine acidification in the rat. HCl was infused during 3 h in rats to determine the parathyroid and renal responses to acute metabolic acidosis. Serum immunoreactive PTH (iPTH) concentration significantly increased and nephrogenous adenosine 3H,5H-cyclic monophosphate tended to increase during HCl loading in intact and adrenalectomized (ADX) rats despite significant increments in plasma ionized calcium. Strong linear relationships existed between serum iPTH concentration and arterial bicarbonate or proton concentration (P less than 0.0001). Serum iPth concentration and NcAMP remained stable in intact time-control rats and decreased in CaCl2-infused, nonacidotic animals. Urinary acidification was markedly reduced in parathyroidectomized (PTX) as compared with intact rats during both basal and acidosis states; human PTH-(1-34) infusion in PTX rats restored in a dose-dependent manner the ability of the kidney to acidify the urine and excrete net acid. Acidosis-induced increase in urinary net acid excretion was observed in intact, PTX, and ADX, but not in ADX-thyroparathyroidectomized rats. We conclude that (a) acute metabolic acidosis enhances circulating PTH activity, and (b) PTH markedly contributes to the renal response against acute metabolic acidosis by enhancing urinary acidification.     

Excision of the imidazole ring-opened form of N-2-aminofluorene-C(8)-guanine adduct in poly(dG-dC) by Escherichia coli formamidopyrimidine-DNA glycosylase

A polynucleotide containing N-(deoxyguanosine-8-yl)-2-aminofluorene residues (dGuo-C8-AF) was obtained by treatment of poly(dG-dC) with [3H]ring-N-hydroxy-2-amino-fluorene. This substrate was further treated under alkaline conditions to convert dGuo-C8-AF residues into their imidazole ring-opened derivative or 1-[6-(2,5-diamino-4-oxo-pyrimidinyl-N-6-deoxyribose]-3-(2-fluorenyl++ +)urea (iro-dGuo-C8-AF). The ring-opening of 50% of the dGuo-C8-AF residues occurs in 24 h at 37 degrees C in the presence of 0.1 N NaOH. This modified polynucleotide was used as substrate for the homogeneous formamidopyrimidine-DNA glycosylase (Fapy-DNA glycosyase) of Escherichia coli. Analysis of the reaction products shows that Fapy-DNA glycosylase releases the imidazole ring-opened derivative (iro-G-C8-AF). In contrast the primary adduct (G-C8-AF) is not removed. These results show that the imidazole ring-opened form of guanine residue modified at the C8 position by a bulky adduct is a substrate for the formamidopyrimidine-DNA glycosylase of E. coli. These observations show that the formamidopyrimidine-DNA glycosylase has a broad substrate specificity including imidazole ring-opened purines either modified at N7 or C8 positions in DNA. Therefore, the Fapy-DNA glycosylase might be involved in the repair of minor lesions induced by many chemical carcinogens.     

Implant-assisted meniscal repair in vivo using a chondrocyte-seeded flexible PLGA scaffold

A cell-based engineered construct can be used for healing of intractable meniscal lesions. Our aims were to assess the culture conditions (static versus dynamic oscillation) and the healing capacity of the chondrocyte-seeded flexible implants in a heterotopic mouse model. Swine articular chondrocytes were labeled with PKH 26 or DiI dye and seeded onto a flexible PLGA scaffold using dynamic oscillating conditions for 24 h. Half of cell-seeded scaffolds were cultured in the same dynamic conditions, while the remaining scaffolds were cultured statically. After 7 days, scaffolds were placed between swine meniscal discs and were implanted subcutaneously in nude mice for 6 weeks. Additional constructs for assessing in vivo cell tracking were implanted for 12 weeks. Live/dead assays demonstrated labeled chondrocytes attached throughout the scaffold in both culture conditions. DNA measurements showed no significant difference between the culture conditions. A continuous fibro-cartilaginous healing tissue was observed between meniscal discs in all 12 dynamically cultured constructs and 9 of 11 statically cultured ones. There was no evidence of meniscal healing using acellular scaffold as well as in meniscal constructs lacking an implant. Both PKH 26- and DiI-labeled cells were identified along the healing interface. We conclude the chondrocyte-seeded flexible PLGA implants induce healing of meniscal discs in nude mice. Culture conditions after seeding have no apparent effects on healing.     

Z-DNA-forming sequences are spontaneous deletion hot spots.

Z-DNA-forming sequences are shown to elicit a biological response in Escherichia coli. Plasmids containing sequences capable of adopting the Z conformation (GC and CA/GT) are shown to be hot spots for spontaneous deletions. All the deletions involve an even number of base pairs. The distribution of the deletion events shows that the process ends when the Z-DNA-forming sequence has been reduced to a size no longer capable of adopting the Z conformation at natural superhelical density.