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Abstracts
IN VIVO VISUALISATION OF THE PARAMETERS OF NORMAL AND CANCER SKIN CELLS USING TWO-PHOTON MICROSCOPY
Darvin, M.E., Ulrich, M., Klemp, M., König, K., Meinke, M.C., and Lademann, J.
Charité - Universitätsmedizin Berlin, Department of Dermatology, Venerology and Allergology, Berlin, Germany
Basal cell carcinoma (BCC) is the most frequent type of skin cancer in humans. The standard procedure of BCC diagnostics is a tissue biopsy sampling with subsequent histological examination. This "gold standard" method is highly invasive and time-consuming. Application of quick, non-invasive methods is of high interest for BCC diagnostics. For example, two-photon microscopy (TPM) is a well suited optical method for in vivo diagnosis of BCC at high resolution.
In the present study the normal and BCC-affected skin of nine BCC patients were measured noninvasively with TPM prior to surgery. Autofluorescence was provided by TPM under the excitation at 760 nm, where the single cells are clearly visible. The main fluorescent contribution for excitation at 760 nm in the skin is due to NAD(P)H, which is involved in cell mitochondrial activity, molecules of melanin, as well as keratin, elastin and collagen. After
the surgery, a histological tissue analysis was additionally performed.
It was found that the nucleus diameter of BCC cells is significantly larger than that of normal cells (granular, spinous and basal cells). The nucleus-to-cytoplasm ratio of BCC cells was found to be significantly lower than that of basal and spinous normal cells. The density of normal cells was found to have increased significantly for granular to basal cell layers, whereas the density of BCC cells is almost equal to that of granular normal cells. It should be taken into consideration that BCC cells were counted in a wide depth range from 10 ^m to 200 ^m, where their diameter did not significantly change, while the normal granular, spinous and basal cells were counted on average at the depths of (21 ± 7) ^m, (39 ± 6) ^m and (56 ± 6) ^m, respectively.
REGULATION OF NA-(K)-CL COTRANSPORT BY SPAK AND OSR1 Delpire, E.
Department of Anesthesiology, Vanderbilt University School of Medicine, Nashville, Tennessee, USA
SPAK (Ste20-MST1-related proline alanine rich kinase), OSR1 (Oxidative stress kinase 1) and WNK (With No K-lysine) kinases modulate distal Na+ transport by affecting the activity of the thick ascending limb of Henle Na-K-2Cl cotransporter and the activity of the distal convoluted tubule Na-Cl cotransporter (NCC). WNK4 affects the cotransporters by regulating their trafficking to the plasma membrane and by phosphorylating and activating SPAK which then phosphorylates and activates the cotransporters. Here we demonstrate that in the presence of Cab39, WNK4 can also directly activate the NaCl cotransporters. We show that whenXenopus laevis oocytes are injected with Cab39a or Cab39p cRNA along with WNK4 and NKCC1, we observe a significant increase in the level of K+ influx. This increase is bumetanide-sensitive indicating that the flux is mediated by the cotransporter. We also show that the stimulation requires the catalytic activity of WNK4 and is not affected by overexpressing dominant negative SPAK. These
data suggest that WNK4 is able to phosphorylate and activate the cotransporter in a SPAK-independent manner. When we mutated a phenylalanine residue in a WNK4 domain that resembles the SPAK/OSR1 binding domain, we prevented the Cab39/WNK4 activation of the cotransporter. We further show that residues that are important to SPAK-Cab39 interaction are not involved in the WNK4-Cab39 interaction, indicating different modes of binding. Finally, we demonstrate that while the WNK4 PHAII mutation, E599K, does not affect the ability of WNK4 to activate SPAK, this mutation completely abrogates the activation of NKCC1 by WNK4. These data were reproduced using a chimeric transporter consisting of the N-terminal regulatory tail of NCC fused to NKCC1, indicating that NCC is similarly activated by the Cab39/WNK4 interaction. Our data therefore demonstrate that WNK4 can interact and activate the Na-(K)-Cl cotransporters in the absence of SPAK or OSR1.
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Бюллетень сибирской медицины, 2013, том 12, № 4, с. 24-68
