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microRNAs (miRNAs) are small regulatory RNAs that are important in development and disease (1-3) and therefore represent a potential new class of targets for therapeutic intervention (4). Despite recent progress [...]

A methodology to quantify the binding of proteins to target DNA molecules based on single-molecule detection and real-time counting of individual free and bound fluorescently tagged molecules flowing past a detection device is presented. The methodology was used for measuring DNA binding by fluorescently tagged domains of four distinct transcription factors and these proteins represent different structural classes, quaternary states and relative affinities.

Renal medullary cells are exposed to elevated and variable osmolarities and low oxygen tension. Despite the harsh environment, these cells are resistant to the effects of many harmful events. To test the hypothesis that this resistance is a consequence of these cells developing a stress tolerance phenotype to survive in this milieu, we created osmotically tolerant cells [hypertonic (HT) cells] by gradually adapting murine inner medullary collecting duct 3 cells to hyperosmotic medium containing NaC1 and urea. HT cells have a reduced DNA synthesis rate, with the majority of cells arrested in the [G.sub.0]/[G.sub.1] phase of the cell cycle, and show constitutive expression of heat shock protein 70 that is proportional to the degree of hyperosmolarity. Unlike acute hyperosmolarity, chronic hyperosmolarity failed to activate MAPKs. Moreover, HT cells acquired protein translational tolerance to further stress treatment, suggesting that HT ceils have an osmotolerant phenotype that is analogous to thermotolerance but is a permanent condition. In addition to osmotic shock, HT cells were more resistant to heat, [H.sub.2][O.sub.2], cyclosporin, and apoptotic inducers, compared with isotonic murine inner medullary duct 3 cells, but less resistant to amphotericin B and cadmium. HT cells demonstrate that in renal medullary cells, hyperosmotic stress activates biological processes that confer cross-tolerance to other stressful conditions. heat shock protein 70; nephrotoxins; thermotolerance; cell cycle; inner medullary collecting duct 3

Osp94 (also known as HSPA4L or HSPH3), a member of the Hsp110/Sse1 family of heat-shock proteins, has a longer C-terminus than found in Hsc70/Hsp70 family proteins, composed of the loop region with a partial substrate-binding domain (SBD) β (L), and the SBDα and the C-terminal extension (H), but the functions of these domains are poorly understood. Here, we found that Osp94 suppressed heat-induced aggregation of luciferase (Luc). Osp94-bound heat-inactivated Luc was reactivated in the presence of rabbit reticulocyte lysate (RRL) and/or a combination of Hsc70 and Hsp40 (also known as HSPA8 and DNAJB1, respectively). Targeted deletion mutagenesis revealed that the SBDβ and H domains of Osp94 are critical for protein disaggregation and RRL-mediated refolding. Reactivation of Hsp90-bound heat-inactivated Luc was abolished in the absence of RRL but compensated for by PA28α (also known as PSME1), a proteasome activator. Interestingly, the LH domain also reactivated heat-inactivated Luc, independently of PA28α. Biotin-tag cross-linking experiments indicated that the LH domain and PA28α interact with Luc bound by Hsp90 during refolding. A chimeric protein in which the H domain was exchanged for PA28α also mediated disaggregation and reactivation of heat-inactivated Luc. These results indicate that Osp94 acts as a holdase, and that the C-terminal region plays a PA28α-like role in the refolding of unfolded proteins. [ABSTRACT FROM AUTHOR]

Author(s): Shan Zhu [1]; Irina G. Stavrovskaya [2]; Martin Drozda [1]; Betty Y. S. Kim [1]; Victor Ona [1]; Mingwei Li [1]; Satinder Sarang [3]; Allen S. Liu [1]; Dean [...]

An experiment was conducted to determine the physiologic effects of hyperosmolar sodium chloride (NaCl) and urea, individually and in combination, on the resistance and response of murine inner medullary collecting duct (mIMCD3) cells. Hyperosmolar NaCl is believed to an activator of mitogen-induced protein kinases while urea stimulates the production of protein kinase C. Results showed that the combination of NaCl and urea was linked with improved resistance and survival of mIMCD3 cells.

A study was undertaken to examine the effects of physiological stresses such as hyperosmolality and heat stress on the mRNA expression of the Hsp110/SSE subfamily of heat and osmotic stress proteins in the murine renal medulla. Hsp/110 stress proteins were also associated with the Hsp70/BiP gene superfamily. Results revealed that Osp94, Hsp70 and Hsp110 were stimulated by hypertonic sodium chloride and heat shock. These stress proteins were discovered in all tissues used in the study. Hypertonicity, rather than hyperosmolality, increased expression of the stress proteins.

Researchers have sought to establish whether hyperosmotic stress leads to expression of molecular chaperones in mouse inner medullary collecting duct (mIMCD3) cells. This cell line is isolated from the terminal IMCD of an SV40 transgenic mouse. It was possible to use a denatured protein (casein)-affinity column with adenosine 5'-triphosphate (ADP) elution to identify several possible molecular chaperones which are upregulated in response to four hours of hyperosmotic NaCl treatment. It is possible that molecular chaperones could be targets for phosphorylation by mitogen-activated protein (MAP) kinase or a downstream effector kinase.

The basolateral Na+-K+-Cl- cotransporter plays a dominant role in secretion of HCl by amphibian gastric mucosa. Northern analysis shows that gastric mucosa express greater than fivefold higher levels of mRNA encoding the basolateral cotransporter than any other tissue in the gastrointestinal tract.

Polymerase chain reaction and mRNA obtained from renal cortex help isolate a renal osmotic stress-induced Na+-Cl(-)-organic solute cotransporter, ROSIT. Amino acid analysis reveals the relation of ROSIT with two recently cloned orphans of the same gene family. Northern analysis reveals the expression of mRNA in renal cortex and outer medulla revealing the involvement of ROSIT in reabsorption of organic solutes by renal proximal tubule. Electrophysiological and labeling studies reveal the absence of involvement of ROSIT in the transport of neurotransmitter amino acids or other organic osmolytes.

Cell volume and K ion influx leak mechanisms decreased during the differentiation of mouse erythroleukamia (MEL) cells from proerythroblasts to young reticulocytes. The Na-K-2Cl cotransporter leads to a fall in cellular K ion levels, which in turn contributes to cell volume reduction. Since water content is not reduced, cell volume reduction is attributed to overall cellular material loss.

The evolution of a murine cell line (mIMCD-3) acquired from separate tubules of the end terminal of the inner medullary collecting duct (IMCD) of mice transgenic for the early area of simian virus SV40 (large T antigen) was described. Functional and antigenic features which are peculiar to the IMCD in vivo, were noted in the cell line mIMCD-3, indicating that various characteristics of this nephron have been retained by these cells.

The effect of urea on immediate-early gene transcription (IEG) factors Egr-1 and c-fos were studied in confluent growth-suppressed Madin-Darby canine kidney (MDCK) cells. A significant time and dose-dependent increase in Egr-1 and c-fos messenger RNA levels were observed in the MDCK cells despite the absence of cytotoxic stimuli and protein synthesis. This demonstrates the ability of hyperosmotic urea to induce an increase in IEG transcription factor expression which is assumed to be specific to epithelial cells.

The effect of urea on deoxyribonucleic acid (DNA) synthesis was studied in confluent growth-suppressed Madin-Darby canine kidney (MDCK) cells. A significant increase in (3H)thymidine incorporation was observed in the MDCK and renal epithelial LLC-PK1 cells. Urea also suppressed sodium chloride-induced inhibition of DNA synthesis. stimuli and protein synthesis. This demonstrates the ability of hyperosmotic urea to induce an increase DNA synthesis despite the absence of cellular proliferation.

The Gitelman's and Bartter's syndromes are disorders resulting from mutations in the sodium chloride cotransporters. These mutations lead to the loss of function of the cotransporters that mediate specific pathways in the kidneys. Using molecular cloning techniques, it has been shown that mutations in the gene coding for the thiazide-sensitive Na-Cl cotransporter causes Gitelman's syndrome, while mutations in the diuretic-sensitive Na-K-2Cl cotransporter gene causes Bartter's syndrome.