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The role of the renal apical brush-border membrane (BBM) endocytic receptors cubilin and megalin in the onset of albuminuria in rats exposed to a single dose of total body irradiation (TBI) has been investigated. Albuminuria was evident as immunoblot (IB) analysis of the urine samples from TBI rats revealed excretion of large amounts of albumin. IB analysis of the BBM proteins did not reveal any significant changes in cubilin or megalin levels, but [sup.125]I-albumin binding to BBM from TBI rats declined by 80% with a fivefold decrease (from 0.5 to 2.5 [micro] M) in the affinity for albumin. IB analysis of cubilin from the BBM demonstrated a 75% loss when purified using albumin, but not intrinsic factor (IF)-cobalamin (Cbl) ligand affinity chromatography. Immunoprecipitation (IP) of Triton X-100 extract of the BBM with antiserum to cubilin followed by IB of the immune complex with an antiserum to megalin revealed a 75% loss of association between megalin and cubilin. IP studies with antiserum to cubilin or megalin and IB with antiserum to the cation-independent mannose 6-phosphate/insulin-like growth factor II-receptor (CIMPR) revealed that CIMPR interacted with both cubilin and megalin. In addition, TBI did not disrupt the association of CIMPR with either cubilin or megalin in BBM. These results suggest that albuminuria noted in TBI rats is due to selective loss of albumin and megalin, but not CIMPR or IF-Cbl binding by cubilin. Furthermore, these results also suggest that albumin and IF-Cbl binding to cubilin occur at distinct sites and that in the rat renal BBM, CIMPR interacts with both cubilin and megalin. endocytic receptors; endocytosis

Obesity, a major risk factor for the development of osteoarthritis (OA), is associated with increased circulating levels of free fatty acids (FFA). However, the role of these FFAs in OA pathophysiology is not clearly understood. In the present study, we found that palmitate treatment of human primary articular chondrocytes increased the expression of ER stress markers [activating transcription factor 4 (ATF4), C/EBP homologous protein (CHOP)] and apoptosis markers [cytochrome c and cleaved caspase-3 (CC3)]. Palmitate treatment also increased the expression of Nuclear protein 1 (Nupr1) and tribbles related protein 3 (TRB3), which are known negative regulators of cell survival pathways. Knockdown of Nupr1 or CHOP expression inhibited palmitate mediated increased expression of TRB3 and CC3, indicating that Nupr1 and CHOP cooperate to regulate cell survival and apoptotic pathways in human chondrocytes. Nupr1 knockdown had no effect on CHOP expression whereas CHOP knockdown abolished the palmitate-mediated Nupr1 expression, indicating that CHOP is functional upstream to Nupr1 in this pathway. Moreover, overexpression of Nupr1 markedly increased the basal expression of pro-apoptotic molecules, including cytochrome c and CC3. Taken together, our study demonstrates that Nupr1 plays a crucial role in palmitate-induced apoptosis in human chondrocytes and Nupr1 as a potential novel drug target for the treatment of OA. [ABSTRACT FROM AUTHOR]

Presents a study which examined the effect of thyroidectomy procedure in cubilin and megalin expression and their interaction in the rat intestine. Materials and methods; Results; Discussion of the results.

Investigates the effect of thyroidectomy on the expression of cubilin receptors in the intestine of rats. Reduction of cobalamin protein level; Impact of thyroxin treatment; Interaction with the protein megalin.

Neural precursor cell‐expressed developmentally downregulated gene 4 (NEDD4) is an E3 ubiquitin ligase that recognizes substrates via protein–protein interactions and takes part in tumor development. This study aims to clarify NEDD4's functions in diffuse large B‐cell lymphoma (DLBCL) and its downstream mechanisms. Collection of 53 DLBCL tissues and adjacent normal lymphoid tissues, and detection of NEDD4 and Forkhead box protein A1 (FOXA1) in the tissues were conducted. The selection of DLBCL cells was for FARAGE, and test of cells' advancement was after transfection. Analysis of NEDD4 and FOXA1's link, and test of Wnt/β‐catenin pathway were implemented. In vivo tumor xenograft experiments were put into effect. Detection of the pathological conditions of tumor tissues and the positive Ki67 in the family was implemented. It came out NEDD4 was reduced in DLBCL tissues and cell lines, and FOXA1 was elevated; Enhancing NEDD4 or repressing FOXA1 refrained DLBCL cells' advancement; NEDD4 could combine with FOXA1 and trigger its ubiquitination and degradation; NEDD4 inactivates the Wnt/β‐catenin pathway by motivating FOXA1 ubiquitination; NEDD4 enhancement refrained DLBCL growth in vivo. In conclusion, the E3 ubiquitin ligase NEDD4 accelerates FOXA1 ubiquitination but refrains DLBCL cell proliferation via the Wnt/β‐Catenin pathway. Highlights: Reduced NEDD4 but elevated FOXA1 are presented in DLBCL tissues and cell lines;Elevating NEDD4 or depressing FOXA1 refrains DLBCL cells' advancement;NEDD4 can combine with FOXA1 and trigger its ubiquitination and degradation;NEDD4 inactivates the Wnt/β‐catenin pathway by accelerating FOXA1 ubiquitination;Elevation of NEDD4 restrains DLBCL's growth in vivo. [ABSTRACT FROM AUTHOR]

Selected residues in a highly conserved 15-residue region, 174SVDTAAMAGLAFTC L188 of human transcobalamin (TC), a cobalamin (Cbl: vitamin B12) binding protein, were subjected to site-directed mutagenesis. The mutant constructs were expressed in TC-deficient fibroblasts or in vitro to assess the effect of these mutations on Cbl binding. Phylogenetic analyses and protein parsimony indicated that TC evolved earlier than other mammalian Cbl-binding proteins, intrinsic factor and haptocorrins, and divergence occurred between mouse/rat and human dispersing TC gene to different chromosomes. These studies show that (a) two of the three polar residues, S174, T177, or D176 and two of the three conserved alanine residues, A179 and A184 present in the 15-residue evolutionary conserved region are essential for Cbl-binding by human TC, and (b) TC gene is transferred in a syntenic manner to different chromosomes, at least before the divergence of mouse/rat and human. [Copyright &y& Elsevier]

Reactive oxygen species (ROS), in particular H2O2, regulate intracellular signaling through reversible oxidation of reactive protein thiols present in a number of kinases and phosphatases. H2O2 has been shown to regulate mitogen-activated protein kinase (MAPK) signaling depending on the cellular context.We report here that in human articular chondrocytes, the MAPK family member c-Jun N-terminal kinase 2 (JNK2) is activated by fibronectin fragments and low physiological levels of H2O2 and inhibited by oxidation due to elevated levels of H2O2. The kinase activity of affinity-purified, phosphorylated JNK2 from cultured chondrocytes was reversibly inhibited by 5-20 µM H2O2. Using dimedone-based chemical probes that react specifically with sulfenylated cysteines (RSOH), we identified Cys-222 in JNK2, a residue not conserved in JNK1 or JNK3, as a redox-reactive site. MS analysis of human recombinant JNK2 also detected further oxidation at Cys-222 and other cysteines to sulfinic (RSO2H) or sulfonic (RSO3H) acid. H2O2 treatment of JNK2 resulted in detectable levels of peptides containing intramolecular disulfides between Cys-222 and either Cys-213 or Cys-177, without evidence of dimer formation. Substitution of Cys-222 to alanine rendered JNK2 insensitive to H2O2 inhibition, unlike C177A and C213A variants. Two other JNK2 variants, C116A and C163A, were also resistant to oxidative inhibition. Cumulatively, these findings indicate differential regulation of JNK2 signaling dependent on H2O2 levels and point to key cysteine residues regulating JNK2 activity. As levels of intracellular H2O2 rise, a switch occurs from activation to inhibition of JNK2 activity, linking JNK2 regulation to the redox status of the cell. [ABSTRACT FROM AUTHOR]

People whom the author would like to thank for their assistance in the creation of the journal includes Khaled Abass, David H. Abbott, and Naohito Abe.

Background: We demonstrated growing evidence supports a protective role of chlorogenic acid of rat hepatocytes elicited by two compounds, i.e. thapsigargin and palmitic acid. Nevertheless, little is known about the mechanisms of palmitic acid induced endoplasmic reticulum (ER) stress and cell death. Methods: The proliferation of primary rat hepatocytes was detected by MTT assay. The expression of GRP78, CHOP and GRP94 was detected by Western blot analyses. Caspase-3 activity was detected by a Caspase-3 substrate kit. Cell apoptosis was detected by Hoechst 33342 staining. Results: We demonstrated that incubation of hepatocytes for 16 h with palmitic acid elevated cell death. Moreover, Western blot analyses demonstrated increased levels of the endoplasmic reticulum stress markers — glucose regulated protein 78 (GRP78), C/EBP homologous protein (CHOP), and glucose regulated protein 94 (GRP94). Chlorogenic acid could inhibit ER stress induced cell death and levels of indicators of ER stress caused by palmitic acid. The effect of thapsigargin, which evokes ER stress were reversed by chlorogenic acid. Conclusions: Altogether, our data indicate that in primary rat hepatocytes, chlorogenic acid prevents ER stress-mediated apoptosis of palmitic acid. [ABSTRACT FROM AUTHOR]