Thymoquinone protects renal tubular cells against tubular injury

In this work the effect of angiotensin II (AT II) on proximal tubular epithelial cells (pTECs) in vitro was studied. AT II was found to activate the nuclear factor kappaB (NF-kappaB) and its controlled genes, for example, interleukin 6 (IL-6) of pTECs in a time-dependent manner. Two points with maximum NF-kappaB activation were found, the first after 12 h and the second after 3.5 days. The first point may be due to activation of NF-kappaB in pTECs in response to AT II while the second may be due to activation of the advanced glycation end product (AGE)/receptor of the AGE (RAGE) system. Thymoquinone (TQ) was found to decrease NF-kappaB activation in a dose-dependant manner with maximum inhibitory effect at a concentration of 500 nM. Also, pre-incubation of pTECs with TQ leads to disappearance of the second peak of NF-kappaB. These data are consistent with results obtained from IL-6 enzyme-linked immunosorbent assay (ELISA) and transient transfection experiments. The results explain the therapeutic value of TQ which can be used to delay end stage renal diseases in diabetics.     

Ca2+ responses to thyrotropin-releasing hormone and angiotensin II: the role of plasma membrane integrity and effect of G11alpha protein overexpression on homologous and heterologous desensitization

The molecular mechanisms involved in GPCR-initiated signaling cascades where the two receptors share the same signaling cascade, such as thyrotropin-releasing hormone (TRH) and angiotensin II (ANG II), are still far from being understood. Here, we analyzed hormone-induced Ca(2+) responses and the process of desensitization in HEK-293 cells, which express endogenous ANG II receptors. These cells were transfected to express exogenously high levels of TRH receptors (clone E2) or both TRH receptors and G(11)alpha protein (clone E2M11). We observed that the characteristics of the Ca(2+) response, as well as the process of desensitization, were both strongly dependent on receptor number and G(11)alpha protein level. Whereas treatment of E2 cells with TRH or ANG II led to significant desensitization of the Ca(2+) response to subsequent addition of either hormone, the response was not desensitized in E2M11 cells expressing high levels of G(11)alpha. In addition, stimulation of both cell lines with THR elicited a clear heterologous desensitization to subsequent stimulation with ANG II. On the other hand, ANG II did not affect a subsequent response to TRH. ANG II-mediated signal transduction was strongly dependent on plasma membrane integrity modified by cholesterol depletion, but signaling through TRH receptors was altered only slightly under these conditions. It may be concluded that the level of expression of G-protein-coupled receptors and their cognate G-proteins strongly influences not only the magnitude of the Ca(2+) response but also the process of desensitization and resistance to subsequent hormone addition.     

Collagen fibrillogenesis in tendon development: current models and regulation of fibril assembly

Tendons are collagen-based fibrous tissues that connect and transmit forces from muscle to bone. These tissues, which are high in collagen type I content, have been studied extensively to understand collagen fibrillogenesis. Although the mechanisms have not been fully elucidated, our understanding has continued to progress. Here, we review two prevailing models of collagen fibrillogenesis and discuss the regulation of the process by candidate cellular and extracellular matrix molecules. Although numerous molecules have been implicated in the regulation of collagen fibrillogenesis, we focus on those that have been suggested to be particularly relevant to collagen type I fibril formation during tendon development, including members of the collagen and small leucine-rich proteoglycan families, as well as other molecules, including scleraxis, cartilage oligomeric matrix protein, and cytoskeletal proteins.     

Adenoviral transduction is more efficient in alginate-derived chondrocytes than in monolayer chondrocytes

Gene transfer into cultured chondrocytes by using adenoviral vectors has potential applications in treating cartilage disorders. The present study was undertaken to compare and optimize two chondrocyte culture conditions for adenoviral transduction efficacy by using primary human articular chondrocytes cultivated either directly in a monolayer condition or as outgrowths from alginate-stored chondrocyte cultures. Isolated primary chondrocytes from human articular cartilage were either immediately transduced with an EGFP (enhanced green fluorescent protein)-gene-bearing adenoviral vector (1,000 and 3,000 virus particles/cell) or cultured in alginate before transduction. Immunohistochemistry and flow cytometric analysis were employed to determine the expression of extracellular matrix proteins and of the αvβ5 integrin receptor involved in adenoviral cell entry. Monolayer chondrocytes exhibited moderate transduction rates (mean 22.2% and 46.9% EGFP-positive cells at 1,000 and 3,000 virus particles/cell by 72 h post-transduction), whereas alginate-derived chondrocytes revealed significantly higher transduction efficacies (95.7% and 99%). Both monolayer and alginate-derived chondrocytes expressed αvβ5 integrin, type II collagen and cartilage proteoglycans. The mean fluorescence intensity of type II collagen was significantly higher in the alginate-derived chondrocytes, whereas that of αvβ5 integrin was higher in the monolayer chondrocytes. Our results indicate that transduction efficacy is independent of αvβ5 integrin expression levels in chondrocytes. Moreover, adenoviral transduction of alginate-derived chondrocytes is more efficient than that for monolayer chondrocytes and may be a suitable tool to achieve sufficient numbers of transduced and differentiated chondrocytes for experimental applications and cartilage repair. Dr. Gundula Schulze-Tanzil is supported by a grant awarded by the Rahel Hirsh Foundation from the Charité Medical Schools Berlin. The study was supported by a grant from the Deutsche Arthrosehilfe e.V.     

Development of sequential multiple comparison procedure for dose response test

We propose a multiple comparison procedure to identify the minimum effective dose level by sequentially comparing each dose level with the zero dose level in the dose finding test. If we can find the minimum effective dose level at an early stage in the sequential test, it is possible to terminate the procedure in the dose finding test after a few group observations up to the dose level. Thus, the procedure is viable from an economical point of view when high costs are involved in obtaining the observations. In the procedure, we present an integral formula to determine the critical values for satisfying a predefined type I familywise error rate. Furthermore, we show how to determine the required sample size in order to guarantee the power of the test in the procedure. In practice, we compare the power of the test and the required sample size for various configurations of the population means in simulation studies and adopt our sequential procedure to the dose response test in a case study.     

Immunization of Rhesus monkeys with a SialylTn–mAb17-1A conjugate vaccine co-formulated with QS-21 induces a temporary systemic cytokine release and NK cytotoxicity against tumor cells

Tumor-associated antigens resulting from aberrant glycosylation, such as the SialylTn carbohydrate antigen, are frequently over-expressed on cancer cells and provide potential targets for cancer vaccination. Immunization of Rhesus monkeys with SialylTn coupled to a highly immunogenic carrier molecule and formulated on aluminum hydroxide induced a strong immune response against the carrier protein but only a moderate IgM immune response against the SialylTn carbohydrate antigen. Co-formulation with QS-21 adjuvant dramatically enhanced the anti-SialylTn immune response and resulted in a SialylTn-specific IgG switch. The kinetics of the carbohydrate-specific IgG response correlated with a temporary release of cytokines such as IFNγ, IL-2, IL-1β, TNFα and GM-CSF which was measurable in the immune serum by xMAP Multiplex technology. Furthermore, tumor cell killing by activated natural killer cells was induced. These data demonstrate that immunization with a tumor-associated carbohydrate antigen in a highly immunogenic formulation results in a temporary release of type 1 cytokines which may be required for the induction of a specific IgG immune response against the carbohydrate antigen as well as for activation of effector cells against tumor cells.     

Structural basis of the collagen-binding mode of discoidin domain receptor 2

Discoidin domain receptor (DDR) is a cell-surface receptor tyrosine kinase activated by the binding of its discoidin (DS) domain to fibrillar collagen. Here, we have determined the NMR structure of the DS domain in DDR2 (DDR2-DS domain), and identified the binding site to fibrillar collagen by transferred cross-saturation experiments. The DDR2-DS domain structure adopts a distorted jellyroll fold, consisting of eight beta-strands. The collagen-binding site is formed at the interloop trench, consisting of charged residues surrounded by hydrophobic residues. The surface profile of the collagen-binding site suggests that the DDR2-DS domain recognizes specific sites on fibrillar collagen. This study provides a molecular basis for the collagen-binding mode of the DDR2-DS domain.     

T cell-induced secretion of MHC class II-peptide complexes on B cell exosomes

Antigen-specific interactions between B cells and T cells are essential for the generation of an efficient immune response. Since this requires peptide–MHC class II complexes (pMHC-II) on the B cell to interact with TCR on antigen-specific T cells, we have examined the mechanisms regulating the persistence, loss, and secretion of specific pMHC-II complexes on activated B cells. Using a mAb that recognizes specific pMHC-II, we found that activated B cells degrade approximately 50% of pMHC-II every day and release 12% of these pMHC-II from the cell on small membrane vesicles termed exosomes. These exosomes directly stimulate primed, but not naïve, CD4 T cells. Interestingly, engagement of antigen-loaded B cells with specific CD4 T cells stimulates exosome release in a manner that can be mimicked by pMHC-II crosslinking. Biochemical studies revealed that the pMHC-II released on exosomes was previously expressed on the plasma membrane of the B cells, suggesting that regulated exosome release from activated B cells is a mechanism to allow pMHC-II to escape intracellular degradation and decorate secondary lymphoid organs with membrane-associated pMHC-II complexes.     

Plasma methylglyoxal and glyoxal are elevated and related to early membrane alteration in young, complication-free patients with Type 1 diabetes

The reactive aldehydes methylglyoxal and glyoxal, arise from enzymatic and non-enzymatic degradation of glucose, lipid and protein catabolism, and lipid peroxidation. In Type 1 diabetes mellitus (T1DM) where hyperglycemia, oxidative stress, and lipid peroxidation are common, these aldehydes may be elevated. These aldehydes form advanced glycation end products (AGEs) with proteins that are implicated in diabetic complications. We measured plasma methylglyoxal and glyoxal in young, complication-free T1DM patients and assessed activity of the ubiquitous membrane enzyme, Na⁺/K⁺ ATPase. A total of 56 patients with TIDM (DM group), 6–22 years, and 18 non-diabetics (ND group), 6–21 years, were enrolled. Mean plasma A1C (%) was higher in the DM group (8.5 ± 1.3) as compared to the ND group (5.0 ± 0.3). Using a novel liquid chromatography-mass spectrophotometry method, we found that mean plasma methylglyoxal (nmol/l) and glyoxal levels (nmol/l), respectively, were higher in the DM group (841.7 ± 237.7, 1051.8 ± 515.2) versus the ND group (439.2 ± 90.1, 328.2 ± 207.5). Erythrocyte membrane Na⁺/K⁺ ATPase activity (nmol NADH oxidized/min/mg protein) was elevated in the DM group (4.47 ± 0.98) compared to the ND group (2.16 ± 0.59). A1C correlated with plasma methylglyoxal and glyoxal, and both aldehydes correlated with each other. A high correlation of A1C with Na⁺/K⁺ ATPase activity, and a regression analysis showing A1C as a good predictor of activity of this enzyme, point to a role for glucose in membrane alteration. In complication-free patients, increased plasma methylglyoxal, plasma glyoxal, and erythrocyte Na⁺/K⁺ ATPase activity may foretell future diabetic complications, and emphasize a need for aggressive management.