Mitochondrial permeability transition pore opening induces the initial process of renal calcium crystallization

Renal tubular cell injury induced by oxidative stress via mitochondrial collapse is thought to be the initial process of renal calcium crystallization. Mitochondrial collapse is generally caused by mitochondrial permeability transition pore (mPTP) opening, which can be blocked by cyclosporine A (CsA). Definitive evidence for the involvement of mPTP opening in the initial process of renal calcium crystallization, however, is lacking. In this study, we examined the physiological role of mPTP opening in renal calcium crystallization in vitro and in vivo. In the in vitro study, cultured renal tubular cells were exposed to calcium oxalate monohydrate (COM) crystals and treated with CsA (2 μM). COM crystals induced depolarization of the mitochondrial membrane potential and generated oxidative stress as evaluated by Cu-Zn SOD and 4-HNE. Furthermore, the expression of cytochrome c and cleaved caspase 3 was increased and these effects were prevented by CsA. In the in vivo study, Sprague-Dawley rats were administered 1% ethylene glycol (EG) to generate a rat kidney stone model and then treated with CsA (2.5, 5.0, and 10.0 mg/kg/day) for 14 days. EG administration induced renal calcium crystallization, which was prevented by CsA. Mitochondrial collapse was demonstrated by transmission electron microscopy, and oxidative stress was evaluated by measuring Cu-Zn SOD, MDA, and 8-OHdG generated by EG administration, all of which were prevented by CsA. Collectively, our results provide compelling evidence for a role of mPTP opening and its associated mitochondrial collapse, oxidative stress, and activation of the apoptotic pathway in the initial process of renal calcium crystallization.     

Corticosteroid enhances heme oxygenase-1 production by circulating monocytes by up-regulating hemoglobin scavenger receptor and amplifying the receptor-mediated uptake of hemoglobin-haptoglobin complex

This study examined the relationship between steroid treatment and CD163-mediated downstream pathways linked to inflammatory resolution. Twelve patients referred for congenital heart disease surgery were divided into two groups based on the severity of intravascular hemolysis during cardiopulmonary bypass surgery. Patients with severe intravascular hemolysis were administered haptoglobin during the procedure. Flow cytometry indicated a peak in monocyte CD163 expression on post-operative day 1 in both groups. Enhanced and prolonged heme oxygenase-1 (HO-1) mRNA expression levels were observed in patients who received haptoglobin. Binding of hemoglobin-haptoglobin complex (Hb/Hp) to CD163 resulted in significant induction of HO-1 by peripheral blood mononuclear cells after exposure to dexamethasone prior to culture. This effect was significantly inhibited by anti-CD163 antibody. Our results demonstrated up-regulation of CD163 expression on the monocyte surface by steroid treatment. Steroid treatment was suggested to facilitate CD163-mediated endocytosis of hemoglobin to monocytes/macrophages and thereby induce acceleration of HO-1 synthesis.     

First observation of incomplete vertical circulation in Lake Biwa

Limnology - Vertical circulation reaches the bottom of the deepest (> 90 m) part of the northern basin of Lake Biwa, a warm monomictic lake, during winter (January to March)...     

Sex-related differences in SLIGRL-induced pruritus in mice

Aims

Pruritus is a common symptom of skin diseases, and is associated with impaired sleep quality and a considerable reduction in the patient's quality of life. Recently, it was reported that there are sex-specific differences in scratching behavior in chronic pruritus patients. Namely, female chronic pruritus patients scratch more and have significantly more scratch lesions than male patients. However, few animal studies have examined sex-related differences in scratching behavior. Thus, the present work investigated sex-related differences in animal pruritus using pruritogens, which are often used to create experimental animal models of itching.

Main methods

Acute pruritus was induced in ICR mice by a single intradermal injection of histamine, 4-methylhistamine, serotonin, compound 48/80, substance P (SP), or the proteinase-activated receptor-2 (PAR-2)-activating peptide SLIGRL-NH₂. Chronic pruritus was induced by 5 weeks of the repeated application of 2,4,6-trinitro-1-chlorobenzene (TNCB) to BALB/c mice.

Key findings

Female mice showed significantly higher scratching counts in SLIGRL-NH₂-induced pruritus than male mice. Conversely, there was no obvious sex-related difference in scratching behavior for the other pruritogens examined.

Significance

These results indicate that sex-related differences may exist in the pruritogen-responsive neurons that transmit the itch signal induced by SLIGRL-NH₂, but not by histamine or 5-HT.     

Distinct Structural Requirements for Interleukin-4 (IL-4) and IL-13 Binding to the Shared IL-13 Receptor Facilitate Cellular Tuning of Cytokine Responsiveness

Both interleukin-4 (IL-4) and IL-13 can bind to the shared receptor composed of the IL-4 receptor α chain and the IL-13 receptor α1 chain (IL-13Rα1); however, the mechanisms by which these ligands bind to the receptor chains are different, enabling the principal functions of these ligands to be different. We have previously shown that the N-terminal Ig-like domain in IL-13Rα1, called the D1 domain, is the specific and critical binding unit for IL-13. However, it has still remained obscure which amino acid has specific binding capacity to IL-13 and why the D1 domain acts as the binding site for IL-13, but not IL-4. To address these questions, in this study we performed mutational analyses for the D1 domain, combining the structural data to identify the amino acids critical for binding to IL-13. Mutations of Lys-76, Lys-77, or Ile-78 in c′ strand in which the crystal structure showed interaction with IL-13, and those of Trp-65 and Ala-79 adjacent to the interacting site, resulted in significant impairment of IL-13 binding, demonstrating that these amino acids generate the binding site. Furthermore, mutations of Val-35, Leu-38, or Val-42 at the N-terminal β-strand also resulted in loss of IL-13 binding, probably from decreased structural stability. None of the mutations employed here affected IL-4 binding. These results demonstrate that the D1 domain of IL-13Rα1 acts as an affinity converter, through direct cytokine interactions, that allows the shared receptor to respond differentially to IL-4 and IL-13.Interleukin-4 (IL-4)² and IL-13 are related cytokines. IL-4 binds to a heterodimeric complex composed of the IL-4R α chain (IL-4Rα) and the common γ chain (γc), or of IL-4Rα and the IL-13R α1 chain (IL-13Rα1), called type I or type II IL-4R, respectively (1, 2). In contrast, IL-13 binds to type II IL-4R, but not type I IL-4R. Therefore, type II IL-4R is also called IL-13R. This means that IL-4 and IL-13 share the same receptor, type II IL-4R·IL-13R, which explains why IL-4 and IL-13 exert similar activities. However, the principal functions of IL-4 and IL-13 are different. As type I IL-4R is mainly expressed on hematopoietic cells, IL-4 acts on these cells, inducing Th2 differentiation in T cells and immunoglobulin class switching to IgE in B cells (1, 3). In contrast, type II IL-4R·IL-13R expresses ubiquitously, including nonhematopoietic cells, and IL-13 plays a central role in the pathogenesis of bronchial asthma by acting on these cells, including epithelial cells and fibroblasts (1, 4). Thus, it can be said that the principal role of IL-4 is an immunoregulatory cytokine, whereas that of IL-13 is an effector cytokine (5).The assembly mechanism for the binding of either IL-4 or IL-13 to type II IL-4R·IL-13R is unique. IL-4 first binds to IL-4Rα with high affinity (K_d = 1 nm), followed by recruitment of IL-13Rα1 with low affinity. In contrast, IL-13 first binds to IL-13Rα1 with low affinity (K_d = 30–37 nm), and then the complex recruits IL-4Rα, forming a high affinity receptor (K_d = 0.03–0.4 nm (6, 7)). This means that, although both IL-4 and IL-13 use IL-4Rα and IL-13Rα1, the roles of IL-4Rα and IL-13Rα1 as the primary or secondary binding unit are the opposite of those for IL-4 and IL-13. Furthermore, these differences in affinity between the ligand, the primary binding unit, and the secondary binding unit can result in that in nonhematopoietic cells on which IL-131 is expressed more abundantly than IL-4α, the number of the IL-13 receptor complex continues to rise as the IL-13 concentration increases, whereas the formation of the IL-4 receptor complex is saturated at a low IL-4 concentration. This can explain why IL-13 transduces stronger signals than IL-4 in nonhematopoietic cell such as epithelial cells and fibroblasts.We previously found that the N-terminal Ig-like domain in IL-13Rα1, called the D1 domain, is the specific and critical binding unit for IL-13, but not for IL-4, using the D1 domain-deleted IL-13Rα1 (8). LaPorte et al. recently described the crystal structure of the IL-13·IL-13Rα1·IL-4Rα, showing that the c′ strand of the D1 domain of IL-13Rα1 and the C-D strand of IL-13 generate an antiparallel β-sheet structure (7). Furthermore, this structural analysis showed that the polar bonds between IL-4 and IL-4Rα were diminished in the IL-13·IL-4Rα complex, possibly suggesting why IL-4Rα has high and no affinity with IL-4 and IL-13, respectively. These results confirmed that the unique assembly mechanism of type II IL-4R·IL-13R for IL-4 and IL-13 is dictated by the D1 domain and indicated that the c′ strand in the D1 domain is the binding site of IL-13Rα1 to IL-13. It is thought that IL-13Rα1 has evolved from γc, which does not have the extra Ig domain, acquiring the D1 domain probably from IL-2Rα or IL-15Rα (7, 9). In other words, the acquisition of the D1 domain enables the cells to respond to IL-13 in addition to IL-4. In this sense, the D1 domain appears to be an affinity converter that has evolved differential interactions with IL-4 and IL-13 to respond to the two cytokines distinctly, based on receptor expression levels and cytokine concentration. Thus, the evolution of distinct interactions of D1 with IL-4 versus IL-13 is an unprecedented example of divergent evolution of function of the same structure. Interestingly, in the structural study, it was observed that the c′ strand of the D1 domain of IL-13Rα1 can also generate an antiparallel β-sheet structure with IL-4 that appears similar to that of IL-13 (7), leaving open the question of whether it is energetically important for IL-13 but not IL-4, and whether direct interactions are required.From these studies, several questions remain unresolved. The structures did not make it clear if this differential effect is indirect, or through direct interaction with the cytokines. Are the c′ contacts with cytokines energetically important and distinct for IL-4 and IL-13? If this is the case, then the second question is which amino acid in the c′ strand has specific binding capacity to IL-13. The third question is why does this portion act as the binding site specific for IL-13, but not IL-4. To address these questions, we took advantage of the mutational approach for the D1 domain, combining data from the structural study, and identified the amino acids critical for binding to IL-13.     

Tricyclic pharmacophore-based molecules as novel integrin alpha(v)beta3 antagonists. Part 2: synthesis of potent alpha(v)beta3/alpha(IIb)beta3 dual antagonists

We synthesized 4-aminopiperidine derivatives of our prototype integrin alpha(v)beta3 antagonist 1 in an attempt to increase the activity and water solubility. Introduction of one or two hydrophilic moieties into the central aromatic ring and/or the benzene ring at the C-terminus of 1 increased water solubility and enhanced inhibition of cell adhesion. The results of a structure-activity relationships (SAR) study indicated that the torsion angle between the central aromatic ring and the piperidine ring, and the acidity at the sulfonamide moiety, might be important for alpha(v)beta3 receptor binding activity. Some of these compounds are novel and potent alpha(v)beta3/alpha(IIb)beta3 dual antagonists with acceptable water solubility and a satisfactory early absorption, distribution, metabolism, excretion, and toxicity (ADMET) profile.     

Comparative biochemical characterization of two GroEL homologs from the cyanobacterium Synechococcus elongatus PCC 7942

Unlike Escherichia coli, cyanobacteria generally contain two GroEL homologs. The chaperone function of cyanobacterial GroELs was examined in vitro for the first time with GroEL1 and GroEL2 of Synechococcus elongatus PCC 7942. Both GroELs prevented aggregation of heat-denatured proteins. The ATPase activity of GroEL1 was approximately one-sixth that of Escherichia coli GroEL, while that of GroEL2 was insignificant. The activities of both GroELs were enhanced by GroES, while that of Escherichia coli GroEL was suppressed. The ATPase activity of GroEL1 was greatly enhanced in the presence of GroEL2, but the folding activities of GroEL1 and GroEL2 were much lower than that of Escherichia coli GroEL, regardless of the co-presence of the counterpart or GroES. Both native and recombinant GroEL1 forms a tetradecamer like Escherichia coli GroEL, while GroEL2 forms a heptamer or dimer, but the GroEL1 and GroEL2 oligomers were extremely unstable. In sum, we concluded that the cyanobacterial GroELs are mutually distinct and different from Escherichia coli GroEL.