Expression of extracellular calcium-sensing receptor in human osteoblastic MG-63 cell line

We have previously shown the expression of the extracellular calcium (Ca2+o)-sensing receptor (CaR) in osteoblast-like cell lines, and others have documented its expression in sections of murine, bovine, and rat bone. The existence of the CaR in osteoblasts remains controversial, however, since some studies have failed to document its expression in the same osteoblast-like cell lines. The goals of the present study were twofold. 1) We sought to determine whether the CaR is expressed in the human osteoblast-like cell line, MG-63, which has recently been reported by others not to express this receptor. 2) We investigated whether the CaR, if present in MG-63 cells, is functionally active, since most previous studies have not proven the role of the CaR in mediating known actions of Ca2+o on osteoblast-like cells. We used immunocytochemistry and Western blotting with the specific, affinity-purified anti-CaR antiserum 4637 as well as Northern blot analysis and RT-PCR using a riboprobe and PCR primers specific for the human CaR, respectively, to show readily detectable CaR protein and mRNA expression in MG-63 cells. Finally, we employed the patch-clamp technique to show that an elevation in Ca2+o as well as the specific, allosteric CaR activator NPS R-467 (0.5 microM), but not its less active stereoisomer NPS S-467 (0.5 microM), activate an outward K+ channel in MG-63 cells, strongly suggesting that the CaR in MG-63 cells is not only expressed but is functionally active.     

Bombesin improves burn-induced intestinal injury in the rat

This study was designed to determine the effect of exogenous bombesin (10 microg/kg/day, subcutaneously, three times a day) on intestinal hypomotility and neutrophil infiltration in the early and late phases of burn injury (partial-thickness, second-degree burn of the skin). In acute (2 h after burn injury) or chronic (3 days after) burn groups, intestinal transit was delayed, which was reversed by bombesin treatment. In the acute burn group, but not in the chronic group, increased MPO activity was also reduced by bombesin treatment. The results demonstrate that bombesin ameliorates the intestinal inflammation due to burn injury, involving a neutrophil-dependent mechanism.     

Dexras1: a G protein specifically coupled to neuronal nitric oxide synthase via CAPON

Because nitric oxide (NO) is a highly reactive signaling molecule, chemical inactivation by reaction with oxygen, superoxide, and glutathione competes with specific interactions with target proteins. NO signaling may be enhanced by adaptor proteins that couple neuronal NO synthase (nNOS) to specific target proteins. Here we identify a selective interaction of the nNOS adaptor protein CAPON with Dexras1, a brain-enriched member of the Ras family of small monomeric G proteins. We find that Dexras1 is activated by NO donors as well as by NMDA receptor-stimulated NO synthesis in cortical neurons. The importance of Dexras1 as a physiologic target of nNOS is established by the selective decrease of Dexras1 activation, but not H-Ras or four other Ras family members, in the brains of mice harboring a targeted genomic deletion of nNOS (nNOS-/-). We also find that nNOS, CAPON, and Dexras1 form a ternary complex that enhances the ability of nNOS to activate Dexras1. These findings identify Dexras1 as a novel physiologic NO effector and suggest that anchoring of nNOS to specific targets is a mechanism by which NO signaling is enhanced.     

Cholesterol movement in Niemann-Pick type C cells and in cells treated with amphiphiles

Cholesterol accumulates to massive levels in cells from Niemann-Pick type C (NP-C) patients and in cells treated with class 2 amphiphiles that mimic NP-C disease. This behavior has been attributed to the failure of cholesterol released from ingested low density lipoproteins to exit the lysosomes. However, we now show that the rate of movement of cholesterol from lysosomes to plasma membranes in NP-C cells is at least as great as normal, as was also found previously for amphiphile-treated cells. Furthermore, the lysosomes in these cells filled with plasma membrane cholesterol in the absence of lipoproteins. In addition, we showed that the size of the endoplasmic reticulum cholesterol pool and the set point of the homeostatic sensor of cell cholesterol were approximately normal in NP-C cells. The plasma membrane cholesterol pools in both NP-C and amphiphile-treated cells were also normal. Furthermore, the build up of cholesterol in NP-C lysosomes was not a physiological response to cholesterol overload. Rather, it appeared that the accumulation in NP-C lysosomes results from an imbalance in the brisk flow of cholesterol among membrane compartments. In related experiments, we found that NP-C cells did not respond to class 2 amphiphiles (e.g. trifluoperazine, imipramine, and U18666A); these agents may therefore act directly on the NPC1 protein or on its pathway. Finally, we showed that the lysosomal cholesterol pool in NP-C cells was substantially and preferentially reduced by incubating cells with the oxysterols, 25-hydroxycholesterol and 7-ketocholesterol; these findings suggest a new pharmacological approach to the treatment of NP-C disease.     

Improvement of vitamin B(12) fermentation by reducing the inhibitory metabolites by cell recycle system and a mixed culture

The major problem in vitamin B(12) production using Propionibacterium is the growth inhibition of the cell due to the accumulation of inhibitory metabolites such as propionic acid and acetic acid. In the present paper, we considered several approaches of controlling the propionic acid concentration at low level. Namely: (1) the periodic cultivation of Propionibacterium where dissolved oxygen (DO) concentration was alternatively changed between 0 and 1ppm; (2) cell recycle system using hollow fiber module; and (3) mixed culture using Propionibacterium and Ralstonia eutropha where the latter microorganism assimilates the propionic acid produced by the former. It was found that the productivity of vitamin B(12) was the highest for the cell recycle system, while if the performance was evaluated based on the amount of vitamin B(12) produced per medium used, the mixed culture system gave the far highest value.     

Identification of iron responsive genes by screening cDNA libraries from suppression subtractive hybridization with antisense probes from three iron conditions

The goal of the present study is to identify genes that respond to iron availability. Suppression subtraction hybridization (SSH) was used to generate cDNA libraries from iron loaded and control human astrocytoma cells (SW1088). The cDNA libraries were screened with antisense cDNA probes obtained from mRNA isolated from astrocytoma cells exposed to three conditions: (i) normal media (control), (ii) deferoxamine treated (iron deficient) or (iii) iron loaded. The screening of the cDNA libraries with antisense probes from the three conditions enhanced the screening efficiency and decreased the number of false positives. Positive clones were identified and sequenced. The genes of interest were further analyzed by determining changes in hybridization signal on northern blots from astrocytoma cells exposed to iron or deferoxamine over different time intervals. Our analysis identified cDNAs corresponding to known iron responsive genes such as L-chain ferritin, but also revealed a number of mRNAs with novel sequences and mRNAs previously not known to be responsive to iron such as one of the ABC transporters and Thy-1 glycoprotein. Thus our results suggest that the expression of a number of genes may be influenced by changes in iron availability.     

Regulation of the NF-kappaB activation pathway by isolated domains of FIP3/IKKgamma, a component of the IkappaB-alpha kinase complex

FIP3, isolated as a type 2 adenovirus E3-14.7-kDa interacting protein, is an essential component of the multimeric IkappaB-alpha kinase (IKK) complex and has been shown to interact with various components (Fas receptor-interacting protein, NF-kappaB-inducing kinase, IKKbeta) of the NF-kappaB activation pathway. FIP3 has also been shown to repress basal and tumor necrosis factor (TNF) alpha-induced NF-kappaB activity as well as to induce cell death when overexpressed. The adenovirus E3-14.7-kDa protein (E3-14.7K) is an inhibitor of TNFalpha-induced cell death. In the current study, we generated deletion mutants to map the domains of FIP3, which are responsible for its various functions. The NF-kappaB inhibitory activity and the E3-14.7K binding domains were mapped at the carboxyl half of the FIP3 protein. We also found that the carboxyl-terminal half of FIP3 blocked TNFalpha-induced IkappaB-alpha phosphorylation and subsequent degradation, which suggests that the stabilization of the cytoplasmic inhibitor of NF-kappaB underlies the FIP3 inhibition of NF-kappaB activity. The amino-terminal 119 amino acids were responsible for the FIP3-IKKbeta and FIP3-IKKalpha interaction, and the middle of the protein (amino acids 201-300) appeared to be both the FIP3 self-association domain as well as the FIP3-Fas receptor-interacting protein interaction domain. Thus, FIP3 might serve as a scaffold protein to organize the various components of the IkappaB-alpha kinase complex. Whereas the full-length protein is required for efficient cell death, the amino-terminal 200 amino acids are sufficient to cause rounding and detachment of the cells from the monolayer.     

Autocrine regulation of interleukin-8 production in human monocytes

Interleukin (IL)-8 is a C-X-C chemokine that plays an important role in acute inflammation through its G protein-coupled receptors CXCR1 and CXCR2. In this study, we investigated the role of IL-8 as an autocrine regulator of IL-8 production and the signaling mechanisms involved in human peripheral blood mononuclear cells (MNCs). Sepharose-immobilized IL-8 stimulated a sevenfold increase in IL-8 production within 2 h. IL-8 induced the expression of its own message, and IL-8 biosynthesis was inhibited by cycloheximide and actinomycin D, indicating de novo RNA and protein synthesis. In contrast to MNCs, polymorphonuclear neutrophils did not respond to the immobilized IL-8 with IL-8 production despite cell surface expression of CXCR1 and CXCR2. Melanoma growth-stimulatory activity/growth-related protein-alpha (MGSA/GROalpha), which binds CXCR2 but not CXCR1, was unable to either stimulate IL-8 secretion in MNCs or desensitize these cells to respond to immobilized IL-8. The involvement of mitogen-activated protein kinase (MAPK) in IL-8-induced IL-8 biosynthesis was suggested by the ability of PD-98059, an inhibitor of MAPK kinase, to block this function. Furthermore, IL-8 induced a significant increase in extracellular signal-regulated kinase 2 phosphorylation, whereas MGSA/GROalpha was much less effective. These findings support the role of IL-8 as an autocrine regulator of IL-8 production and suggest that this function is mediated by CXCR1 through activation of MAPK.     

A second cytotoxic proteolytic peptide derived from amyloid beta-protein precursor

The amyloid beta-protein precursor gives rise to the amyloid beta-protein, the principal constituent of senile plaques and a cytotoxic fragment involved in the pathogenesis of Alzheimer disease. Here we show that amyloid beta-protein precursor was proteolytically cleaved by caspases in the C terminus to generate a second unrelated peptide, called C31. The resultant C31 peptide was a potent inducer of apoptosis. Both caspase-cleaved amyloid beta-protein precursor and activated caspase-9 were present in brains of Alzheimer disease patients but not in control brains. These findings indicate the possibility that caspase cleavage of amyloid beta-protein precursor with the generation of C31 may be involved in the neuronal death associated with Alzheimer disease.