Purification and Biological Function of Caldecrin

Blood calcium homeostasis is critical for biological function. Caldecrin, or chymotrypsin-like elastase, was originally identified in the pancreas as a serum calcium-decreasing factor. The serum calcium-decreasing activity of caldecrin requires the trypsin-mediated activation of the protein. Protease activity-deficient mature caldecrin can also reduce serum calcium concentration, indicating that structural processing is necessary for serum calcium-decreasing activity. Caldecrin suppresses the differentiation of bone-resorbing osteoclasts from bone marrow macrophages (BMMs) by inhibiting receptor activator of NF-κB ligand (RANKL)-induced nuclear factor of activated T-cell cytoplasmic 1 expression via the Syk–PLCγ–Ca2+ oscillation-calcineurin signaling pathway. It also suppresses mature osteoclastic bone resorption by RANKL-stimulated TRAF6–c-Src–Syk–calcium entry and actin ring formation. Caldecrin inhibits lipopolysaccharide (LPS)-induced osteoclast formation in RANKL-primed BMMs by inducing the NF-κB negative regulator A20. In addition, caldecrin suppresses LPS-mediated M1 macrophage polarization through the immunoreceptor triggering receptor expressed on myeloid cells (TREM) 2, suggesting that caldecrin may function as an anti-osteoclastogenic and anti-inflammatory factor via TREM2. The ectopic intramuscular expression of caldecrin cDNA prevents bone resorption in ovariectomized mice, and the administration of caldecrin protein also prevents skeletal muscle destruction in dystrophic mice. In vivo and in vitro studies have indicated that caldecrin is a unique multifunctional protease and a possible therapeutic target for skeletal and inflammatory diseases

Long-Time Treatment by Low-Dose <i>N</i>-Acetyl-L-Cysteine Enhances Proinflammatory Cytokine Expressions in LPS-Stimulated Macrophages

<div><p><i>N</i>-acetyl-L-cysteine is known to act as a reactive oxygen species scavenger and used in clinical applications. Previous reports have shown that high-dose <i>N</i>-acetyl-L-cysteine treatment inhibits the expression of proinflammatory cytokines in activated macrophages. Here, we have found that long-time <i>N</i>-acetyl-L-cysteine treatment at low-concentration increases phosphorylation of extracellular signal-regulated kinase 1/2 and AKT, which are essential for the induction of proinflammatory cytokines including interleukin 1β and interleukin 6 in lipopolysaccharide-stimulated RAW264.7 cells. Furthermore, long-time <i>N</i>-acetyl-L-cysteine treatment decreases expressions of protein phosphatases, catalytic subunit of protein phosphatase-2A and dual specificity phosphatase 1. On the other hand, we have found that short-time <i>N</i>-acetyl-L-cysteine treatment at low dose increases p53 expression, which inhibits expressions of proinflammatory cytokines. These observations suggest that long-time low-dose <i>N</i>-acetyl-L-cysteine treatment increases expressions of proinflammatory cytokines through enhancement of kinase phosphorylation.</p></div

Dichloroacetyl Amides of 3,5-Bis(benzylidene)-4-piperidones Displaying Greater Toxicity to Neoplasms than to Non-Malignant Cells

A series of 3,5-bis(benzylidene)-1-dichloroacetyl-4-piperidones 1a–l was evaluated against Ca9-22, HSC-2, HSC-3, and HSC-4 squamous cell carcinomas. Virtually all of the compounds displayed potent cytotoxicity, with 83% of the CC50 values being submicromolar and several CC50 values being in the double digit nanomolar range. The compounds were appreciably less toxic to human HGF, HPLF, and HPC non-malignant cells, which led to some noteworthy selectivity index (SI) figures. From these studies, 1d,g,k emerged as the lead molecules in terms of their potencies and SI values. A Quantitative Structure-Activity Relationship (QSAR) study revealed that cytotoxic potencies and potency–selectivity expression figures increased when the magnitude of the sigma values in the aryl rings was elevated. The modes of action of the representative cytotoxins in Ca9-22 cells were found to include G2/M arrest and stimulation of the cells to undergo mitosis and cause poly(ADP-ribose) polymerase (PARP) and procaspase 3 cleavage

Reduction of interleukin expressions in LPS-stimulated RAW264.7 cells by over-expression of p53.

<p>RAW264.7 Tet-On stable cells were stably transfected with pTRE2Hyg-tp53, and treated with or without doxycycline. 48 hours later, the cells were treated with or without 100 ng/ml LPS for 3 hours, and the cell lysates and total RNA were extracted. A. Protein levels of p53 in the cell lysates were analyzed by western blot analysis. B. IL-1β and IL-6 mRNAs were detected by real time RT-PCR in total RNA from RAW264.7 Tet-On cells stably transfected with pTRE2Hyg-tp53. The mRNA levels were normalized by Ubc mRNA levels. (C) RAW264.7 Tet-On cells stably transfected with pTRE2Hyg-p53 were further transfected with pGV-IL1p. The resultant cells were treated with doxycycline for 48 hours. Luciferase activity in the cell lysates was measured and normalized by the protein content.</p

Effects of NAC treatment on p53 protein levels in the nuclear fraction of LPS-stimulated macrophages.

<p>A, B. RAW264.7 cells were pretreated with 2(A) or 20 mM (B) NAC for the indicated time. Cell lysates and nuclear extracts were prepared before (upper panels) and after (lower panels) stimulation with 100 ng/ml LPS for 3 hours, and then subjected to western blot analyses with anti-p53, anti-TBP, or anti-GAPDH antibody. C. RAW264.7 cells, pretreated with 2 mM NAC for the indicated time, were stimulated with 100 ng/ml LPS for further 3 hours, and then total RNA was extracted from the cells. p53 mRNA expression was analyzed by real time RT-PCR. The mRNA levels were normalized by Ubc mRNA levels. *<i>p</i><0.05 versus time 0.</p

Search of Neuroprotective Polyphenols Using the “Overlay” Isolation Method

Previous studies of the neuroprotective activity of polyphenols have used ununiform culture systems, making it difficult to compare their neuroprotective potency. We have established a new and simple method for preparing differentiated PC12 cells by removing the toxic coating step. Cells were induced to differentiate with the nerve growth factor (NGF) in a serum-free medium, without a medium change, but with a one-time overlay supplementation of NGF. The optimal inoculation density of the cells was 6&ndash;12 &times; 103 cells/cm2, and the presence of serum inhibited the differentiation. Neuroprotective activity could be quantified by the specific index (SI) value, that is, the ratio of the 50% cytotoxic concentration to the 50% effective concentration. Alkaline extract from the leaves of Sasa senanensis Rehder (SE), having had hormetic growth stimulation, showed the highest SI value, followed by epigallocatechin gallate. The SI value of curcumin and resveratrol was much lower. This simple overly method, that can prepare massive differentiated neuronal cells, may be applicable for the study of the differentiation-associated changes in intracellular metabolites, and the interaction between neuronal cells and physiological factors

Reduction of phosphatase expressions by long-time NAC treatment.

<p>RAW264.7 cells were treated with 2(A) or 20 mM (B) NAC following the time schedule shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087229#pone-0087229-g001" target="_blank">Fig. 1A</a>. Total RNA and cell lysates were extracted from the cells. The gene expression of PP2Ac and DUSP1 was detected by real time RT-PCR. The mRNA levels were normalized by Ubc mRNA levels. *<i>p</i><0.05 versus time 0.</p

The effect of NAC treatment on ROS production in RAW264.7 cells.

<p>RAW264.7 cells were treated with 2 µM H₂DCFDA-containing HBSS for 2 hours. The fluorescence emission of the cell suspensions was measured. *<i>p</i><0.05 versus time 0, relatively. RFU, relative fluorescence units.</p