In vivo NIRF imaging of tumor targetability of nanosized liposomes in tumor-bearing mice

To optimize tumor targetability of nanosized liposomes for application as drug carriers, various liposomes are prepared by incorporating different amounts (10, 30, and 50?wt%) of cationic, anionic, and PEGylated lipids into neutral lipid. In vivo near-infrared fluorescence images reveal that PEG-PE/PC liposomes display high tumor accumulation in tumor-bearing mice, while large amounts of DOTAP/PC liposomes are rapidly captured in the liver, resulting in poor tumor accumulation. These results demonstrate that optimization of the surface properties of liposomes is very important for their tumor targetability, and that in vivo imaging techniques are useful in developing and optimizing nanosized liposome-based drug carriers.     

Berberine inhibits TPA-induced MMP-9 and IL-6 expression in normal human keratinocytes

Berberine is a plant ingredient that has anti-inflammatory and anti-oxidative effects. Matrix metalloproteinase-9 (MMP-9) and interleukin-6 (IL-6) are known to be highly induced by ultraviolet (UV) light and may play important roles in UV-induced skin inflammation and the skin aging process. In this study, we investigated the effects of berberine on MMP-9 and IL-6 expression in normal human keratinocytes (NHK). Our results demonstrated that berberine dose-dependently inhibited basal and TPA-induced expression and activity of MMP-9, and also suppressed TPA-induced IL-6 expression. Berberine prevented TPA-induced ERK activation and AP-1 DNA binding activity. Therefore, berberine may be used as an effective ingredient for anti-skin aging products, which can prevent skin inflammation and the degradation of extracellular matrix proteins, including collagen, by MMPs.     

Oxidized NADH Oxidase Inhibits Activity of an ATP/NAD Kinase from a Thermophilic Archaeon

NADH oxidases (NOXs) are important enzymes in detoxifying oxidative stress and regenerating oxidized pyridine nucleotides. In the present study, a NOX from Thermococcus kodakarensis KOD1 (NOXtk) was recombinantly expressed in Escherichia coli and purified to homogeneity. NOXtk displayed NADH oxidase activity that was inhibited by oxidization. Under physiological conditions, unoxidized and oxidized NOXtk formed dimers and hexamers, respectively. Mutating the single cysteine residue Cys45 to alanine (NOXtkC45A) decreased NADH oxidase activity without affecting dimerization or hexamerization, suggesting that oligomerization does not occur through disulfide bond formation. Pull-down assay results indicated that an ATP/NAD kinase from T. kodakarensis KOD1 (ANKtk) binds to NOXtk. Use of several assays revealed that ANKtk can only bind to oxidized hexameric NOXtk, through which it inhibits ANKtk activity. Because ANKtk converts NADH to NADPH (an important factor in oxidative stress protection), a model based on in vitro result was proposed in which NOXtk hexamerization under oxic conditions inhibits both NOXtk and ANKtk activities, thereby sensitizing cells to oxidative stress-induced death.     

EGF-induced MMP-9 expression is mediated by the JAK3/ERK pathway,but not by the JAK3/STAT-3 pathway in a SKBR3 breast cancer cell line

The number of epidermal growth factor receptors (EGFRs) and their ligands are highly expressed in malignant tumor cells. The EGF signaling pathway is also activated in up to one-third of patients with breast cancer. In this study, we investigated the novel function of the JAK3 inhibitor, WHI-P131, on EGF-induced MMP-9 expression and the regulatory mechanism of EGF-induced MMP-9 expression in SKBR3 cells. We observed that EGF increased MMP-9 mRNA and protein expression in a dose-dependent manner. EGF also induced the phosphorylation of EGFR, ERK, and STAT-3, and these effects were inhibited by the EGFR inhibitor, AG1478. To investigate the involvement of the STAT-3 pathway on EGF-induced MMP-9 expression, we pretreated SKBR3 cells with JAK1, JAK2, and JAK3 inhibitors prior to EGF treatment. The results showed that the JAK3 inhibitor, WHI-P131, as well as JAK3 siRNA transfection, but not the JAK1 and JAK2 inhibitors, significantly decreased EGF-induced MMP-9 expression. In addition, EGF-induced STAT-3 phosphorylation was only inhibited by WHI-P131. We then transfected cells with adenoviral STAT-3 (Ad-STAT-3), followed by treatment with EGF. Interestingly, EGF-induced MMP-9 expression was decreased by Ad-STAT-3 overexpression in a dose-dependent manner, while it was significantly increased by STAT-3 siRNA transfection. Our results also showed that basal levels of MMP-9 expression were significantly increased by constitutive active-MEK (CA-MEK) overexpression. EGF-induced ERK phosphorylation was prevented by WHI-P131, but not by JAK1 and JAK2 inhibitors. On the other hand, EGF-induced MMP-9 expression was decreased by the MEK1/2 inhibitor, UO126. Therefore, for the first time, we suggest that the JAK3 inhibitor, WHI-P131, inhibits EGF-induced STAT-3 phosphorylation as well as ERK phosphorylation. The JAK3/ERK pathway may play an important role in EGF-induced MMP-9 expression in SKBR3 cells.     

Role of Lipocalin-2-Chemokine Axis in the Development of Neuropathic Pain following Peripheral Nerve Injury

Lipocalin 2 (LCN2), which is also known as 24p3 and neutrophil gelatinase-associated lipocalin (NGAL), binds small, hydrophobic ligands and interacts with cell surface receptor 24p3R to regulate diverse cellular processes. In the present study, we examined the role of LCN2 in the pathogenesis of neuropathic pain using a mouse model of spared nerve injury (SNI). Lcn2 mRNA levels were significantly increased in the dorsal horn of the spinal cord after SNI, and LCN2 protein was mainly localized in neurons of the dorsal and ventral horns. LCN2 receptor 24p3R was expressed in spinal neurons and microglia after SNI. Lcn2-deficient mice exhibited significantly less mechanical pain hypersensitivity during the early phase after SNI, and an intrathecal injection of recombinant LCN2 protein elicited mechanical pain hypersensitivity in naive animals. Lcn2 deficiency, however, did not affect acute nociceptive pain. Lcn2-deficient mice showed significantly less microglial activation and proalgesic chemokine (CCL2 and CXCL1) production in the spinal cord after SNI than wild-type mice, and recombinant LCN2 protein induced the expression of these chemokines in cultured neurons. Furthermore, the expression of LCN2 and its receptor was detected in neutrophils and macrophages in the sciatic nerve following SNI, suggesting the potential role of peripheral LCN2 in neuropathic pain. Taken together, our results indicate that LCN2 plays a critical role in the development of pain hypersensitivity following peripheral nerve injury and suggest that LCN2 mediates neuropathic pain by inducing chemokine expression and subsequent microglial activation.     

Architecture and characterization of a thermostable MoxR family AAA+ ATPase from Thermococcus kodakarensis KOD1

AAA⁺ ATPases are ubiquitous enzymes that can function as molecular chaperones, employing the energy obtained from ATP hydrolysis to remodel macromolecules. In this report, the MoxR enzyme from Thermococcus kodakarensis KOD1 (TkMoxR) was shown to have two native forms: a two-stack hexameric ring and a hexameric structure, under physiological conditions and cold stress, respectively. TkMoxR was altered to a microtubule-like form in the presence of ATP and tightly interacted with dsDNA molecules of various lengths. In addition, the two-stack hexameric protein catalyzed dsDNA decomposition to form and then release ssDNA, whereas the hexamer TkMoxR structure interacted with but did not release dsDNA. These results suggest that TkMoxR has DNA helicase activity involved in gene expression control.     

Activation tagging of an Arabidopsis SHI-RELATED SEQUENCE gene produces abnormal anther dehiscence and floral development

The tapetum is a layer of cells covering the inner surface of pollen sac wall. It contributes to anther development by providing enzymes and materials for pollen coat biosynthesis and nutrients for pollen development. At the end of anther development, the tapetum is degenerated, and the anther is dehisced, releasing mature pollen grains. In Arabidopsis, several genes are known to regulate tapetum formation and pollen development. However, little is known about how tapetum degeneration and anther dehiscence are regulated. Here, we show that an activation-tagged mutant of the S HI-R ELATED S EQUENCE 7 (SRS7) gene exhibits disrupted anther dehiscence and abnormal floral organ development in addition to its dwarfed growth with small, curled leaves. In the mutant hypocotyls, cell elongation was reduced, and gibberellic acid sensitivity was diminished. Whereas anther development was normal, its dehiscence was suppressed in the dominant srs7-1D mutant. In wild-type anthers, the tapetum disappeared at anther development stages 11 and 12. In contrast, tapetum degeneration was not completed at these stages, and anther dehiscence was inhibited, causing male sterility in the mutant. The SRS7 gene was expressed mainly in the filaments of flowers, where the DEFECTIVE-IN-ANTHER-DEHISCENCE 1 (DAD1) enzyme catalyzing jasmonic acid (JA) biosynthesis is accumulated immediately before flower opening. The DAD1 gene was induced in the srs7-1D floral buds. In fully open flowers, the SRS7 gene was also expressed in pollen grains. It is therefore possible that the abnormal anther dehiscence and floral development of the srs7-1D mutant would be related with JA.