Wee1 is a tyrosine kinase that phosphorylates and inactivates CDC2 and is involved in G 2 checkpoint signaling. Because p53 is a key regulator in the G 1 checkpoint, p53-deficient tumors rely only on the G 2 checkpoint after DNA damage. Hence, such tumors are selectively sensitized to DNA-damaging agents by Wee1 inhibition. Here, we report the discovery of a potent and selective smallmolecule inhibitor of Wee1 kinase, MK-1775. This compound inhibits phosphorylation of CDC2 at Tyr15 (CDC2Y15), a direct substrate of Wee1 kinase in cells. MK-1775 abrogates G 2 DNA damage checkpoint, leading to apoptosis in combination with DNA-damaging chemotherapeutic agents such as gemcitabine, carboplatin, and cisplatin selectively in p53-deficient cells. In vivo, MK-1775 potentiates tumor growth inhibition by these agents, and cotreatment does not significantly increase toxicity. The enhancement of antitumor effect by MK-1775 was well correlated with inhibition of CDC2Y15 phosphorylation in tumor tissue and skin hair follicles. Our data indicate that Wee1 inhibition provides a new approach for treatment of multiple human malignancies.

Neutrophils (PMN) have been described as critical effector cells in the host's antibacterial innate immunities. However, the classification of murine PMNs remains unclear. Here, we show that in addition to normal PMN (PMN-N), there are at least two distinct subsets of PMNs (PMN-I and PMN-II) distinguished as follows: (1) cytokine and chemokine production (PMN-I, IL-12/CCL3; PMN-II, IL-10/CCL2; PMN-N, no cytokine/chemokine production), (2) macrophage activation (PMN-I, classically activated macrophages; PMN-II, alternatively activated macrophages; PMN-N, no effect on macrophage activation), (3) Toll-like receptor (TLR) expression (PMN-I, TLR2/TLR4/TLR5/TLR8; PMN-II, TLR2/TLR4/TLR7/TLR9; PMN-N, TLR2/TLR4/TLR9), and (4) surface antigen expression (PMN-I, CD49d(+)CD11b-; PMN-II, CD49d(-)CD11b+; PMN-N, CD49d(-)CD11b-). PMN-I was obtained from MRSA (methicillin-resistant Staphylococcus aureus)-resistant hosts, while MRSA-sensitive hosts were a source of PMN-II. PMN-N was obtained from naive mice. Anti-MRSA innate immunities might be influenced differently by these biochemically and physically distinguished PMNs. PMN-N may convert to PMN-I or PMN-II in response to host circumstance.

Classically activated macrophages (CAMφ) have been described as a major effector cell on the host’s innate immunities. However, CAMφ are not generated in immunocompromised hosts whose alternatively activated macrophages (AAMφ) predominate. In this study, the mechanism by which AAMφ suppress the ability of resident macrophages (RMφ) to generate CAMφ was investigated. AAMφ were isolated from peritoneal exudates of mice 2 days after third-degree thermal injuries affecting 15% total body surface area. CAMφ were generated from RMφ (peritoneal Mφ from normal mice) through stimulation with CpG DNA, a typical CAMφ inducer. RMφ did not polarize to CAMφ when they were cultured with AAMφ in a dual-chamber Transwell even when supplemented with CpG DNA. In addition, RMφ stimulated with CpG DNA did not convert to CAMφ when they were cultured with the culture fluids of AAMφ (AAMφ Culture-Sup). AAMφ Culture-Sup contained IL-6, IL-10, CCL17, PGE2, and TGF-β. Among these, CCL17 and IL-10 inhibited CAMφ generation. The ability of AAMφ Culture-Sup to inhibit CAMφ generation was eliminated when the Culture-Sup was treated with a mixture of mAbs directed against CCL17 and IL-10. These results indicate that CCL17 and IL-10 released from AAMφ inhibit CAMφ generation from RMφ stimulated with CpG DNA.

No abstract

TLRs recognize microbial products. Their subcellular distribution is optimized for microbial recognition. Little is known, however, about mechanisms regulating the subcellular distribution of TLRs. LPS is recognized by the receptor complex consisting of TLR4 and MD-2. Although MD-2, a coreceptor for TLR4, enhances cell surface expression of TLR4, an additional mechanism regulating TLR4 distribution has been suggested. We show here that PRAT4A, a novel protein associated with TLR4, regulates cell surface expression of TLR4. PRAT4A is associated with the immature form of TLR4 but not with MD-2 or TLR2. PRAT4A knockdown abolished LPS responsiveness in a cell line expressing TLR4/MD-2, probably due to the lack of cell surface TLR4. PRAT4A knockdown down-regulated cell surface TLR4/MD-2 on dendritic cells. These results demonstrate a novel mechanism regulating TLR4/MD-2 expression on the cell surface.

The antiviral effect of glycyrrhizin (GR), an active component of licorice roots, was investigated in mice infected with influenza virus A2 (H2N2). When mice that had been exposed to 10 50% lethal doses of the virus were treated intraperitoneally with 10 mg of GR per kg of body weight 1 day before infection and 1 and 4 days postinfection, all of the mice survived over the 21-day experimental period. At the end of this period, the mean survival time (in days) for control mice treated with saline was 10.5 days, and there were no survivors. The grade of pulmonary consolidations and the virus titers in the lung tissues of infected mice treated with GR were significantly lower than those in the lung tissues of infected mice treated with saline. GR did not show any effects on the viability or replication of influenza virus A2 in vitro. When splenic T cells from GR-treated mice were adoptively transferred to mice exposed to influenza virus, 100% of the recipients survived, compared to 0% survival for recipient mice inoculated with naive T cells or splenic B cells and macrophages from GR-treated mice. In addition, the antiviral activities of GR on influenza virus infection in mice were not demonstrated when it was administered to infected mice in combination with anti-gamma interferon (anti-IFN-gamma) monoclonal antibody. These results suggest that GR may protect mice exposed to a lethal amount of influenza virus through the stimulation of IFN-gamma production by T cells, because T cells have been shown to be producer cells of IFN-gamma stimulated with the compound.

Thick film piezoelectric ceramic sensors have been successfully deposited on different metallic substrates with different shapes by a sol–gel spray technique. The ball-milled bismuth titanate fine powders were dispersed into PZT solution to achieve the gel. The films with desired thickness up to 200 µm have been obtained through the multilayer coating approach. These thick films were also effectively coated onto thin sheet metals of thickness down to 25 µm. Self-support films with flat and shell geometries were made. Piezoelectricity was achieved using the corona discharge poling method. The area of the top silver paste electrode was also optimized. The center frequencies of ultrasonic signals generated by these films ranged from 3.6 to 30 MHz and their bandwidth was broad as well. The ultrasonic signals generated and received by these ultrasonic transducers (UTs) operated in the pulse/echo mode had a signal to noise ratio more than 30 dB. The main advantages of such sensors are that they (1) do not need couplant, (2) can serve as piezoelectric and UT, (3) can be coated onto curved surfaces and (4) can operate up to 440 °C. The capability of these thick film UTs for non-destructive evaluation of materials at 440 °C has been demonstrated.

LPS, a principal membrane component in Gram-negative bacteria, is recognized by a receptor complex consisting of TLR4 and MD-2. MD-2 is an extracellular molecule that is associated with the extracellular domain of TLR4 and has a critical role in LPS recognition. MD-2 directly interacts with LPS, and the region from Phe119 to Lys132 (Arg132 in mice) has been shown to be important for interaction between LPS and TLR4/MD-2. With mouse MD-2 mutants, we show in this study that Gly59 was found to be a novel critical amino acid for LPS binding outside the region 119–132. LPS signaling is thought to be triggered by ligand-induced TLR4 clustering, which is also regulated by MD-2. Little is known, however, about a region or an amino acid in the MD-2 molecule that regulates ligand-induced receptor clustering. MD-2 mutants substituting alanine for Phe126 or Gly129 impaired LPS-induced TLR4 clustering, but not LPS binding to TLR4/MD-2, demonstrating that ligand-induced receptor clustering is differentially regulated by MD-2 from ligand binding. We further show that dissociation of ligand-induced receptor clustering and of ligand-receptor interaction occurs in a manner dependent on TLR4 signaling and requires endosomal acidification. These results support a principal role for MD-2 in LPS recognition.