Aluminum Doping and Nanostructuring Enabled Designing of Magnetically Recoverable Hexaferrite Catalysts.

We demonstrate an approach based on substituting a magnetic cation with a carefully chosen isovalent non-magnetic cation to derive catalytic activity from otherwise catalytically inactive magnetic materials. Using the model system considered, the results illustratively present that the catalytically inactive but highly magnetic strontium hexaferrite (SrFe12O19; SFO) system can be transformed into a catalytically active system by simply replacing some of the magnetic cation Fe3+ by a non-magnetic cation Al3+ in the octahedral coordination environment in the SFO nanocrystals. The intrinsic SFO and Al-doped SrFe12O19 (SrFe11.5Al0.5O19; Al-SFO) nanomaterials were synthesized using a simple, eco-friendly tartrate-gel technique, followed by thermal annealing at 850 °C for 2 h. The SFO and Al-SFO were thoroughly characterized for their structure, phase, morphology, chemical bonding, and magnetic characteristics using X-ray diffraction, Fourier-transform infrared spectroscopy, and vibrating sample magnetometry techniques. Catalytic performance evaluated toward 4-nitrophenol, which is the toxic contaminant at pharmaceutical industries, reduction reaction using NaBH4 (mild reducing agent), the Al-doped SFO samples exhibit a reasonably good performance compared to intrinsic SFO. The results indicate that the catalytic activity of Al-SFO is due to Al-ions occupying the octahedral sites of the hexaferrite lattice; as these sites are on the surface of the catalyst, they facilitate electron transfer. Furthermore, surface/interface characteristics of nanocrystalline Al-SFO coupled with magnetic properties facilitate the catalyst recovery by simple, inexpensive methods while readily allowing the reusability. Moreover, the activity remains the same even after five successive cycles of experiments. Deriving the catalytic activity from otherwise inactive compounds as demonstrated in the optimized, engineered nanoarchitecture of Al-doped-Sr-hexaferrite may be useful in adopting the approach in exploring further options and designing inexpensive and recyclable catalytic materials for future energy and environmental technologies.

Size and Chemistry Controlled Cobalt-Ferrite Nanoparticles and Their Anti-proliferative Effect against the MCF-7 Breast Cancer Cells.

Engineering cobalt ferrites for application in health and biomedical science poses a challenge in terms of nanoscale morphology with a controlled size, shape, and thermochemical stability coupled with controlled properties for biocompatibility. Here, we report a simple one-step, low temperature approach to produce crystalline, nanosized cobalt ferrites (CFO) with a size ∼4.7 nm and demonstrate their applicability in breast cancer treatment. Inherent physiochemical and magnetic properties, which are quite important for biomedical applications, along with cytotoxicity of CFO nanoparticles (NPs) are investigated in detail. X-ray diffraction analyses confirm the cubic spinel phase with the tensile strain in crystalline CFO NPs. Chemical bonding analyses using infrared and Raman spectroscopic studies also support the cubic spinel phase. Electron microscopy and small-angle X-ray scattering revealed the narrow particle-size distribution and spherical-shape morphology. The as-synthesized CFO NPs exhibit superparamagnetic character. Unsaturated magnetization behavior suggests the existence of disordered spins in the surface layers. The temperature dependence of the magnetic parameters, namely, saturation magnetization, coercivity, retentivity, and squareness ratio, also supports the surface-localized spins. Cytotoxic activity of the as-synthesized CFO NPs against the human breast cancer (MCF-7) cell line and normal human peripheral blood mononuclear cells (PBMC) has been evaluated. The mild response of CFO NPs in terms of their antiproliferative nature against cancer cells and negligible Cytotoxicity reflecting their human-safe-and-friendly nature makes them suitable for bioapplications. Moreover, assessment of toxicity toward human red blood cells (RBC) revealed (<3%) hemolysis as compared to the positive control, suggesting potential applications of CFO NPs for human cells.

Small molecule inhibition of phosphatidylinositol-3,4,5-triphosphate (PIP3) binding to pleckstrin homology domains.

The PI3-kinase (PI3K) pathway regulates many cellular processes, especially cell metabolism, cell survival, and apoptosis. Phosphatidylinositol-3,4,5-trisphosphate (PIP3), the product of PI3K activity and a key signaling molecule, acts by recruiting pleckstrin-homology (PH) domain-containing proteins to cell membranes. Here, we describe a new structural class of nonphosphoinositide small molecule antagonists (PITenins, PITs) of PIP3-PH domain interactions (IC(50) ranges from 13.4 to 31 µM in PIP3/Akt PH domain binding assay). PITs inhibit interactions of a number of PIP3-binding PH domains, including those of Akt and PDK1, without affecting several PIP2-selective PH domains. As a result, PITs suppress the PI3K-PDK1-Akt pathway and trigger metabolic stress and apoptosis. A PIT-1 analog displayed significant antitumor activity in vivo, including inhibition of tumor growth and induction of apoptosis. Overall, our studies demonstrate the feasibility of developing specific small molecule antagonists of PIP3 signaling.

Requirement for tumor necrosis factor-receptor 2 in alveolar chemokine expression depends upon the form of the ligand.

Respiratory virus infection evokes a potent T-cell response that may result in a considerable insult to the structural and functional integrity of the gas exchange units of the lung. Alveolar antigen recognition by CD8+ T lymphocytes results in significant injury that is critically dependent upon tumor necrosis factor (TNF)-alpha expressed by the CD8+ T cells and is largely dependent upon TNF-receptor 1 expression on the alveolar epithelial target cells. TNF-receptor 2 (TNF-R2)-deficient mice were used to demonstrate that CD8+ T-cell-mediated lung injury associated with clearance of experimental influenza requires TNF-R2 for full expression of immunopathology. In vitro analysis indicates that alveolar cell expression of TNF-R2 is critical in the induction of epithelial monocyte chemoattractant protein (MCP)-1 expression specifically in response to soluble TNF-alpha, suggesting an important role for this receptor in bystander lung injury. However, TNF-R2 was dispensable for induction of alveolar MCP-1 expression in response to transmembrane TNF-alpha expressed by antigen-specific CD8+ T cells, and the effects of the two receptors seem to be additive. Because TNF-R2 may be rapidly shed as part of feedback inhibition of bystander inflammation, this suggests a mechanism by which immunopathology in respiratory virus infection may be regulated and by which T-cell receptor-dependent TNF-alpha activity might bypass such negative regulation for contact-dependent antiviral activities.

Superior vena cava syndrome secondary to saphenous venous graft aneurysm with right atrial fistula.

Mega-aneurysms of saphenous vein grafts (SVGs) to coronary arteries are rare complications of bypass surgery. We report the development of superior vena cava syndrome secondary to an SVG mega-aneurysm with concomitant fistulous communication to the right atrium. Successful treatment was achieved by coil embolization and chronic anticoagulation.

Roles of phosphatidylinositol 3-kinase in interferon-gamma-dependent phosphorylation of STAT1 on serine 727 and activation of gene expression.

STAT1 must be phosphorylated on serine 727 to be fully active in transcription. We show that phosphatidylinositol 3-kinase (PI3K) and its effector kinase Akt play an important role in the serine phosphorylation of STAT1 and in the activation of gene expression in response to interferon-gamma (IFN gamma). IFN gamma activates PI3K as well as Akt in a variety of cell lines. Specific inhibition of PI3K abrogates IFN gamma-induced, but not interleukin-1- or tumor necrosis factor-alpha-induced, phosphorylation of STAT1 on serine and reduces STAT1-dependent transcription and gene expression by approximately 7-fold. Constitutively active forms of PI3K or Akt activate and their dominant-negative derivatives inhibit STAT1-driven transactivation in response to IFN gamma. In addition to PI3K and Akt, JAK1, JAK2, and the tyrosine 440 STAT1 docking residue of IFNGR1 are required for STAT1 to be phosphorylated on serine. Taken together, these results suggest that the following events lead to the activation of STAT1 upon IFN gamma stimulation: 1) PI3K and Akt are activated by the occupied receptor and Tyr-440 is phosphorylated by the activated JAKs; 2) STAT1 docks to Tyr-440; and 3) Tyr-701 is phosphorylated by the JAKs and Ser-727 is phosphorylated by a kinase downstream of Akt.

Regulation of p53 expression by the RAS-MAP kinase pathway.

Activation of MAP kinase leads to the activation of p53-dependent pathways, and vice-versa. Although the amount of p53 protein increases in response to MAP kinase-dependent signaling, the basis of this increase is not yet fully understood. We have isolated the mutant cell line AP14, defective in p53 expression, from human HT1080 fibrosarcoma cells, which have an activated ras allele. The expression of p53 mRNA and protein is approximately 10-fold lower in AP14 cells than in the parental cells. The high constitutive phosphorylation and activities of the MAP kinases ERK1 and ERK2 in HT1080 cells are greatly reduced in AP14 cells, although the levels of these proteins are unchanged, suggesting that the defect in the mutant cells affects the steady-state phosphorylation of ERK1 and ERK2. Overexpression of ERK2 in AP14 cells restored both MAP kinase activity and p53 expression, and incubation of the mutant cells with the phosphatase inhibitor orthovanadate resulted in strong coordinate elevation of MAP kinase activity and p53 expression. The levels of expression of the p53-regulated gene p21 parallel those of p53 throughout, showing that basal p21 expression depends on p53. The levels of p53 mRNA increased by 5-8-fold when activated ras was introduced into wild-type cells, and the levels of the p53 and p21 proteins decreased substantially in wild-type cells treated with the MEK inhibitor U0216. We conclude that MAP kinase-dependent pathways help to regulate p53 levels by regulating the expression of p53 mRNA.

Stat1-independent regulation of gene expression in response to IFN-gamma.

Although Stat1 is essential for cells to respond fully to IFN-gamma, there is substantial evidence that, in the absence of Stat1, IFN-gamma can still regulate the expression of some genes, induce an antiviral state and affect cell growth. We have now identified many genes that are regulated by IFN-gamma in serum-starved Stat1-null mouse fibroblasts. The proteins induced by IFN-gamma in Stat1-null cells can account for the substantial biological responses that remain. Some genes are induced in both wild-type and Stat1-null cells and thus are truly Stat1-independent. Others are subject to more complex regulation in response to IFN-gamma, repressed by Stat1 in wild-type cells and activated in Stat1-null cells. Many genes induced by IFN-gamma in Stat1-null fibroblasts also are induced by platelet-derived growth factor in wild-type cells and thus are likely to be involved in cell proliferation. In mouse cells expressing the docking site mutant Y440F of human IFN-gamma receptor subunit 1, the mouse Stat1 is not phosphorylated in response to human IFN-gamma, but c-myc and c-jun are still induced, showing that the Stat1 docking site is not required for Stat1-independent signaling.

Biologic consequences of Stat1-independent IFN signaling.

Although Stat1 is required for many IFN-dependent responses, recent work has shown that IFNgamma functions independently of Stat1 to affect the growth of tumor cells or immortalized fibroblasts. We now demonstrate that both IFNgamma and IFNalpha/beta regulate proliferative responses in cells of the mononuclear phagocyte lineage derived from Stat1-null mice. Using both representational difference analysis and gene arrays, we show that IFNgamma exerts its Stat1-independent actions on mononuclear phagocytes by regulating the expression of many genes. This result was confirmed by monitoring changes in expression and function of the corresponding gene products. Regulation of the expression of these genes requires the IFNgamma receptor and Jak1. The physiologic relevance of IFN-dependent, Stat1-independent signaling was demonstrated by monitoring antiviral responses in Stat1-null mice. Thus, the IFN receptors engage alternative Stat1-independent signaling pathways that have important physiological consequences.

Complex roles of Stat1 in regulating gene expression.

Stat1 is a fascinating and complex protein with multiple, yet contrasting transcriptional functions. Upon activation, it drives the expression of many genes but also suppresses the transcription of others. These opposing characteristics also apply to its role in facilitating crosstalk between signal transduction pathways, as it participates in both synergistic activation and inhibition of gene expression. Stat1 is a functional transcription factor even in the absence of inducer-mediated activation, participating in the constitutive expression of some genes. This review summarizes the well studied involvement of Stat1 in IFN-dependent and growth factor-dependent signaling and then describes the roles of Stat1 in positive, negative and constitutive regulation of gene expression as well as its participation in crosstalk between signal transduction pathways. Oncogene (2000).

Regulation of c-myc expression by IFN-gamma through Stat1-dependent and -independent pathways.

Interferons (IFNs) inhibit cell growth in a Stat1-dependent fashion that involves regulation of c-myc expression. IFN-gamma suppresses c-myc in wild-type mouse embryo fibroblasts, but not in Stat1-null cells, where IFNs induce c-myc mRNA rapidly and transiently, thus revealing a novel signaling pathway. Both tyrosine and serine phosphorylation of Stat1 are required for suppression. Induced expression of c-myc is likely to contribute to the proliferation of Stat1-null cells in response to IFNs. IFNs also suppress platelet-derived growth factor (PDGF)-induced c-myc expression in wild-type but not in Stat1-null cells. A gamma-activated sequence element in the promoter is necessary but not sufficient to suppress c-myc expression in wild-type cells. In PKR-null cells, the phosphorylation of Stat1 on Ser727 and transactivation are both defective, and c-myc mRNA is induced, not suppressed, in response to IFN-gamma. A role for Raf-1 in the Stat1-independent pathway is revealed by studies with geldanamycin, an HSP90-specific inhibitor, and by expression of a mutant of p50(cdc37) that is unable to recruit HSP90 to the Raf-1 complex. Both agents abrogated the IFN-gamma-dependent induction of c-myc expression in Stat1-null cells.

Activation of human O6-methylguanine-DNA methyltransferase gene by glucocorticoid hormone.

O6-methylguanine-DNA methyltransferase (MGMT), a ubiquitous DNA repair protein, removes the mutagenic DNA adduct O6-alkylguanine, which is synthesized both endogenously and after exposure to alkylnitrosamines and alkylating antitumor drugs such as 2-chloroethyl-N-nitrosourea (CNU). The MGMT gene is highly regulated in mammalian cells and its overexpression, observed in many types of tumor cells, is often associated with cellular resistance to CNU. Dexamethasone, a synthetic glucocorticoid hormone, was found to increase MGMT expression in HeLa S3 cells, concomitant with their increased resistance to CNU. Two putative glucocorticoid responsive elements (GREs) were identified in the human MGMT (hMGMT) promoter. Transient expression of the luciferase reporter gene driven by an hMGMT promoter fragment containing these GREs was activated by dexamethasone. DNase I footprinting assays demonstrated the binding of glucocorticoid receptor to these sequences. In vitro transcription experiment showed that these DNA sequences are functional in glucocorticoid receptor signal-mediated activation of transcription. These results suggest glucocorticoid-mediated induction of the MGMT gene contributes to high level expression of MGMT.

Regulation of expression of the DNA repair gene O6-methylguanine-DNA methyltransferase via protein kinase C-mediated signaling.

O6-Alkylguanine is the major mutagenic and cytotoxic DNA lesion induced by alkylating agents, including 2-chloroethyl-N-nitrosourea-based antitumor drugs. This lesion is repaired by O6-methylguanine-DNA methyltransferase (MGMT), the expression of which is highly variable in both normal tissues and in tumor cells. The promoter of the human MGMT gene was found to contain two putative activator protein (AP)-1 sites. Here, we show that the level of MGMT mRNA in HeLa S3 cells was increased 3-5-fold by phorbol-12-myristate-13-acetate (TPA) and 1,2-diacyl-sn-glycerol (DAG), which are activators of protein kinase C (PKC), as well as by okadaic acid, an inhibitor of protein phosphatases. The PKC inhibitor 1-(5-isoquinoline sulfonyl)-2-methylpiperazine-HCl eliminated MGMT activation by TPA and DAG but not by OA. Prior down-regulation of PKC abolished subsequent effects of TPA or DAG. The results indicate AP-1 to be involved in regulation of MGMT expression. This hypothesis was supported by showing AP-1 binding to two target sequences of the MGMT promoter and transactivation of the MGMT promoter upon cotransfection with c-fos and c-jun in F9 cells. That TPA-mediated induction of MGMT caused increased cellular resistance to 2-chloroethyl-N-nitrosourea suggests a therapeutic significance for PKC-mediated MGMT modulation.

Activation of apurinic/apyrimidinic endonuclease in human cells by reactive oxygen species and its correlation with their adaptive response to genotoxicity of free radicals.

Apurinic/apyrimidinic (AP) endonuclease (APE; EC 4.2.99.18) plays a central role in repair of DNA damage due to reactive oxygen species (ROS) because its DNA 3'-phosphoesterase activity removes 3' blocking groups in DNA that are generated by DNA glycosylase/AP-lyases during removal of oxidized bases and by direct ROS reaction with DNA. The major human APE (APE-1) gene is activated selectively by sublethal levels of a variety of ROS and ROS generators, including ionizing radiation, but not by other genotoxicants-e.g., UV light and alkylating agents. Increased expression of APE mRNA and protein was observed both in the HeLa S3 tumor line and in WI 38 primary fibroblasts, and it was accompanied by translocation of the endonuclease to the nucleus. ROS-treated cells showed a significant increase in resistance to the cytotoxicity of such ROS generators as H2O2 and bleomycin, but not to UV light. This "adaptive response" appears to result from enhanced repair of cytotoxic DNA lesions due to an increased activity of APE-1, which may be limiting in the base excision repair process for ROS-induced toxic lesions.

Stabilization and activation of p53 are regulated independently by different phosphorylation events.

Treatment of mouse or human cells with the protein kinase C (PKC) inhibitors H7 or bisindolylmaleimide I induced an increase in the lifetime of p53, leading to its accumulation. In inhibitor-treated cells, p53 translocated to the nuclei and bound to DNA but was not competent to induce transcription. However, transactivation could be induced by subsequent DNA damage. Phorbol ester, a potent activator of PKC, significantly inhibited the accumulation of p53 after DNA damage. Therefore, constitutive PKC-dependent phosphorylation of p53 itself, or of a protein that interacts with p53, is required for the rapid degradation of p53 in untreated cells. Furthermore, an increase in the lifetime of p53 is not accompanied necessarily by its activation. Treatment with the PKC inhibitors decreased the overall level of p53 phosphorylation but led to the appearance of a phosphopeptide not seen in tryptic digests of p53 from untreated cells. Therefore, the lifetime and activities of p53 are likely to be regulated by distinct alterations of the phosphorylation pattern of p53, probably caused by the actions of different kinases.

Structural organization of the mouse DNA repair gene, N-methylpurine-DNA glycosylase.

N-Methylpurine-DNA glycosylase (MPG), a ubiquitous DNA repair enzyme, removes N-alkylpurines and other purine lesions induced in DNA by simple alkylating carcinogens. A mouse MPG cDNA clone was isolated from a lambda recombinant phage library of BALB/c mouse lung cell and characterized. Using the mouse MPG cDNA as a probe, the complete mouse MPG gene was isolated in two overlapping lambda recombinant genomic clones. The 6-kb gene has four exons containing 1,002 bp of coding sequence. The transcription start site was identified in the genomic sequence by primer extension of MPG mRNA from a mouse lung fibroblast cell line. The location of this transcription start site was confirmed by in vitro transcription with the promoter-containing plasmid template. Promoter function of the sequence 5' upstream of the transcription initiation site was shown by transient expression of the firefly luciferase reporter gene under the control of this sequence in transfected human and mouse cells. The mouse MPG promoter contains no TATA box, but has a CAAT element and is G.C-rich with putative AP2 elements and SP1-complementary sequences.