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A new metal–organic framework, [Pb4(bpdc)4(H2O)2]·3H2O (1) (H2bpdc = 2,2′-bipyridine-3,3′-dicarboxylate), has been synthesized under hydrothermal conditions and characterized by single crystal X-ray diffraction, powder X-ray diffraction, IR spectroscopy, TGA and elemental analyses. Compound 1 shows 4-connected 2-D lattice structures which are assembled by novel [Pb4(bpdc)4(H2O)2] tetranuclear units and bpdc ligands with different coordination modes that have never been reported. In addition, compound 1 exhibits blue fluorescent emissions at λmax = 421 nm in the solid state at room temperature.

Perovskite KTa0.65Nb0.35O3 (KTN) thin films were grown by pulsed laser deposition (PLD) on single crystal SrTiO3 (100) substrates. X-ray diffraction (XRD) analyses illustrate epitaxially grown of KTN thin films along the (100) orientation. The surface morphology of films observed by atomic force microscope (AFM) showed that, the surface of films was smooth and uniform built from regular, ordered and dense grains with the root mean square (RMS) roughness of 5.602 nm. Linear and third-order nonlinear optical properties of the films were investigated by using transmission spectra as well as the Z-scan technique with femtosecond laser pulses, respectively. The open-aperture and closed-aperture Z-scan curves of KTN thin films were obtained in the first measurement. The calculated nonlinear refractive index was , the nonlinear absorption coefficient is = , and the real part and imaginary part of the third order nonlinear susceptibility are and respectively.

The urokinase plasminogen activator system is involved in angiogenesis and tumor growth of malignant gliomas, which are highly neovascularized and so may be amenable to antiangiogenic therapy. In this paper, we describe the activity of Å6, an octamer capped peptide derived from the non-receptor-binding region of urokinase plasminogen activator. Å6 inhibited human microvascular endothelial cell migration but had no effect on the proliferation of human microvascular endothelial cells or U87MG glioma cells in vitro . In contrast, Å6 or cisplatin (CDDP) alone suppressed subcutaneous tumor growth in vivo by 48% and 53%, respectively, and, more strikingly, the combination of Å6 plus CDDP inhibited tumor growth by 92%. Such combination treatment also greatly reduced the volume of intracranial tumor xenografts and increased survival of tumor-bearing animals when compared with CDDP or Å6 alone. Tumors from the combination treatment group had significantly reduced neovascularization, suggesting a mechanism involving Å6-mediated inhibition of endothelial cell motility, thereby eliciting vascular sensitivity to CDDP-mediated toxicity. These data suggest that the combination of an angiogenesis inhibitor that targets endothelial cells with a cytotoxic agent may be a useful therapeutic approach.

The peroxiredoxin (Prx) protein is expressed widely in animal tissues and serves an antioxidant function associated with removal of cellular peroxides. We have cloned two Prx genes and observed differential expression of Prx-I and Prx-II (formerly NKEF-A and NKEF-B) in purified rat brain cell cultures (Sarafian et al. [1998] Mol. Chem. Neuropathol. 34:39-51). We have examined regional and cell-type-specific expression of Prx-I and Prx-II in paraffin sections of human brain using immunohistochemical methods. These studies revealed a clear segregation of expression of these two gene products in different brain cell types. In the cerebral cortex, cerebellum, basal ganglia, substantia nigra, and spinal cord, Prx-I was expressed primarily in astrocytes, while Prx-II was expressed exclusively in neurons. Prx-I was also prominently expressed in ependymal cells and subependymal matrix of substantia nigra and basal ganglia. Prx-II was not expressed at uniform density in all neurons. In general, small neurons such as cerebellar granule neurons displayed little or no staining, while large neurons, such as hippocampal pyramidal and Purkinje neurons were heavily stained. The absence of expression of Prx-I in neurons and the selective expression of Prx-II in large neurons suggest that these antioxidant enzymes serve distinct functional roles that may reflect the different functions and biochemical activities of these cell types. Restricted expression of these genes may also contribute to the selective vulnerability of these cells to a wide variety of neuropathologic conditions.

Several monoclonal antibodies (mAbs) and novel mAb-based assays for the androgen receptors (AR) have been developed. Large amounts of the recombinant human AR protein produced by a baculovirus expression system were used as an antigen to produce mAbs. Twenty-nine AR-specific mAbs were first confirmed by Western blot analysis and were then characterized for their immunoglobulin isotypes, epitopes, and epitope localization in AR. Novel assays using flow cytometry and sandwich enzyme-linked immunosorbent assays (ELISA) were established to detect AR-expressing cells and to quantify soluble AR protein, respectively. Using immunostaining, we identified several anti-AR mAbs exclusively recognizing AR within the nuclei of the prostate cancer cell line LNCaP and of prostate tissues in both frozen and paraffin-embedded sections, whereas other mAbs could detect AR in both nuclear and cytoplasmic compartments. Interestingly, certain mAbs, such as G122-25 and G122-77, could distinguish the androgen-bound AR from the unoccupied AR. In sum, many purified AR protein and anti-AR mAbs, together with the assays developed, could be powerful tools for the study of functional AR and for the diagnosis of prostatic cancers.

Serotonin (5-hydroxytryptamine, 5-HT) mediates many functions of the central and peripheral nervous systems by its interaction with specific neuronal and glial receptors. Fourteen serotonin receptors belonging to seven families have been identified through physiological, pharmacological, and molecular cloning studies. Monoclonal antibodies (MAbs) specific for each of these receptor subtypes are needed to characterize their expression, distribution, and function in embryonic, adult, and pathological tissues. In this article we report the development and characterization of MAbs specific to the serotonin 5-HT2Areceptor. To generate MAbs against 5-HT2AR, mice were immunized with the N-terminal domain of the receptor. The antigens were produced as glutathionine S-transferase (GST) fusion proteins in insect cells using a Baculovirus expression system. The hybridomas were initially screened by ELISA against the GST-5-HT2AR recombinant proteins and subsequently against GST control proteins to eliminate clones with unwanted reactivity. They were further tested by Western blotting against recombinant GST-5-HT2AR, rat and human brain lysate, and lysate from cell lines transfected with 5-HT2AR cDNA. One of the MAbs G186-1117, which recognizes a portion of the 5-HT2AR N-terminus, was selected for further characterization. G186-1117 reacted with a band of molecular size 55 kD corresponding to the predicted size of 5-HT2AR in lysates from rat brain and a 5-HT2AR-transfected cell line. Its specificity was further confirmed by adsorption of immunoreactivity with recombinant 5-HT2AR but not with recombinant 5-HT2BR and 5-HT2CR. Rat brain sections and Schwann cell cultures were immunohistochemically labeled with this MAb. G186-1117 showed differential staining in various regions of the rat brain, varying from regions with no staining to regions of intense reactivity. In particular, staining of cell bodies and dendrites of the pyramidal neurons in the cortex was observed, which is in agreement with observations of electrophysiological studies.

Deregulation of signaling by the epidermal growth factor receptor (EGFR) is common in human malignancy progression. One mutant EGFR (variously named DeltaEGFR, de2-7 EGFR, or EGFRvIII), which occurs frequently in human cancers, lacks a portion of the extracellular ligand-binding domain due to genomic deletions that eliminate exons 2 to 7 and confers a dramatic enhancement of brain tumor cell tumorigenicity in vivo. In order to dissect the molecular mechanisms of this activity, we analyzed location, autophosphorylation, and attenuation of the mutant receptors. The mutant receptors were expressed on the cell surface and constitutively autophosphorylated at a significantly decreased level compared with wild-type EGFR activated by ligand treatment. Unlike wild-type EGFR, the constitutively active DeltaEGFR were not down-regulated, suggesting that the altered conformation of the mutant did not result in exposure of receptor sequence motifs required for endocytosis and lysosomal sorting. Mutational analysis showed that the enhanced tumorigenicity was dependent on intrinsic tyrosine kinase activity and was mediated through the carboxyl terminus. In contrast with wild-type receptor, mutation of any major tyrosine autophosphorylation site abolished these activities suggesting that the biological functions of DeltaEGFR are due to low constitutive activation with mitogenic effects amplified by failure to attenuate signaling by receptor down-regulation.

The development and neoplastic progression of human astrocytic tumors appears to result through an accumulation of genetic alterations occurring in a relatively defined order. One such alteration is amplification of the epidermal growth factor receptor (EGFR) gene. This episomal amplification occurs in 40-50% of glioblastomas, which also normally express endogenous receptors. Moreover, a significant fraction of amplified genes are rearranged to specifically eliminate a DNA fragment containing exons 2-7 of the gene, resulting in an in-frame deletion of 801 bp of the coding sequence of the extracellular domain. Here we used retroviral transfer of such a mutant receptor (de 2-7 EGFR) into glioblastoma cells expressing normal endogenous receptors to test whether the mutant receptor was able to augment their growth and malignancy. Western blotting analysis showed that these cells expressed endogenous EGFR of 170 kDa as well as the exogenous de 2-7 EGFR of 140-155 kDa. Although holo-EGFRs were phosphorylated on tyrosine residues only after exposure of the cells to ligand, de 2-7 EGFRs were constitutively phosphorylated. In tissue culture neither addition of EGF nor expression of the mutant EGFR affected the rate of cell growth. However, when cells expressing mutant EGFR were implanted into nude mice subcutaneously or intracerebrally, tumorigenic capacity was greatly enhanced. These results suggest that a tumor-specific alteration of the EGFR plays a significant role in tumor progression perhaps by influencing interactions of tumor cells with their microenvironment in ways not easily assayed in vitro.

We study QCD radiative corrections to the nucleon's transverse spin-dependent structure function {ital g}{sub 2}({ital x},{ital Q}{sup 2}) in the leading-logarithmic order for large {ital Q}{sup 2}. The anomalous-dimension matrix for the nonsinglet twist-3 operators appearing in the operator-product expansion for {ital g}{sub 2}({ital x},{ital Q}{sup 2}) is calculated and the experimental implications are discussed.

Signal Transducer and Activator of Transcription 1 (STAT1) is phosphorylated at tyrosine 701 (pY701) upon cellular activation by interferons. This site-specific phosphorylation activates the signaling activity of STAT1 that translocates to the nucleus and regulates target gene transcription, which determines cellular growth, proliferation, and effector functions. An antibody that specifically recognizes STAT1 (pY701) was developed to study STAT1 roles played in experimental systems. Human tonsil sections, briefly cultured with a phosphatase inhibitor, revealed enhanced immunohistochemical (IHC) staining of nuclear STAT1 (pY701) in lymphocytes and epithelial cells when compared with untreated or phosphatase-treated controls. Similarly, in an in vivo model, liver sections prepared from rats treated with a phosphatase inhibitor revealed enhanced IHC staining of nuclear STAT1 (pY701) in hepatocytes compared with controls. The Ab also served as a specific probe in multicolor flow cytometric analyses designed to characterize the nature of lymphoid cells that express STAT1 (pY701) in response to cytokines. Exogenous interferon-alpha treated CD4+ and CD8+ T cells, B cells, monocytes and NK cells all were induced to express STAT1 (pY701) as determined by multicolor flow cytometric analysis. The applications of this Ab, as well as additional Abs specific for phosphorylated STATs 3-6, will be presented and discussed in terms of their use for cell analysis across multiple applications.

Unlike conventional biochemical methods for cell signaling analysis, analyzing protein levels by flow cytometry allows measurements to be made at the single-cell level. Multiparameter flow cytometry provides a powerful tool for the analysis of protein expression and phosphorylation status within complex populations, such as those found in lymphoid tissues or blood. However, different intracellular and cell surface proteins often require different conditions for optimal detection, creating challenges for analyzing multiple protein types in a single sample. To facilitate the design of multiparameter staining panels for analysis of intracellular and cell surface proteins, we tested over 250 fluorescently conjugated antibodies in cells fixed and permeabilized under various conditions. Multiple fluorescent conjugates of each antibody were tested in human PBMCs, human whole blood, mouse spleen, and/or mouse bone marrow following fixation with BD Phosflow™ Lyse/Fix or BD Cytofix™ Fixation Buffer and permeabilization with one of the four BD Phosflow™ Perm buffers. These studies demonstrated that certain intracellular epitopes required harsh permeabilization conditions for optimal detection, whereas others were detectable under milder conditions. Surface marker stains often required optimization, particularly under harsh permeabilization conditions. Factors such as antibody clone, fluorophore, and concentration had a considerable impact on staining resolution, and alternative staining protocols were required for optimal staining of some surface markers. To facilitate the optimization of fixation, permeabilization, and staining conditions for multiparameter flow cytometry, experimental data and protocols from this study are available through an interactive database freely accessible at www.cytobank.org/facselect. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 157. doi:1538-7445.AM2012-157