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J Shao

Publications and source records attributed to J Shao.

At least 19 recordsLinked to original sources
PubMed2006

Antisense Smo under the control of the PTCH1 promoter delivered by an adenoviral vector inhibits the growth of human pancreatic cancer.

Hedgehog (Hh) signaling pathway is crucial in growth and patterning during embryonic development. Recent data have shown an association of its activation with cancer formation and maintenance. A ligand-dependent activation, where Hh components (SHH, PTCH1, Smo and GLi1) are aberrantly expressed with PTCH1 being a negative feedback regulator, is a newly identified mechanism for pancreatic carcinogenesis. In this study, we developed a cell-specific cytotoxic model for the treatment of human pancreatic cancer (HPC) in which expression of antisense Smo (SAS) was under the control of the PTCH1 promoter (ptch/p) delivered by an adenoviral vector (Ad-ptch/p-SAS). We observed that the cell-specific cytotoxicity in HPC cells depended on the expressions of inherent PTCH1, Smo and GLi1 in the target cells in which the Hh pathway was presumed to be activated. Fluorescence-activated cell sorting analysis indicated that the cell death was apoptosis. Western blot showed that Smo protein in the infected cells significantly decreased. Furthermore, an in vivo experiment demonstrated that such Hh activity-cell-specific cytotoxicity was achieved by daily intratumoral injection of Ad-ptch/p-SAS (10(9) plaque-forming unit) for 5 days. Our study suggests that targeting at the Hh signaling pathway may be an effective novel gene therapeutic strategy alone or in combination with other agents for the treatment of pancreatic cancer.

Adenoviridae↗
PubMed2006

Alterations in intracerebral hemorrhage-induced brain injury in the iron deficient rat.

BACKGROUND: Iron contributes to brain edema and cellular toxicity after intracerebral hemorrhage (ICH). Knowledge regarding ICH in the context of iron deficiency anemia (IDA), a common nutritional disorder, is limited. OBJECTIVE: To determine the effect of IDA on brain and behavioral outcome after ICH in rats. METHODS: Six-week-old male rats (n = 75) were randomized to non-IDA or IDA groups. After 1 month of iron sufficient or deficient diets, 100 microl autologous blood was infused into the right basal ganglia (BG). Brains were assessed for iron concentration, regional water content, BG transferrin, and transferrin receptor concentrations after ICH. Recovery of upper extremity sensorimotor function was assessed. Brain and behavioral variables were compared by diet group. Significance was set at p < 0.05. RESULTS: Whole brain iron was decreased and water content was increased for IDA rats in injured cortex and BG at day 3 (p < 0.05) compared with non-IDA rats. Transferrin and transferrin receptor content were increased in injured BG for IDA compared to non-IDA in the first week after ICH (p < 0.05). IDA rats had greater left vibrissae-stimulated forelimb-placing deficits and forelimb-use asymmetry than non-IDA after ICH (p < 0.05). CONCLUSIONS: Brain iron status may be an important determinant of injury severity and recovery after ICH.

Anemia, Iron-Deficiency↗
PubMed2006

Ribonucleotide reductase inhibitors and future drug design.

Ribonucleotide reductase (RR) is a multisubunit enzyme responsible for the reduction of ribonucleotides to their corresponding deoxyribonucleotides, which are building blocks for DNA replication and repair. The key role of RR in DNA synthesis and cell growth control has made it an important target for anticancer therapy. Increased RR activity has been associated with malignant transformation and tumor cell growth. Efforts for new RR inhibitors have been made in basic and translational research. In recent years, several RR inhibitors, including Triapine, Gemcitabine, and GTI-2040, have entered clinical trial or application. Furthermore, the discovery of p53R2, a p53-inducible form of the small subunit of RR, raises the interest to develop subunit-specific RR inhibitors for cancer treatment. This review compiles recent studies on (1) the structure, function, and regulation of two forms of RR; (2) the role in tumorigenesis of RR and the effect of RR inhibition in cancer treatment; (3) the classification, mechanisms of action, antitumor activity, and clinical trial and application of new RR inhibitors that have been used in clinical cancer chemotherapy or are being evaluated in clinical trials; (4) novel approaches for future RR inhibitor discovery.

Animals↗
PubMed2005

Therapeutic actions of an insulin receptor activator and a novel peroxisome proliferator-activated receptor gamma agonist in the spontaneously hypertensive obese rat model of metabolic syndrome X.

Insulin resistance clusters with hyperlipidemia, impaired glucose tolerance, and hypertension as metabolic syndrome X. We tested a low molecular weight insulin receptor activator, demethylasterriquinone B-1 (DMAQ-B1), and a novel indole peroxisome proliferator-activated receptor gamma agonist, 2-(2-(4-phenoxy-2-propylphenoxy)ethyl)indole-5-acetic acid (PPEIA), in spontaneously hypertensive obese rats (SHROB), a genetic model of syndrome X. Agents were given orally for 19 days. SHROB showed fasting normoglycemia but impaired glucose tolerance after an oral load, as shown by increased glucose area under the curve (AUC) [20,700 mg x min/ml versus 8100 in lean spontaneously hypertensive rats (SHR)]. Insulin resistance was indicated by 20-fold excess fasting insulin and increased insulin AUC (6300 ng x min/ml versus 990 in SHR). DMAQ-B1 did not affect glucose tolerance (glucose AUC = 21,300) but reduced fasting insulin 2-fold and insulin AUC (insulin AUC = 4300). PPEIA normalized glucose tolerance (glucose AUC = 9100) and reduced insulin AUC (to 3180) without affecting fasting insulin. PPEIA also increased food intake, fat mass, and body weight gain (81 +/- 12 versus 45 +/- 8 g in untreated controls), whereas DMAQ-B1 had no effect on body weight but reduced subscapular fat mass. PPEIA but not DMAQ-B1 reduced blood pressure. In skeletal muscle, insulin-stimulated phosphorylation of the insulin receptor and insulin receptor substrate protein 1-associated phosphatidylinositol 3-kinase activity were decreased by 40 to 55% in SHROB relative to lean SHR. PPEIA, but not DMAQ-B1, enhanced both insulin actions. SHROB also showed severe hypertriglyceridemia (355 +/- 42 mg/dl versus 65 +/- 3 in SHR) attenuated by both agents (DMAQ-B1, 228 +/- 18; PPEIA, 79 +/- 3). Both these novel antidiabetic agents attenuate insulin resistance and hypertriglyceridemia associated with metabolic syndrome but via distinct mechanisms.

Acetates↗
PubMed2005

Biofilm Formation by Neisseria gonorrhoeae.

Studies were performed in continuous-flow chambers to determine whether Neisseria gonorrhoeae could form a biofilm. Under these growth conditions, N. gonorrhoeae formed a biofilm with or without the addition of 10 microM sodium nitrite to the perfusion medium. Microscopic analysis of a 4-day growth of N. gonorrhoeae strain 1291 revealed evidence of a biofilm with organisms embedded in matrix, which was interlaced with water channels. N. gonorrhoeae strains MS11 and FA1090 were found to also form biofilms under the same growth conditions. Cryofield emission scanning electron microscopy and transmission electron microscopy confirmed that organisms were embedded in a continuous matrix with membranous structures spanning the biofilm. These studies also demonstrated that N. gonorrhoeae has the capability to form a matrix in the presence and absence of CMP-N-acetylneuraminic acid (CMP-Neu5Ac). Studies with monoclonal antibody 6B4 and the lectins soy bean agglutinin and Maackia amurensis indicated that the predominate terminal sugars in the biofilm matrix formed a lactosamine when the biofilm was grown in the absence of CMP-Neu5Ac and sialyllactosamine in the presence of CMP-Neu5Ac. N. gonorrhoeae strain 1291 formed a biofilm on primary urethral epithelial cells and cervical cells in culture without loss of viability of the epithelial cell layer. Our studies demonstrated that N. gonorrhoeae can form biofilms in continuous-flow chambers and on living cells. Studies of these biofilms may have implications for understanding asymptomatic gonococcal infection.

Biofilms↗
PubMed2004

Nontypeable Haemophilus influenzae strain 2019 produces a biofilm containing N-acetylneuraminic acid that may mimic sialylated O-linked glycans.

Previous studies suggested that nontypeable Haemophilus influenzae (NTHI) can form biofilms during human and chinchilla middle ear infections. Microscopic analysis of a 5-day biofilm of NTHI strain 2019 grown in a continuous-flow chamber revealed that the biofilm had a diffuse matrix interlaced with multiple water channels. Our studies showed that biofilm production was significantly decreased when a chemically defined medium lacking N-acetylneuraminic acid (sialic acid) was used. Based on these observations, we examined mutations in seven NTHI strain 2019 genes involved in carbohydrate and lipooligosaccharide biosynthesis. NTHI strain 2019 with mutations in the genes encoding CMP-N-acetylneuraminic acid synthetase (siaB), one of the three NTHI sialyltransferases (siaA), and the undecaprenyl-phosphate alpha-N-acetylglucosaminyltransferase homolog (wecA) produced significantly smaller amounts of biofilm. NTHI strain 2019 with mutations in genes encoding phosphoglucomutase (pgm), UDP-galactose-4-epimerase, and two other NTHI sialyltransferases (lic3A and lsgB) produced biofilms that were equivalent to or larger than the biofilms produced by the parent strain. The biofilm formed by the NTHI strain 2019pgm mutant was studied with Maackia amurensis fluorescein isothiocyanate (FITC)-conjugated and Sambucus nigra tetramethyl rhodamine isocyanate (TRITC)-conjugated lectins. S. nigra TRITC-conjugated lectin bound to this biofilm, while M. amurensis FITC-conjugated lectin did not. S. nigra TRITC-conjugated lectin binding was inhibited by incubation with alpha2,6-neuraminyllactose and by pretreatment of the biofilm with Vibrio cholerae neuraminidase. Matrix-assisted laser desorption ionization-time of flight mass spectometry analysis of lipooligosaccharides isolated from a biofilm, the planktonic phase, and plate-grown organisms showed that the levels of most sialylated glycoforms were two- to fourfold greater when the lipooligosaccharide was derived from planktonic or biofilm organisms. Our data indicate that NTHI strain 2019 produces a biofilm containing alpha2,6-linked sialic acid and that the sialic acid content of the lipooligosaccharides increases concomitant with the transition of organisms to a biofilm form.

Biofilms↗
PubMed2003

Phosphatidylinositol 3-kinase mediates proliferative signals in intestinal epithelial cells.

BACKGROUND AND AIMS: Determination of intracellular signalling pathways that mediate intestinal epithelial proliferation is fundamental to the understanding of the integrity and function of the intestinal tract under normal and diseased conditions. The phosphoinositide 3-kinase (PI3K)/Akt pathway transduces signals initiated by growth factors and is involved in cell proliferation and differentiation. In this study, we assessed the role of PI3K/Akt in transduction of proliferative signals in intestinal epithelial cells. METHODS: A rat intestinal epithelial (RIE) cell line and human colorectal cancer HCA-7 and LS-174 cell lines served as in vitro models. The Balb/cJ mouse was the in vivo model. RESULTS: PI3K activation was critical for G1 cell cycle progression of intestinal epithelial cells. Ectopic expression of either active p110alpha or Akt-1 increased RIE cell proliferation. In vivo experiments demonstrated that PI3K activation was closely associated with the proliferative activity of intestinal mucosa. Treatment of mice with PI3K inhibitors blocked induction of PI3K activity and attenuated intestinal mucosal proliferation associated with oral intake. Epidermal growth factor and transforming growth factor alpha stimulated PI3K activation which was required for growth factor induced expression of cyclin D1. CONCLUSIONS: The PI3K/Akt pathway transduces mitogenic signals from growth factor receptors to the cell cycle machinery and plays a critical role in regulation of intestinal epithelial proliferation.

Animals↗
PubMed2002

Apical barriers to airway epithelial cell gene transfer with amphotropic retroviral vectors.

Gene transfer to airway epithelia with amphotropic pseudotyped retroviral vectors is inefficient following apical vector application. To better understand this inefficiency, we localized the expression of Pit2, the amphotropic receptor, in polarized human airway epithelia. Pit2 was expressed on both the apical and basolateral surfaces of the cells, suggesting that factors other than receptor abundance may limit apical gene transfer efficiency. Binding studies performed with radiolabeled amphotropic MuLV suggested that the apically applied virus binds to Pit2. Hypothetical barriers to retroviral gene transfer include the apical glycocalyx and other secreted products of epithelia. In this study, we demonstrated that sialic acid, keratan sulfate and collagen type V are present on the apical surface of well-differentiated human airway epithelia. While enzyme treatment reduced the abundance of these components, the treatment also decreased the transepithelial resistance to approximately 35% of the controls, suggesting that the epithelial integrity was impaired. To attain an airway epithelial culture with a modified apical surface and intact epithelial integrity, we utilized 100 mM 2-deoxy-D-glucose, a glycosylation inhibitor, to prevent the glycocalyx from reforming following enzyme treatment. This approach allowed the resistance, but not the apical glycocalyx to recover. Despite this physical modification of the cell surface, the amphotropic retroviral vector failed to transduce airway epithelia following apical application. These results suggest that factors other than apical receptor abundance and the glycocalyx inhibit amphotropic retroviral gene transfer in human airway epithelia.

Cell Polarity↗
PubMed2001

Heat shock inhibits IL-12 p40 expression through NF-kappa B signalling pathway in murine macrophages.

We report the effect of heat shock on lipopolysaccharide (LPS)-induced interleukin 12 (IL-12) expression. The augmentation of LPS-induced IL-12 p40 mRNA and p70 protein was significantly suppressed in both peritoneal macrophages and RAW264.7 cells after heat shock at 43 degrees C. The binding activity of nuclear factor kappa B (NF-kappa B) was reduced by prior heat shock. LPS did not induce degradation of the inhibitory protein I-kappa B alpha in the shocked cells, which might be a potential mechanism to block NF-kappa B activation. Furthermore, transient transfection assay in RAW264.7 cells demonstrated that LPS-induced activation of DM703 and DM138 (contains NF-kappa B motif) was highly sensitive to heat shock. These data suggest that heat shock influences expression of IL-12 through the I-kappa B/NF-kappa B pathway.

Animals↗
PubMed2001

Interaction of acetylene and cyanide with the resting state of nitrogenase alpha-96-substituted MoFe proteins.

The nitrogenase MoFe protein contains the active site metallocluster called FeMo-cofactor [7Fe-9S-Mo-homocitrate] that exhibits an S = 3/2 EPR signal in the resting state. No interaction with FeMo-cofactor is detected when either substrates or inhibitors are incubated with MoFe protein in the resting state. Rather, the detection of such interactions requires the incubation of the MoFe protein together with its obligate electron donor, called the Fe protein, and MgATP under turnover conditions. This indicates that a more reduced state of the MoFe protein is required to accommodate substrate or inhibitor interaction. In the present work, substitution of an arginine residue (alpha-96(Arg)) located next to the active site FeMo-cofactor in the MoFe protein by leucine, glutamine, alanine, or histidine is found to result in MoFe proteins that can interact with acetylene or cyanide in the as-isolated, resting state without the need for the Fe protein, or MgATP. The dithionite-reduced, resting states of the alpha-96(Leu)-, alpha-96(Gln)-, alpha-96(Ala)-, or alpha-96(His)-substituted MoFe proteins show an S = 3/2 EPR signal (g = 4.26, 3.67, 2.00) similar to that assigned to FeMo-cofactor in the wild-type MoFe protein. However, in contrast to the wild-type MoFe protein, the alpha-96-substituted MoFe proteins all exhibit changes in their EPR spectra upon incubation with acetylene or cyanide. The alpha-96(Leu)-substituted MoFe protein was representative of the other alpha-96-substituted MoFe proteins examined. The incubation of acetylene with the alpha-96(Leu) MoFe protein decreased the intensity of the normal FeMo-cofactor signal with the appearance of a new EPR signal having inflections at g = 4.50 and 3.50. Incubation of cyanide with the alpha-96(Leu) MoFe protein also decreased the FeMo-cofactor EPR signal with concomitant appearance of a new EPR signal having an inflection at g = 4.06. The acetylene- and cyanide-dependent EPR signals observed for the alpha-96(Leu)-substituted MoFe protein were found to follow Curie law 1/T dependence, consistent with a ground-state transition as observed for FeMo-cofactor. The microwave power dependence of the EPR signal intensity is shifted to higher power for the acetylene- and cyanide-dependent signals, consistent with a change in the relaxation properties of the spin system of FeMo-cofactor. Finally, the alpha-96(Leu)-substituted MoFe protein incubated with (13)C-labeled cyanide displays a (13)C ENDOR signal with an isotropic hyperfine coupling of 0.42 MHz in Q-band Mims pulsed ENDOR spectra. This indicates the existence of some spin density on the cyanide, and thus suggests that the new component of the cyanide-dependent EPR signals arise from the direct bonding of cyanide to the FeMo-cofactor. These data indicate that both acetylene and cyanide are able to interact with FeMo-cofactor contained within the alpha-96-substituted MoFe proteins in the resting state. These results support a model where effective interaction of substrates or inhibitors with FeMo-cofactor occurs as a consequence of both increased reactivity and accessibility of FeMo-cofactor under turnover conditions. We suggest that, for the wild-type MoFe protein, the alpha-96(Arg) side chain acts as a gatekeeper, moving during turnover in order to permit accessibility of acetylene or cyanide to a specific [4Fe-4S] face of FeMo-cofactor.

Acetylene↗
PubMed2001

Photochemical and electrochemical properties of zinc chlorin-C60 dyad as compared to corresponding free-base chlorin-C60, free-base porphyrin-C60, and zinc porphyrin-C60 dyads.

The photochemical and electrochemical properties of four chlorin-C60 or porphyrin-C60 dyads having the same short spacer between the macrocycle and the fullerene are examined. In contrast with all the previous results on porphyrin-fullerene dyads, the photoexcitation of a zinc chlorin-C60 dyad results in an unusually long-lived radical ion pair which decays via first-order kinetics with a decay rate constant of 9.1 x 10(3) x s(-1). This value is 2-6 orders of magnitude smaller than values reported for all other porphyrin or chlorin donor-acceptor of the molecule dyad systems. The formation of radical cations of the donor part and the radical anion of the acceptor part was also confirmed by ESR measurements under photoirradiation at low temperature. The photoexcitation of other dyads (free-base chlorin-C60, zinc porphyrin-C60, and free-base porphyrin-C60 dyads) results in formation of the ion pairs which decay quickly to the triplet excited states of the chlorin or porphyrin moiety via the higher lying radical ion pair states as is expected from the redox potentials.

Carbon↗
PubMed2001

Alkyl and aryl substituted corroles. 1. Synthesis and characterization of free base and cobalt containing derivatives. x-ray structure of (Me(4)Ph(5)Cor)Co(py)(2).

The synthesis, spectroscopic properties, and electrochemistry of six different alkyl- and aryl-substituted Co(III) corroles are presented. The investigated compounds contain methyl, ethyl, phenyl, or substituted phenyl groups at the eight beta-positions of the corrole macrocycle and four derivatives also contain a phenyl group at the 10-meso position of the macrocycle. Each cobalt corrole undergoes four reversible oxidations in CH(2)Cl(2) containing 0.1 M tetra-n-butylammonium perchlorate and exists as a dimer in its singly and doubly oxidized forms. The difference in potential between the first two oxidations is associated with the degree of interaction between the two corrole units of the dimer and ranges from an upper value of 0.62 V, in the case of (Me(6)Et(2)Cor)Co, to a lower value of about 0.17 V, in the case of four compounds which have a phenyl group located at the 10-meso position of the macrocycle. These Co(III) corroles strongly coordinate two pyridine molecules or one carbon monoxide molecule in CH(2)Cl(2) media, and ligand binding constants were evaluated using spectroscopic and electrochemical methods. The structure of (Me(4)Ph(5)Cor)Co(py)(2) was also determined by X-ray diffraction. Crystal data: (Me(4)Ph(5)Cor)Co(py)(2).3CH(2)Cl(2).H(2)O, orthorhombic, a = 19.5690(4) A, b = 17.1070(6) A, c = 15.9160(6) A, V = 5328.2(5) A(3), space group Pna2(1), Z = 2, 35 460 observations, R(F) = 0.069.

Journal Article↗
PubMed2001

Alkyl and aryl substituted corroles. 2. Synthesis and characterization of linked "face-to-face" biscorroles. X-ray structure of (BCA)Co(2)(py)(3), where BCA represents a biscorrole with an anthracenyl bridge.

The synthesis, spectroscopic properties, and electrochemistry of (BCA)Co(2) and (BCB)Co(2) are described where BCA and BCB represent biscorroles linked by an anthracenyl (A) or a biphenylenyl (B) bridge. The pyridine and CO binding properties of (BCA)Co(2) and (BCB)Co(2) are also presented, and one of the compounds in its pyridine-ligated form, (BCA)Co(2)(py)(3), is structurally characterized. The data on the biscorroles are compared on one hand to the monocorrole having the same substitution pattern and on the other hand to bisporphyrins having two Co(II) ions and the same anthracenyl or biphenylenyl linkers in order to better understand the interaction which occurs between the two corrole macrocycles. A parallel study on five different Co(III) phenyl-substituted corroles showed that bis-pyridine and mono-CO adducts are readily formed from the complexes in CH(2)Cl(2). This present paper examines how the ligand binding properties and electrochemistry of these Co(III) corroles are modified by the anthracenyl or biphenylenyl bridge which links the two macrocycles in a face to face orientation. An X-ray crystal structure was obtained for the tris-pyridine adduct of the anthracenyl bridged derivative, (BCA)Co(2)(py)(3), and gives the following results: C(127)H(99)Co(2)N(11).2CHCl(3), M = 2135.90, triclinic, space group P&onemacr;, a = 13.2555(5) A, b = 18.6406(8) A, c = 22.2140(9) A, alpha = 94.186(9) degrees, beta = 102.273(9) degrees, gamma = 94.205(9) degrees, V = 5326.8(4) A(3), 9293 independent reflections collected, R(F) = 0.066.

Journal Article↗
PubMed2001

Copper(II) complexes of novel N-alkylated derivatives of cis,cis-1,3,5-triaminocyclohexane. 1. Preparation and structure.

Novel N,N',N' '-trialkylated derivatives of cis,cis-1,3,5-triaminocyclohexane (tach), designated tach-R(3), were prepared through alkylation of N-protected tach with subsequent acid deprotection, to afford N-methyl, N-ethyl, and N-n-propyl derivatives as their trihydrobromide salts. The tach-neopentyl(3) and tach-furan(3) derivatives were prepared by formation of the imine from tach and pivaldehyde or furan-2-carboxaldehyde, respectively, followed by reduction of the imine. Complexes [Cu(tach-R(3))Cl(2)] (R = Me, Et, n-Pr, CH(2)-2-thienyl, and CH(2)-2-furanyl) were prepared from CuCl(2) in MeOH or MeOH-Et(2)O solvent. Crystallographic characterization of [Cu(tach-Et(3))Br(0.8)Cl(1.2)] (Pnma, a = 8.2265(1) A, b = 12.5313(1) A, c = 15.3587(3) A, Z = 4) reveals a square-based pyramidal CuN(3)X(2) coordination sphere in which one nitrogen donor occupies the apical position at a slightly longer distance (Cu-N = 2.218(5) A) than those of the basal nitrogens (Cu-N = 2.053(2) A). The solution-phase (pH 7.4 buffered and methanol) and solid-phase structures of [Cu(tach-R(3))Cl(2)] have been studied extensively by EPR and visible-near-IR spectroscopies. The square-based pyramidal structure is retained in solution, according to correspondence of solution and solid-state data. In aqueous solution, halide is replaced by water, as indicated by the high-energy UV-vis spectral shifts and bonding parameters of [Cu(tach-Et(3))](2+)(aq) derived from EPR data. The proposed aqueous-phase species, in the pH range 7.4 to 10.1, is [Cu(tach-Et(3))(H(2)O)(2)](2+). The complex [Cu(tach-Me(3))](2+)(aq) does not appear to dimerize or form metal-hydroxo species at pH 7.4, in contrast to other Cu(II)-triamine complexes, e.g., [Cu(1,4,7-triazacyclononane)](2+) (aq) and [Cu(tach-H(3))](2+)(aq) (the complex of unalkylated tach). This difference is attributed to the steric effect of the N-alkyl groups in the tach-R(3) series.

Alkylation↗
PubMed2001

Copper(II) complexes of novel N-alkylated derivatives of cis,cis-1,3,5-triaminocyclohexane. 2. Metal-promoted phosphate diester hydrolysis.

Aqueous copper(II) N,N',N' '-trimethyl-cis,cis-1,3,5-triaminocyclohexane (Cu(tach-Me(3))(2+)(aq)) promotes the hydrolysis of activated phosphate diesters in aqueous medium at pH 7.2. This complex is selective for cleavage of the phosphate diester sodium bis(p-nitrophenyl) phosphate (BNPP), the rate of hydrolysis of the monoester disodium p-nitrophenyl phosphate being 1000 times slower. The observed rate acceleration of BNPP hydrolysis is slightly greater than that observed for other Cu(II) complexes, such as [Cu([9]aneN(3))Cl(2)] ([9]aneN(3) identical with 1,4,7-triazacyclononane). The rate of hydrolysis is first-order in phosphate ester at low ester concentration and second-order in [Cu(tach-Me(3))](2+)(aq), suggesting the involvement of two metal complexes in the mechanism of substrate hydrolysis. The reaction exhibits saturation kinetics with respect to BNPP concentration according to a modified Michaelis-Menten mechanism: 2CuL + S <==> LCu-S-CuL --> 2CuL + products (K(M) = 12.3 +/- 1.8 mM(2), k(cat) = (4.0 +/- 0.4) x 10(-)(4) s(-1), 50 degrees C) where CuL (triple bond) [Cu(tach-Me(3))](2+), S (triple bond) BNPP, and LCu-S-CuL is a substrate-bridged dinuclear complex. EPR data indicate that the dicopper complex is formed only in the presence of BNPP; the active LCu-S-CuL intermediate species then slowly decays to products, regenerating monomeric CuL.

Copper↗
PubMed2001

Transforming growth factor-beta1 promotes invasiveness after cellular transformation with activated Ras in intestinal epithelial cells.

Invasion is a defining event in carcinoma progression. In general, invasive carcinoma is characterized by an epithelial-fibroblastoid conversion associated with loss of cell-cell adhesion receptors such as E-cadherin and beta-catenin. We report here that TGF-beta1 promotes the invasiveness by modulating the alterations of cellular plasticity including a loss of cell-cell contact in Ras-transformed epithelial cells. In order to examine the role of TGF-beta1 in the Ras-induced responses, intestinal epithelial cells expressing a conditionally activated Ha-Ras(Val12) (RIE-iRas cells) were used in this study. Induced expression of activated Ha-Ras(Val12) caused morphologic transformation of the RIE-iRas cells with an increase in vimentin expression and a decrease of E-cadherin levels. There was also redistribution of beta-catenin from the cytoplasm to the nucleus after the induction of Ras. TGF-beta1 treatment enhanced both the decrease in E-cadherin levels and the redistribution of beta-catenin. Interestingly, the activation of Ras markedly decreased the level of TGF-beta receptor type II (TbetaRII) in RIE-iRas cells. However, the expression of plasminogen activator inhibitor-1, which is known to be transcriptionally induced by TGF-beta1, was strongly induced by TGF-beta1 despite the marked downregulation of TbetaRII. The induction of Ha-Ras(Val12) markedly increased the invasiveness in RIE-iRas cells, as evaluated by a collagen type I-coated Boyden-chamber assay, and the Ras-mediated invasiveness was significantly enhanced by TGF-beta1 treatment. Expression of a dominant-negative form of TbetaRII in the RIE-iRas cells abrogated both growth-inhibitory and invasion responses to TGF-beta1. Collectively, these results suggest that TGF-beta1 and oncogenic Ras collaborate in promoting cellular invasiveness in intestinal epithelial cells. The enhancement of invasiveness was correlated with decreased E-cadherin levels and subcellular distribution of beta-catenin. The enhancement of oncogenic Ras-mediated cell transformation by TGF-beta1 occurs via TbetaRII.

Adherens Junctions↗
PubMed2001

Two-phase shelf-life estimation.

Unlike most drug products, some drug products must be stored at several temperatures, such as -20 degrees C, 5 degrees C and 25 degrees C, in order to maintain the stability of the drug products. Drug products of this kind are usually referred to as frozen drug products. The determination of shelf-life for frozen drug products involves the estimation of drug shelf-lives at different temperatures, which requires multiple phase linear regression. Mellon suggested obtaining a combined shelf-life by determining shelf-lives based on data available at different temperatures. This method, however, does not account for the fact that the shelf-life at the second-phase would depend on the shelf-life at the first phase. As an alternative, we propose a method for determination of drug shelf-lives for the two phases using a two-phase regression analysis based on the statistical principle described in the FDA guideline and the ICH guideline. A numerical example is presented to illustrate the proposed method.

Confidence Intervals↗