Biomedical subjects

F Zhang

Publications and source records attributed to F Zhang.

At least 19 recordsLinked to original sources

PubMed2000

Benzo[a]pyrene induces the transcription of cyclooxygenase-2 in vascular smooth muscle cells. Evidence for the involvement of extracellular signal-regulated kinase and NF-kappaB.

Polycyclic aromatic hydrocarbons, such as benzo[a]pyrene (B[a]P) present in tobacco smoke and tar, have been implicated in the development of atherosclerosis as well as cancer. Increased expression of cyclooxygenase-2 (COX-2) has been detected both in atherosclerotic lesions and in epithelial cancers. To determine whether polycyclic aromatic hydrocarbons might directly affect COX expression in vascular cells, we investigated the effects of B[a]P on COX-2 expression in human and rat arterial smooth muscle cells (SMC). Treatment with B[a]P increased levels of COX-2 protein and mRNA and enhanced prostaglandin synthesis. Nuclear runoff assays and transient transfections revealed increased COX-2 gene transcription after treatment with B[a]P. Experiments were done to define the signaling mechanism by which B[a]P induced COX-2. B[a]P caused a rapid increase in phosphorylation of extracellular signal-regulated kinase (ERK); pharmacologic inhibition of mitogen-activated protein kinase kinase blocked B[a]P-mediated induction of COX-2. Depletion of the intracellular antioxidant, glutathione, with buthionine sulfoximine significantly increased B[a]P-mediated induction of COX-2 while exposure to N-acetylcysteine, a precursor of glutathione, suppressed the induction of COX-2 by B[a]P. Several lines of evidence suggest that the induction of COX-2 by B[a]P is mediated, at least in part, by NF-kappaB. Treatment with B[a]P increased binding of NF-kappaB to DNA. Moreover, B[a]P-mediated stimulation of COX-2 promoter activity was blocked when a construct containing a mutagenized NF-kappaB site was used. Pharmacological inhibitors of NF-kappaB blocked the induction of COX-2 protein and the stimulation of COX-2 promoter activity by B[a]P. Taken together, these data are likely to be important for understanding the atherogenic effects of tobacco smoke.

Animals↗

PubMed2000

Combinatorial analysis of the structural requirements of the Escherichia coli hemolysin signal sequence.

We have investigated the substrate specificity of the Escherichia coli hemolysin transporter system. Translocation of hemolysin is dependent on a C-terminal signal sequence located within the last 60 amino acids of this protein. Previous comparative studies of the signal sequence have revealed a conserved helix(alpha1)-linker-helix(alpha2) motif, suggesting that secondary structure is important for transport. In this study, we generated three random libraries in the alpha1, linker, and alpha2 regions, as well as an alpha1-amphiphilic helical library to identify features buried within the structural motif that contribute to transport. Combinatorial variants were generated by altering the primary sequence of specific regions, and correlation between the genotype and phenotype of the mutant populations allowed us to objectively identify any functional features involved. It was found that the alpha1-amphiphilic helix and the linker are both important for function. To our surprise, the second helix of the conserved structural motif was not essential for transport. The finding that a predicted amphiphilic helix and hydrophobicity, rather than primary sequence, contribute to transport in the alpha1 region allows us to speculate on the mechanism of multiple substrate recognition. This may have implications for understanding the broad substrate specificity common among other ATP-binding cassette transporters.

Amino Acid Motifs↗

PubMed2000

Sp1 binding is critical for promoter assembly and activation of the MCP-1 gene by tumor necrosis factor.

The monocyte chemoattractant protein-1 gene (MCP-1) is induced by the inflammatory cytokine tumor necrosis factor through the coordinate assembly of an NF-kappaB-dependent distal regulatory region and a proximal region that has been suggested to bind Sp1 as well as other factors. To provide a genetic correlation for Sp1 activity in this system, a cell line homozygous for a targeted truncation of the Sp1 gene was derived and examined. We found that the lack of Sp1 binding activity resulted in the inability of both the distal and proximal regions to assemble in vivo even though the binding of NF-kappaB to distal region DNA was unaffected in vitro. We also found that Sp1 and NF-kappaB were the minimal mammalian transcription factors required for efficient activity when transfected into Drosophila Schneider cells. Additionally, Sp3 was able to compensate for Sp1 in the Drosophila tissue cell system but not in the Sp1(-/-) cell line suggesting that Sp1 usage is site-specific and is likely to depend on the context of the binding site. Together, these data provide genetic and biochemical proof for Sp1 in regulating the MCP-1 gene.

3T3 Cells↗

PubMed2000

Free vascularized fibular bone flap in the rat.

Free vascularized fibula bone flap has been widely used in reconstruction of the mandible, long segment defect, congenital pseudarthroses, and osteomyelitis. Such applications stirred an interest in basic studies of bone biology, bone healing process, and incorporation of recipient bone defect. An experimental free vascularized fibula rat model is presented here for such investigations. We performed 16 angiograms and anatomic dissections in eight rats for collecting data on fibular length, blood supply, and the caliber of significant vessels. The fibula was harvested with part of the flexor hallucis longus muscle with an average length of 28 mm. The pedicle can be taken in continuity with the popliteal vessels (average diameter of 0.8 mm and 0.9 mm of artery and vein, respectively, with an average pedicle length of 14 mm). This vascularized fibula bone was harvested and transferred to the groin area of the same rat and anastomosed to the saphenous vessels. Twelve transplantations were performed, with a 7-day flap survival rate of 100%. The free fibula vascularized bone flap in the rat is a reliable model for further investigations.

Animals↗

PubMed2000

Learning curve of microvascular venous anastomosis: a never ending struggle?

In this study, a simple protocol based on the rat femoral venous anastomosis was established to provide a quantitative representation of the progress. The learning curve is based on the patency rate in each consecutive group of five anastomoses. Two groups of surgeons were observed. The inexperienced group encountered a tough time in the first 25 anastomoses. However, the progress was fast and is represented by the steep slope of the curve. A plateau was reached whereby the avearge patency rate matches that of the experienced group. As expected, there was no learning curve for the experienced group. Despite every effort to attempt to maintain a perfect 100% patency on this model, the best achievable patency was only 88%. The results and its implication are discussed.

Anastomosis, Surgical↗

PubMed2000

4-Hydroxylated metabolites of the antiestrogens tamoxifen and toremifene are metabolized to unusually stable quinone methides.

Tamoxifen is widely prescribed for the treatment of hormone-dependent breast cancer, and it has recently been approved by the Food and Drug Administration for the chemoprevention of this disease. However, long-term usage of tamoxifen has been linked to increased risk of developing endometrial cancer in women. One of the suggested pathways leading to the potential toxicity of tamoxifen involves its oxidative metabolism to 4-hydroxytamoxifen, which may be further oxidized to an electrophilic quinone methide. The resulting quinone methide has the potential to alkylate DNA and may initiate the carcinogenic process. To further probe the chemical reactivity and toxicity of such an electrophilic species, we have prepared the 4-hydroxytamoxifen quinone methide chemically and enzymatically, examined its reactivity under physiological conditions, and quantified its reactivity with GSH. Interestingly, this quinone methide is unusually stable; its half-life under physiological conditions is approximately 3 h, and its half-life in the presence of GSH is approximately 4 min. The reaction between 4-hydroxytamoxifen quinone methide and GSH appears to be a reversible process because the quinone methide GSH conjugates slowly decompose over time, regenerating the quinone methide as indicated by LC/MS/MS data. The tamoxifen GSH conjugates were detected in microsomal incubations with 4-hydroxytamoxifen; however, none were observed in breast cancer cell lines (MCF-7) perhaps because very little quinone methides is formed. Toremifene, which is a chlorinated analogue of tamoxifen, undergoes similar oxidative metabolism to give 4-hydroxytoremifene, which is further oxidized to the corresponding quinone methide. The toremifene quinone methide has a half-life of approximately 1 h under physiological conditions, and its rate of reaction in the presence of excess GSH is approximately 6 min. More detailed analyses have indicated that the 4-hydroxytoremifene quinone methide reacts with two molecules of GSH and loses chlorine to give the corresponding di-GSH conjugates. The reaction mechanism likely involves an episulfonium ion intermediate which may contribute to the potential cytotoxic effects of toremifene. Similar to what was observed with 4-hydroxytamoxifen, 4-hydroxytoremifene was metabolized to di-GSH conjugates in microsomal incubations at about 3 times the rate of 4-hydroxytamoxifen, although no conjugates were detected with MCF-7 cells. Finally, these data suggest that quinone methide formation may not make a significant contribution to the cytotoxic and genotoxic effects of tamoxifen and toremifene.

Animals↗

PubMed2000

Synthesis and reactivity of a potential carcinogenic metabolite of tamoxifen: 3,4-dihydroxytamoxifen-o-quinone.

Although tamoxifen is approved for the treatment of hormone-dependent breast cancer as well as for the prevention of breast cancer in high-risk women, several studies in animal models have shown that tamoxifen is heptocarcinogenic, and in humans, tamoxifen has been associated with an increased risk of endometrial cancer. One potential mechanism of tamoxifen carcinogenesis could involve metabolism of tamoxifen to 3,4-dihydroxytamoxifen followed by oxidation to a highly reactive o-quinone which has the potential to alkylate and/or oxidize cellular macromolecules in vivo. In the study presented here, we synthesized the 3,4-dihydroxytamoxifen, prepared its o-quinone chemically and enzymatically, and studied the reactivity of the o-quinone with GSH and deoxynucleosides. The E (trans) and Z (cis) isomers of 3,4-dihydroxytamoxifen were synthesized using a concise synthetic pathway (four steps). This approach is based on the McMurry reaction between the key 4-(2-chloroethoxy)-3,4-methylenedioxybenzophenone and propiophenone, followed by selective removal of the methylenedioxy ring of (E, Z)-1-[4-[2-(N,N-dimethylamino)ethoxy]phenyl]-1-(3, 4-methylenedioxyphenyl)-2-phenyl-1-butene with BCl(3). Oxidation of 3,4-dihydroxytamoxifen by activated silver oxide or tyrosinase gave 3,4-dihydroxytamoxifen-o-quinone as a mixture of E and Z isomers. The resulting o-quinone has a half-life of approximately 80 min under physiological conditions. Reaction of the o-quinone with GSH gave two di-GSH conjugates and three mono GSH conjugates. Incubation of 3,4-dihydroxytamoxifen with GSH in the presence of microsomal P450 gave the same GSH conjugates which were also detected in incubations with human breast cancer cells (MCF-7). Reaction of 3, 4-dihydroxytamoxifen-o-quinone with deoxynucleosides gave only thymidine and deoxyguanosine adducts; neither deoxyadenosine nor deoxycytosine adducts were detected. Preliminary studies conducted with human breast cancer cell lines showed that 3, 4-dihydroxytamoxifen exhibited cytotoxic potency similar to that of 4-hydroxytamoxifen and tamoxifen in an estrogen receptor negative (ER(-)) cell line (MDA-MB-231); however, in the ER(+) cell line (MCF-7), the catechol metabolite was about half as toxic as the other two compounds. Finally, in the presence of microsomes and GSH, 4-hydroxytamoxifen gave predominantly quinone methide GSH conjugates as reported in the previous paper in this issue [Fan, P. W., et al. (2000) Chem. Res. Toxicol. 13, XX-XX]. However, in the presence of tyrosinase and GSH, 4-hydroxytamoxifen was primarily converted to o-quinone GSH conjugates. These results suggest that the catechol metabolite of tamoxifen has the potential to cause cytotoxicity in vivo through formation of 3,4-dihydroxytamoxifen-o-quinone.

Animals↗

PubMed2000

Biliary tract reconstruction using an autologous vein graft in rats.

Numerous biologic and synthetic materials have been used with limited success as an interposed graft to repair segmental common bile duct (CBD) defects. The authors report here that an autologous vein graft can be successfully used to correct a CBD deficit contingent on accurate microsurgical technique immediate stenting and rapid graft vascularization. Thirty Sprague-Dawley rats underwent laparotomy and the experimental group (n=25) had a 3-mm segment of the CBD excised. The CBD defect was repaired using an interposed femoral vein graft aided by a plastic stent. The control group (n=5) had the CBD cut and repaired by means of primary anastomosis. The experimental group was subdivided into three sub-groups each examined at three different postoperative intervals: 1, 4 and 12 weeks. The results showed that inflammation was apparent in the venous wall following the first postoperative week. A progressive loss of the vascular endothelium and replacement with the columnar epithelium typical of the CBD was seen in the vein graft. Nineteen of the 25 experimental rats (76 percent) of the animals survived without complication from the surgery and there were no abnormalities in the liver biochemical tests of these animals. Any biliary tract obstruction that developed was attributed to dislocation of the stent leading to collapse of the vein graft (experimental group), or constriction of the anastomosis (control group). This study demonstrates that biliary tract reconstruction using an autologous vein graft can be successfully performed in a rat model of CBD repair. The application of this method to the clinical setting is also discussed.

Animals↗

PubMed2000

E2F is required to prevent inappropriate S-phase entry of mammalian cells.

E2F is a family of transcription factors that regulates the cell cycle. It is widely accepted that E2F-mediated transactivation of a set of genes is the critical activity that governs cellular progression through G(1) into S phase. In contrast to this hypothesis, we demonstrate that E2F actually suppresses the onset of S phase in two cell types when the cells are arrested by gamma irradiation. Our findings indicate that in these cells, the critical event triggering progression from G(0)/G(1) arrest into S phase is the release of E2F-mediated transrepression of cell cycle genes, not transactivation by E2F. Furthermore, our data suggest that E2F-mediated transactivation is not necessary for the G(1)/S-phase transition in these cells.

Animals↗

PubMed2000

GAP-43 mediates retinal axon interaction with lateral diencephalon cells during optic tract formation.

GAP-43 is an abundant intracellular growth cone protein that can serve as a PKC substrate and regulate calmodulin availability. In mice with targeted disruption of the GAP-43 gene, retinal ganglion cell (RGC) axons fail to progress normally from the optic chiasm into the optic tracts. The underlying cause is unknown but, in principle, can result from either the disruption of guidance mechanisms that mediate axon exit from the midline chiasm region or defects in growth cone signaling required for entry into the lateral diencephalic wall to form the optic tracts. Results here show that, compared to wild-type RGC axons, GAP-43-deficient axons exhibit reduced growth in the presence of lateral diencephalon cell membranes. Reduced growth is not observed when GAP-43-deficient axons are cultured with optic chiasm, cortical, or dorsal midbrain cells. Lateral diencephalon cell conditioned medium inhibits growth of both wild-type and GAP-43-deficient axons to a similar extent and does not affect GAP-43-deficient axons more so. Removal or transplant replacement of the lateral diencephalon optic tract entry zone in GAP-43-deficient embryo preparations results in robust RGC axon exit from the chiasm. Together these data show that RGC axon exit from the midline region does not require GAP-43 function. Instead, GAP-43 appears to mediate RGC axon interaction with guidance cues in the lateral diencephalic wall, suggesting possible involvement of PKC and calmodulin signaling during optic tract formation.

Animals↗

PubMed1999

Design and synthesis of novel alpha(1)(a) adrenoceptor-selective antagonists. 1. Structure-activity relationship in dihydropyrimidinones.

Dihydropyrimidinones such as compound 12 exhibited high binding affinity and subtype selectivity for the cloned human alpha(1a) receptor. Systematic modifications of 12 led to identification of highly potent and subtype-selective compounds such as (+)-30 and (+)-103, with high binding affinity (K(i) = 0.2 nM) for alpha(1a) receptor and greater than 1500-fold selectivity over alpha(1b) and alpha(1d) adrenoceptors. The compounds were found to be functional antagonists in human, rat, and dog prostate tissues. Compound (+)-103 exhibited excellent selectively to inhibit intraurethral pressure (IUP) as compared to lowering diastolic blood pressure (DBP) in mongrel dogs (K(b)(DBP)/K(b)(IUP) = 40) suggesting uroselectivity for alpha(1a)-selective compounds.

Adrenergic alpha-1 Receptor Antagonists↗

PubMed1999

Design and synthesis of novel alpha(1)(a) adrenoceptor-selective antagonists. 2. Approaches to eliminate opioid agonist metabolites via modification of linker and 4-methoxycarbonyl-4-phenylpiperidine moiety.

We have previously described compound 1a as a high-affinity subtype selective alpha(1a) antagonist. In vitro and in vivo evaluation of compound 1a showed its major metabolite to be a mu-opioid agonist, 4-methoxycarbonyl-4-phenylpiperidine (3). Several dihydropyrimidinone analogues were synthesized with the goal of either minimizing the formation of 3 by modification of the linker or finding alternative piperidine moieties which when cleaved as a consequence of metabolism would not give rise to mu-opioid activity. Modification of the linker gave several compounds with good alpha(1a) binding affinity (K(i) = < 1 nM) and selectivity (>300-fold over alpha(1b) and alpha(1d)). In vitro analysis in the microsomal assay revealed these modifications did not significantly affect N-dealkylation and the formation of the piperidine 3. The second approach, however, yielded several piperidine replacements for 3, which did not show significant mu-opioid activity. Several of these compounds maintained good affinity at the alpha(1a) adrenoceptor and selectivity over alpha(1b) and alpha(1d). For example, the piperidine fragments of (+)-73 and (+)-83, viz. 4-cyano-4-phenylpiperidine and 4-methyl-4-phenylpiperidine, were essentially inactive at the mu-opioid receptor (IC(50) > 30 microM vs 3 microM for 3). Compounds (+)-73 and (+)-83 were subjected to detailed in vitro and in vivo characterization. Both these compounds, in addition to their excellent selectivity (>880-fold) over alpha(1b) and alpha(1d), also showed good selectivity over several other recombinant human G-protein coupled receptors. Compounds (+)-73 and (+)-83 showed good functional potency in isolated human prostate tissues, with K(b)s comparable to their in vitro alpha(1a) binding data. In addition, compound (+)-73 also exhibited good uroselectivity (DBP K(b)/IUP K(b) > 20-fold) in the in vivo experiments in dogs, similar to 1a.

Adrenergic alpha-1 Receptor Antagonists↗

PubMed1999

Design and synthesis of novel alpha(1)(a) adrenoceptor-selective antagonists. 3. Approaches to eliminate opioid agonist metabolites by using substituted phenylpiperazine side chains.

Dihydropyrimidinones, such as 1, represent a novel class of alpha(1a) adrenoceptor antagonists with potential for the treatment of benign prostatic hyperplasia (BPH) (see part 1 of this series). Analysis of the metabolites of 1 revealed that 4-methoxycarbonyl-4-phenylpiperidine is formed as the major metabolite and is an agonist at the mu-opioid receptor. To circumvent any potential liability resulting from the metabolite, we decided to identify alternate templates devoid of agonist activity at the mu-opioid receptor to replace the 4-methoxycarbonyl-4-phenylpiperidine moiety. The present study describes the synthesis and SAR of dihydropyrimidinones linked to substituted 4-phenylpiperazine containing side chains. Compound (+)-38 was identified as a lead compound with a binding and functional profile comparable to that of 1. The putative metabolite 2-carboxamidophenylpiperazine has negligible affinity for the mu-opioid receptor.

Adrenergic alpha-Antagonists↗

PubMed1999

Design and synthesis of novel alpha(1)(a) adrenoceptor-selective antagonists. 4. Structure-activity relationship in the dihydropyrimidine series.

We have previously disclosed dihydropyridines such as 1a,b as selective alpha(1a) antagonists as a potential treatment for benign prostatic hyperplasia (BPH). The propensity of dihydropyridines toward an oxidation led us to find suitable replacements of the core unit. The accompanying papers describe the structure-activity relationship (SAR) of dihydropyrimidinones 2a,b as selective alpha(1a) antagonists. We report herein the SAR of dihydropyrimidines such as 4 and highlight the similarities and differences between the dihydropyrimidine and dihydropyrimidinone series of compounds.

Administration, Oral↗

PubMed1999

Determination of glyphosate by ion chromatography.

An ion chromatography system for the determination of glyphosate was described. Ion chromatograph was carried out by suppressed conductivity detection (DX-100). The eluent contained 9 mmol l-1 Na2CO3 and 4 mmol l-1 NaOH. The detection limit was 0.042 microgram ml-1 (S/N = 3). The relative standard deviation was 1.99% and the correlation coefficient of the calibration curve for area was 0.9995. The linear range was 0.042-100 micrograms ml-1. Common inorganic ion and organic acids did not interfere. The recovery was 96.4-103.2%. The method was simple, rapid, reliable and inexpensive.

Chromatography, Ion Exchange↗

PubMed1999

Calcitonin driven v-Ha-ras induces multilineage pulmonary epithelial hyperplasias and neoplasms.

We initiated a transgenic model for primary pulmonary neuroendocrine cell (PNEC) hyperplasia/neoplasia using v-Ha-ras driven by the neural/neuroendocrine (NE)-specific calcitonin promoter (rascal). Previously, we showed that nitrosamine treated rodents develop PNEC hyperplasia but non-NE lung tumors, with variable outcomes presumably reflecting ras activation in multiple cell lineages. Interestingly, all rascal transgenic mouse lineages develop hyperplasias of NE and non-NE cells but mostly non-NE lung carcinomas, with rascal mRNA in differentiated PNECs and tumor cells. Analyses of embryonic lung demonstrate rascal mRNA in undifferentiated epithelium, consistent with expression in a common pluripotent precursor cell. These unexpected observations indicate that v-Ha-ras can lead to both NE and non-NE hyperplasia/neoplasia in vivo, opening new avenues for studies of lung carcinogenesis.

Adenocarcinoma↗

PubMed1999

TCR and IL-12 receptor signals cooperate to activate an individual response element in the IFN-gamma promoter in effector Th cells.

IFN-gamma is a key regulatory cytokine of the immune system. Reporter transgenic mice expressing the luciferase gene under the control of separate TCR-response elements (TCR-RE) from the IFN-gamma promoter or expressing the green fluorescent protein gene under the control of an IFN-gamma "minigene" were employed to explore the basis for IL-12 regulation of IFN-gamma gene transcription. In the absence of TCR stimulation, IL-12 did not activate the TCR-REs but did induce green fluorescent protein expression. TCR plus IL-12R stimulation of effector Th cells resulted in: 1) enhanced activation of the proximal, but not the distal, TCR-RE, and 2) increased induction of cJun-proximal TCR-RE complexes and c-Jun protein expression. Overexpression of cJun, but not cFos, increased activity of the proximal TCR-RE in T cells. These results suggest that IL-12R signaling affects IFN-gamma gene transcription by at least two separate mechanisms; IL-12R signaling without TCR signaling targets promoter regions outside of the approximately 100-bp IFN-gamma TCR-RE, and IL-12R signaling also stimulates TCR-induced activity of the proximal TCR-RE.

Animals↗

PubMed1999

De novo autoimmunity to cardiac myosin after heart transplantation and its contribution to the rejection process.

Allograft rejection is initiated by an immune response to donor MHC proteins. We recently reported that this response can result in breakdown of immune tolerance to a recipient self Ag. However, the contribution of this autoimmune response to graft rejection has yet to be determined. Here, we found that after mouse allogeneic heart transplantation, de novo CD4+ T cell and B cell autoimmune response to cardiac myosin (CM), a major contractile protein of cardiac muscle, is elicited in recipients. Importantly, CM is the autoantigen that causes autoimmune myocarditis, a heart autoimmune disease whose histopathological features resemble those observed in rejected cardiac transplants. Furthermore, T cell responses directed to CM peptide myhcalpha 334-352, a known myocarditogenic determinant, were detected in heart-transplanted mice. No responses to CM were observed in mice that had received an allogeneic skin graft or a syngeneic heart transplant, demonstrating that this response is tissue specific and that allogeneic response is necessary to break tolerance to CM. Next, we showed that sensitization of recipient mice with CM markedly accelerates the rejection of allogeneic heart. Therefore, posttransplant autoimmune response to CM is relevant to the rejection process. We conclude that transplantation-induced autoimmune response to CM represents a new mechanism that may play a significant role in cardiac transplant rejection.

Amino Acid Sequence↗