Fauq, Abdul H.

The structure-activity relationship studies of neurotensin and its analogs have revealed that i) the C-terminal hexapeptide NT (8-13) (H-ArgB­ Arg9-Pro1o_Tyr1LIIe12-Leu13.QH) is equipotent to or more potent than the native NT(1-13) and retains all the intrinsic biological properties of neurotensin, ii) the N-terminal peptide fragment up to NT(1-7) does not significantly contribute to the binding potency, iii) Pro1o that can form a reverse-turn conformation is required, iv) an aromatic amino acid is important at Tyr11 position, v) the lipophilic side chain of Leu 13 is also crucial to the biological activities, and, vi) L-form amino acids are essential at critical positions for the binding potency. In addition to many peptidic neurotensin analogs, highly potent, orally active, and selective non-peptidic analogs as antagonists of NT receptors, which have long lasting effects and the ability to cross the blood­ brain barrier (e. g. SR 142948 A and SR 48692), have been discovered.

AbstractBackground: Measurement of porphobilinogen (PBG) is useful in the diagnosis of the acute neurologic porphyrias. Currently used colorimetric assays lack analytical and clinical sensitivity and specificity.Methods: We developed a liquid chromatography-electrospray tandem mass spectrometry (LC-MS/MS) method for the measurement of PBG in 1 mL of urine, using 5-(aminoethyl)-4-(carboxymethyl) 1H-2,4-[13C]pyrolle-3-propanoic acid ([2,4-13C]PBG; 2.75 μg) as internal standard. After solid-phase extraction, LC-MS/MS analysis was performed in the selected-reaction monitoring (SRM) mode. PBG and [2,4-13C]PBG were monitored through their own precursor and product ion settings (m/z 227 to 210 and m/z 229 to 212, respectively). The retention time of PBG and [2,4-13C]PBG was 1.0 min in a 2.3-min analysis.Results: Daily calibrations (n = 6) between 0.1 and 2.0 mg/L were linear and reproducible. Inter- and intraassay CVs were 3.2–3.5% and 2.6–3.1%, respectively, at mean concentrations of 0.24, 1.18, and 2.15 mg/L. The regression equation for the comparison between an anion-exchange column method (y) and the LC-MS/MS method (x) was: y = 0.84x + 0.74 (Sy|x = 5.8 mg/24 h; r = 0.85; n = 100). In 47 volunteers, PBG excretion was 0.02–0.42 mg/24 h, lower than reported reference intervals (up to 2.0 mg/24 h) based on colorimetric methods. In 85 samples with PBG ≤0.5 by LC-MS/MS, 8 (9.4%) had values ≥2.0 mg/24 h by the anion-exchange method (mean ± SD, 4.3 ± 1.8 mg/24 h). In 11 patients with confirmed diagnoses of acute porphyria and increased PBG by LC-MS/MS, 2 had values within the reported reference intervals by a quantitative anion-exchange method.Conclusions: The quantitative LC-MS/MS method for PBG measurement exhibits greater analytical specificity and improved clinical sensitivity and specificity than currently available methods.

Amyloid-β (Aβ) accumulates in several types of retinal degeneration and in Alzheimer’s disease (AD), but its source has been unclear. We detected the neuronal 695 amino acid form of amyloid-β protein precursor (AβPP) in the normal retina and AβPP751 in the retinal pigment epithelium (RPE) and anterior eye tissues. Similar to the brain, a- and β-secretases cleaved AβPP to soluble derivatives (sAβPP) a or β and membrane-bound C-terminal fragments a or β in the retina and RPE. Levels of sAβPP were particularly high in the vitreous and low in aqueous humor revealing a molecular barrier for AβPP. In contrast, Aβ40 and Aβ42 levels were only 50% lower in the aqueous than the vitreous humor, indicating relatively barrier-free movement of Aβ. These studies demonstrated a relatively high yield of AβPP and Aβ in the ocular fluids, which may serve as a trackable marker for AD. In addition, failure of free clearance from the eye may trigger retina degeneration in a manner similar to Aβ-related neurodegeneration in AD.

Severe aplastic anemia (AA) is a bone marrow (BM) failure (BMF) disease frequently caused by aberrant immune destruction of blood progenitors. Although a Th1-mediated pathology is well described for AA, molecular mechanisms driving disease progression remain ill defined. The NOTCH signaling pathway mediates Th1 cell differentiation in the presence of polarizing cytokines, an action requiring enzymatic processing of NOTCH receptors by γ-secretase. Using a mouse model of AA, we demonstrate that expression of both intracellular NOTCH1IC and T-BET, a key transcription factor regulating Th1 cell differentiation, was increased in spleen and BM-infiltrating T cells during active disease. Conditionally deleting Notch1 or administering γ-secretase inhibitors (GSIs) in vivo attenuated disease and rescued mice from lethal BMF. In peripheral T cells from patients with untreated AA, NOTCH1IC was significantly elevated and bound to the TBX21 promoter, showing NOTCH1 directly regulates the gene encoding T-BET. Treating patient cells with GSIs in vitro lowered NOTCH1IC levels, decreased NOTCH1 detectable at the TBX21 promoter, and decreased T-BET expression, indicating that NOTCH1 signaling is responsive to GSIs during active disease. Collectively, these results identify NOTCH signaling as a primary driver of Th1-mediated pathogenesis in AA and may represent a novel target for therapeutic intervention.

Abstract The nonsteroid antiestrogen tamoxifen [trans-1-(4-b-dimethylaminoethoxyphenyl)-1,2-diphenylbut-1-ene] is the most commonly used endocrine treatment for estrogen receptor α (ERα)-positive breast cancer in pre- and post-menopausal women, and it has helped to reduce breast cancer death rate by one third. Tamoxifen is extensively metabolized, and several metabolites have been detected in human serum. It is metabolized to 4-hydroxytamoxifen and N-desmethyltamoxifen by the action of CYP2D6 and CYP3A4/5 enzymes, respectively. N-desmethyltamoxifen and 4-hydroxy-tamoxifen are further converted to endoxifen by the action of these enzymes. Steady state levels of serum tamoxifen/metabolite are achieved within 4 weeks of continuous administration of the drug. The therapeutic efficacy of tamoxifen is determined by the distribution of the drug into tissues and the availability of the parent drug and its active metabolites in target tissues. Recent reports indicate that endoxifen might act as an antiestrogen. In order to examine the mechanism of action of endoxifen on an ERα-positive breast cancer cell line, MCF-7, we determined the effects of 100, 250, 500 and 1000 nM concentrations of this agent on cell growth, c-myc oncogene expression and activity of the polyamine biosynthetic enzymes. MCF-7 breast cancer cells were treated for 1, 3 or 5 days with endoxifen. Endoxifen had a growth inhibitory effect on MCF-7 cells by day 3 and 5 of treatment, as measured by cell number. Estradiol increased cell number by >2-fold compared to untreated control by day 5 of treatment, whereas endoxifen inhibited the estrogenic effect. c-myc gene expression was increased by a significant 2- to 3-fold by 4 nM estradiol in 2 and 4 hours of treatment, while 250 and 500 nM endoxifen suppressed the increase in c-myc gene expression, as measured by real-time qPCR experiment. In the absence of estradiol, endoxifen slightly (1.5 fold) increased c-myc gene expression. Endoxifen decreased ornithine decarboxylase (ODC) and S-adenosine methyl decarboxylase (AdoMetDC) activities in a concentration- and time-dependent manner; however, it had little effect on spermidine/spermine N1-acetyl transferase (SSAT) activity. The change in biosynthetic enzyme activities were reflected in polyamine pools, as decreased putrescine and spermidine levels. There were no changes in spermine level. As expected, treatment of the cells with 4 nM estradiol induced ODC and AdoMetDC activities, and slightly decreased SSAT activity. Addition of endoxifen in combination with 4 nM estradiol led to even more dramatic decrease in ODC and AdoMetDC activities and reduction of putrescine and spermidine levels. These results support our hypothesis that endoxifen can act as an antiestrogen and suppress cell growth stimulatory effects of estradiol in ERα-positive breast cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4603.

Abstract Background: Glomerular filtration rate (GFR) can be determined by measuring renal clearance of the radiocontrast agent iothalamate. Current analytic methods for quantifying iothalamate concentrations in plasma and urine using liquid chromatography or capillary electrophoresis have limitations such as long analysis times and susceptibility to interferences. We developed a liquid chromatography–tandem mass spectrometry (LC-MS/MS) method to overcome these limitations. Methods: Urine and plasma samples were deproteinized using acetonitrile and centrifugation. The supernatant was diluted in water and analyzed by LC-MS/MS using a water:methanol gradient. We monitored 4 multiple reaction monitoring transitions: m/z 614.8–487.0, 614.8–456.0, 614.8–361.1, and 614.8–177.1. We compared the results to those obtained via our standard capillary electrophoresis (CE-UV) on samples from 53 patients undergoing clinical GFR testing. Results: Mean recovery was 90%–110% in both urine and plasma matrices. Imprecision was ≤15% for the m/z 614.8–487.0 and 614.8–456.0 transitions over a 10-day period at 1 mg/L. Method comparison for 159 patient samples (53 clearances) provided the following Passing–Bablok regressions: plasma iothalamate LC-MS/MS (y) vs CE-UV (x), y = 0.99x + 0.36; urine iothalamate LC-MS/MS vs CE-UV, y = 1.01x + 0.31; corrected GFR LC-MS/MS vs CE-UV, y = 1.00x + 0.00. Interfering substances prevented accurate iothalamate quantification by CE-UV in 2 patients, whereas these samples could be analyzed by LC-MS/MS. Conclusions: Iothalamate can be quantified by LC-MS/MS for GFR measurement. This method circumvents potential problems with interfering substances that occasionally confound accurate GFR determinations.

Abstract The maturation of naive CD8+ T cells into effector CTLs is a critical feature of a functional adaptive immune system. Development of CTLs depends, in part, upon the expression of the transcriptional regulator eomesodermin (EOMES), which is thought to regulate expression of two key effector molecules, perforin and granzyme B. Although EOMES is important for effector CTL development, the precise mechanisms regulating CD8+ effector cell maturation remains poorly understood. In this study, we show that Notch1 regulates the expression of EOMES, perforin, and granzyme B through direct binding to the promoters of these crucial effector molecules. By abrogating Notch signaling, both biochemically as well as genetically, we conclude that Notch activity mediates CTL activity through direct regulation of EOMES, perforin, and granzyme B.

Abstract Notch signaling plays a role in normal lymphocyte development and function. Activating Notch1-mutations, leading to aberrant downstream signaling, have been identified in human T-cell acute lymphoblastic leukemia (T-ALL). While this highlights the contribution of Notch signaling to T-ALL pathogenesis, the mechanisms by which Notch regulates proliferation and survival in normal and leukemic T cells are not fully understood. Our findings identify a role for Notch signaling in G1-S progression of cell cycle in T cells. Here we show that expression of the G1 proteins, cyclin D3, CDK4, and CDK6, is Notch-dependent both in vitro and in vivo, and we outline a possible mechanism for the regulated expression of cyclin D3 in activated T cells via CSL (CBF-1, mammals; suppressor of hairless, Drosophila melanogaster; Lag-1, Caenorhabditis elegans), as well as a noncanonical Notch signaling pathway. While cyclin D3 expression contributes to cell-cycle progression in Notch-dependent human T-ALL cell lines, ectopic expression of CDK4 or CDK6 together with cyclin D3 shows partial rescue from γ-secretase inhibitor (GSI)-induced G1 arrest in these cell lines. Importantly, cyclin D3 and CDK4 are highly overexpressed in Notch-dependent T-cell lymphomas, justifying the combined use of cell-cycle inhibitors and GSI in treating human T-cell malignancies.