bluuebunny/crossref_metadata_2025 huggingface dataset:
A philosophy for scientific computing tools
Authors: Coughran W. M. , Grosse E.
Published: April 2007

We argue that subroutine libraries are not enough but need to be supplemented by common file formats and directly executable commands. A concrete example, tensor spline primitives, illustrates this style of programming.
Perturbation analysis of Horner's method for nice cases
Authors: Dunham C. B.
Published: April 2007

Under favourable conditions, evaluation of polynomials by Homer's rule has an error not exceeding a few units in the last place.It has been observed that for moderate sized
x
and coefficients decreasing rapidly in magnitude that floating point evaluation of polynomials by Homer's method gives results accurate to around one or two units in the last place. A hand calculated example together with an informal justification is given by Fike (1968, pp. 52--53). In this note the observation is justified by a Wilkinson-type backward error analysis. It would be helpful for the reader to be familiar with the approach of Wilkinson (1963, Chap. 1), in which
fl
(expr) denotes the effect of evaluation of expression expr in floating point.We will assume that floating point is done using guard digits (Johnston, 1982, p. 11: Sterbenz, 1974) in which case we have by analysis similar to those of Wilkinson (1963, Chap. 1).
Comment on "Integer Arithmetic" by Hodge
Authors: Kenison Paul E.
Published: April 2007

The other day, I finally got to read some back issues of journals to which I subscribe. One of these journals is
SIGSMALL/PC Notes.
In the most recent issue (May 1989) I read your article about "Integer Arithmetic." In looking at the article I noted that you had indicated that you hadn't proven the induction assumption that you had used. I thought that I would take a few minutes and look at a possible proof. I believe that I found a proof that is rather simple. It all falls out rather easily when the expression is represented in factorial notation.
Role of the scaffold protein RACK1 in apical expression of CFTR
Authors: Auerbach Michael , Liedtke Carole M.
Published: April 2007

Previous studies from this laboratory demonstrated a role for protein kinase C (PKC)ε in the regulation of cAMP-dependent cystic fibrosis transmembrane regulator (CFTR) Cl channel function via binding of PKCε to RACK1, a receptor for activated C kinase, and of RACK1 to human Na+/H+ exchanger regulatory factor (NHERF1). In the present study, we investigated the role of RACK1 in regulating CFTR function in a Calu-3 airway epithelial cell line. Confocal microscopy and biotinylation of apical surface proteins demonstrate apical localization of RACK1 independent of actin. Mass spectrometric analysis of NHERF1 revealed copurification of tubulin, which, in in vitro binding assays, selectively binds to NHERF1, but not RACK1, via a PDZ1 domain. In binding and pulldown assays, we show direct binding of a PDZ2 domain to NHERF1, pulldown of endogenous NHERF1 by a PDZ2 domain, and inhibition of NHERF1-tubulin binding by a PDZ1 domain. Downregulation of RACK1 using double-stranded silencing RNA reduced the amount of RACK1 by 77.5% and apical expression of biotinylated CFTR by 87.4%. Expression of CFTR, NHERF1, and actin were not altered by treatment with siRACK1 or by nontargeting control silencing RNA, which, in addition, did not affect RACK1 expression. On the basis of these results, we model a RACK1 proteome consisting of PKCε-RACK1-NHERF1-NHERF1-tubulin with a role in stable expression of CFTR in the apical plasma membrane of epithelial cells.
The deleterious effects of bed rest on human skeletal muscle fibers are exacerbated by hypercortisolemia and ameliorated by dietary supplementation
Authors: Fitts R. H. , Romatowski J. G. , Peters J. R. , Paddon-Jones D. , Wolfe R. R. , Ferrando A. A.
Published: April 2007

Prolonged inactivity associated with bed rest in a clinical setting or spaceflight is frequently associated with hypercortisolemia and inadequate caloric intake. Here, we determined the effect of 28 days of bed rest (BR); bed rest plus hypercortisolemia (BRHC); and bed rest plus essential amino acid (AA) and carbohydrate (CHO) supplement (BRAA) on the size and function of single slow- and fast-twitch muscle fibers. Supplementing meals, the BRAA group consumed 16.5 g essential amino acids and 30 g sucrose at 1100, 1600, and 2100 h, and the BRHC subjects received 5 daily doses of 10–15 mg of oral hydrocortisone sodium succinate throughout bed rest. Bed rest induced atrophy and loss of force (mN) and power (μN·FL·s−1) in single fibers was exacerbated by hypercortisolemia where soleus peak force declined by 23% in the type I fiber from a prevalue of 0.78 ± 0.02 to 0.60 ± 0.02 mN post bed rest (compared to a 7% decline with bed rest alone) and 27% in the type II fiber (1.10 ± 0.08 vs. 0.81 ± 0.05 mN). In the BRHC group, peak power dropped by 19, 15, and 11% in the soleus type I, and vastus lateralis (VL) type I and II fibers, respectively. The AA/CHO supplement protected against the bed rest-induced loss of peak force in the type I soleus and peak power in the VL type II fibers. These results provide evidence that an AA/CHO supplement might serve as a successful countermeasure to help preserve muscle function during periods of relative inactivity.
Effects of acute and chronic sleep loss on immune modulation of rats
Authors: Zager A. , Andersen M. L. , Ruiz F. S. , Antunes I. B. , Tufik S.
Published: April 2007

Sleep deprivation is now recognized as an increasingly common condition inherent to modern society, and one that in many ways, is detrimental to certain physiological systems, namely, immune function. Although sleep is now viewed by a significant body of researchers as being essential for the proper working of a host of defense systems, the consequences of a lack of sleep on immune function remains to be fully comprehended. The aim of the current study was to investigate how paradoxical sleep deprivation (PSD) for 24 and 96 h and sleep restriction (SR) for 21 days by the modified multiple-platform method, and their respective 24-h recovery periods, affect immune activation in rats. To this end, we assessed circulating white blood cell counts, lymphocyte count within immune organs, as well as Ig and complement production. The data revealed that PSD for 96 h increased complement C3 and corticosterone concentration in relation to the control group. In contrast, the spleen weight, total leukocytes, and lymphocytes decreased during SR for 21 days when compared with the control group, although production of a certain class of immunoglobulin, the IgM, did increase. After recovery sleep, lymphocyte count in axillary lymph nodes grew when rats had rebound sleep after PSD for 24 h, neutrophils increased after PSD 96 h and lymphocytes numbers were higher after SR 21 days. Such alterations during sleep deprivation suggest only minor alterations of nonspecific immune parameters during acute PSD, and a significant impairment in cellular response during chronic SR.
Spontaneous sleep and homeostatic sleep regulation in ghrelin knockout mice
Authors: Szentirmai Éva , Kapás Levente , Sun Yuxiang , Smith Roy G. , Krueger James M.
Published: April 2007

Ghrelin is well known for its feeding and growth hormone-releasing actions. It may also be involved in sleep regulation; intracerebroventricular administration and hypothalamic microinjections of ghrelin stimulate wakefulness in rats. Hypothalamic ghrelin, together with neuropeptide Y and orexin form a food intake-regulatory circuit. We hypothesized that this circuit also promotes arousal. To further investigate the role of ghrelin in the regulation of sleep-wakefulness, we characterized spontaneous and homeostatic sleep regulation in ghrelin knockout (KO) and wild-type (WT) mice. Both groups of mice exhibited similar diurnal rhythms with more sleep and less wakefulness during the light period. In ghrelin KO mice, spontaneous wakefulness and rapid-eye-movement sleep (REMS) were slightly elevated, and non-rapid-eye-movement sleep (NREMS) was reduced. KO mice had more fragmented NREMS than WT mice, as indicated by the shorter and greater number of NREMS episodes. Six hours of sleep deprivation induced rebound increases in NREMS and REMS and biphasic changes in electroencephalographic slow-wave activity (EEG SWA) in both genotypes. Ghrelin KO mice recovered from NREMS and REMS loss faster, and the delayed reduction in EEG SWA, occurring after sleep loss-enhanced increases in EEG SWA, was shorter-lasting compared with WT mice. These findings suggest that the basic sleep-wake regulatory mechanisms in ghrelin KO mice are not impaired and they are able to mount adequate rebound sleep in response to a homeostatic challenge. It is possible that redundancy in the arousal systems of the brain or activation of compensatory mechanisms during development allow for normal sleep-wake regulation in ghrelin KO mice.
Phosphorylation of extracellular signal-regulated kinases in urinary bladder in rats with cyclophosphamide-induced cystitis
Authors: Corrow Kimberly A. , Vizzard Margaret A.
Published: April 2007

Phosphorylated ERK expression has been demonstrated in the central and peripheral nervous system after various stimuli, including visceral stimulation. Changes in the activation (i.e., phosphorylation) of extracellular signal-regulated kinases (pERK) were examined in the urinary bladder after 4 h (acute), 48 h (intermediate), or chronic (10 day) cyclophosphamide (CYP) treatment. CYP-induced cystitis significantly ( P ≤ 0.01) increased pERK expression in the urinary bladder with intermediate (48 h) and chronic CYP treatment. Immunohistochemistry for pERK immunoreactivity revealed little pERK-IR in control or acute (4 h) CYP-treated rat urinary bladders. However, pERK expression was significantly ( P ≤ 0.01) upregulated in the urothelium after 48 h or chronic CYP treatment. Whole mount preparations of urothelium/lamina propria or detrusor smooth muscle from control (noninflamed) rats showed no pERK-IR in PGP9.5-labeled nerve fibers in the suburothelial plexus. However, with CYP-treatment (48 h, chronic), a few pERK-IR nerve fibers in the suburothelial plexus of whole mount preparations of bladder and at the serosal edge of urinary bladder sections were observed. pERK-IR cells expressing the CD86 antigen were also observed in urinary bladder from CYP-treated rats (48 h, chronic). Treatment with the upstream inhibitor of ERK phosphorylation, U0126, significantly ( P ≤ 0.01) increased bladder capacity in CYP-treated rats (48 h). These studies suggest that therapies targeted at pERK pathways may improve urinary bladder function in CYP-treated rats.
Localization of broadly selective equilibrative and concentrative nucleoside transporters, hENT1 and hCNT3, in human kidney
Authors: Damaraju Vijaya L. , Elwi Adam N. , Hunter Charlene , Carpenter Pat , Santos Cheryl , Barron Gerry M. , Sun Xuejun , Baldwin Stephen A. , Young James D. , Mackey John R. , Sawyer Michael B. , Cass Carol E.
Published: April 2007

Nucleoside transporters in kidney mediate renal reabsorption and secretion of nucleosides. Using RT-PCR, we demonstrated mRNAs encoding hENT1, hENT2, hCNT1, hCNT2, and hCNT3 in both cortex and medulla. Immunoblotting with crude membrane preparations revealed abundant hENT1 and hCNT3 in both cortex and medulla, and little, if any, hENT2, hCNT1, or hCNT2, indicating that the latter were either absent or below limits of detection of immunoassays. hENT1 immunostaining was observed on apical surfaces of proximal tubules and on both apical and basal surfaces of thick ascending loops of Henle and collecting ducts. Prominent hCNT3 immunostaining was observed on apical surfaces of proximal tubules and thick ascending loops of Henle in addition to some cytoplasmic staining. Equilibrium binding of [3H]nitrobenzylmercaptopurine ribonucleoside (NBMPR), a high-affinity inhibitor of hENT1, to brush-border membrane vesicles from cortex confirmed the presence of hENT1 on apical surfaces of proximal tubules. Uptake of [3H]uridine by polarized renal proximal tubule cells exhibited a sodium-dependent component that was inhibited by thymidine and inosine as well as a sodium-independent component that was partially inhibited by NBMPR and completely inhibited by dilazep, indicating high levels of hENT1 and hCNT3 and low levels of hENT2 activities. The presence of 1) transcripts for hENT1/2 and hCNT1/2/3 and the hENT1 and hCNT3 proteins in human kidneys and 2) hENT1, hENT2, and hCNT3 activities in cultured proximal tubule cells suggest involvement of hENT1, hCNT3, and possibly also hENT2 in renal handling of nucleosides and nucleoside drugs.
NHE3 phosphorylation at serines 552 and 605 does not directly affect NHE3 activity
Authors: Kocinsky Hetal S. , Dynia Diane W. , Wang Tong , Aronson Peter S.
Published: April 2007

Direct phosphorylation of sodium hydrogen exchanger type 3 (NHE3) is a well-established physiological phenomenon; however, the exact role of NHE3 phosphorylation in its regulation remains unclear. The objective of this study was to evaluate whether NHE3 phosphorylation at serines 552 and 605 is physiologically regulated in vivo and, if so, whether changes in phosphorylation at these sites are tightly coupled to changes in transport activity. To this end, we directly compared PKA-induced NHE3 inhibition with site-specific changes in NHE3 phosphorylation in vivo and in vitro. In vivo, PKA was activated using an intravenous infusion of parathyroid hormone in Sprague-Dawley rats. In vitro, PKA was activated directly in opossum kidney (OKP) cells using forskolin and IBMX. NHE3 activity was assayed in microvillar membrane vesicles in the rat model and by22Na uptake in the OKP cell model. In both cases, NHE3 phosphorylation at serines 552 and 605 was determined using previously characterized monoclonal phosphospecific antibodies directed to these sites. In vivo, we found dramatic changes in NHE3 phosphorylation at serines 552 and 605 with PKA activation but no corresponding alteration in NHE3 activity. This dissociation between NHE3 phosphorylation and activity was further verified in OKP cells in which phosphorylation clearly preceded transport inhibition. We conclude that although phosphorylation of NHE3 at serines 552 and 605 is regulated by PKA both in vivo and in vitro, phosphorylation of these sites does not directly alter Na+/H+exchange activity.
WISE 2005: stroke volume changes contribute to the pressor response during ischemic handgrip exercise in women
Authors: Shoemaker J. K. , Mattar L. , Kerbeci P. , Trotter S. , Arbeille P. , Hughson R. L.
Published: April 2007

The mechanism of the pressor response to small muscle mass (e.g., forearm) exercise and during metaboreflex activation may include elevations in cardiac output (Q̇) or total peripheral resistance (TPR). Increases in Q̇ must be supported by reductions in visceral venous volume to sustain venous return as heart rate (HR) increases. Therefore, this study tested the hypothesis that increases in Q̇, supported by reductions in splanchnic volume (portal vein constriction), explain the pressor response during handgrip exercise and metaboreflex activation. Seventeen healthy women performed 2 min of static ischemic handgrip exercise and 2 min of postexercise circulatory occlusion (PECO) while HR, stroke volume and superficial femoral artery flow (Doppler), blood pressure (Finometer), portal vein diameter (ultrasound imaging), and muscle sympathetic nerve activity (MSNA; microneurography) were measured followed by the calculation of Q̇, TPR, and leg vascular resistance (LVR). Compared with baseline, mean arterial blood pressure (MAP) ( P < 0.001) and Q̇ ( P < 0.001) both increased in each minute of exercise accompanied by a ∼5% reduction in portal vein diameter ( P < 0.05). MAP remained elevated during PECO, whereas Q̇ decreased below exercise levels. MSNA was elevated above baseline during the second minute of exercise and through the PECO period ( P < 0.05). Neither TPR nor LVR was changed from baseline during exercise and PECO. The data indicate that the majority of the blood pressure response to isometric handgrip exercise in women was due to mobilization of central blood volume and elevated stroke volume and Q̇ rather than elevations in TVR or LVR resistance.
Impact of Coordinate Transformation Uncertainty on Human Sensorimotor Control
Authors: Schlicht Erik J. , Schrater Paul R.
Published: April 2007

Humans build representations of objects and their locations by integrating imperfect information from multiple perceptual modalities (e.g., visual, haptic). Because sensory information is specified in different frames of reference (i.e., eye- and body-centered), it must be remapped into a common coordinate frame before integration and storage in memory. Such transformations require an understanding of body articulation, which is estimated through noisy sensory data. Consequently, target information acquires additional coordinate transformation uncertainty (CTU) during remapping because of errors in joint angle sensing. As a result, CTU creates differences in the reliability of target information depending on the reference frame used for storage. This paper explores whether the brain represents and compensates for CTU when making grasping movements. To address this question, we varied eye position in the head, while participants reached to grasp a spatially fixed object, both when the object was in view and when it was occluded. Varying eye position changes CTU between eye and head, producing additional uncertainty in remapped information away from forward view. The results showed that people adjust their maximum grip aperture to compensate both for changes in visual information and for changes in CTU when the target is occluded. Moreover, the amount of compensation is predicted by a Bayesian model for location inference that uses eye-centered storage.
Neurokinin Receptor-Expressing Pre-Bötzinger Complex Neurons in Neonatal Mice Studied In Vitro
Authors: Hayes John A. , Del Negro Christopher A.
Published: April 2007

Breathing in mammals depends on inspiratory-related neural activity generated in the pre-Bötzinger complex (preBötC), where neurokinin receptor-expressing neurons (NKR+) have been hypothesized to play a critical rhythmogenic role. Currently, the extent to which the preBötC is populated by rhythmogenic NKR+ neurons and whether neurons without neurokinin receptor expression (NKR−) share similar electrical properties with NKR+ neurons are not well understood. These interrelated problems must be resolved to understand the widespread excitatory effects of neuropeptides and the mechanism of respiratory rhythmogenesis. We recorded and imaged inspiratory neurons in neonatal mouse slices that isolate the preBötC and generate respiratory motor output in vitro. Using tetramethylrhodamine conjugated to the endogenous NKR agonist substance P (TMR-SP) to tag neurons that express NKRs, we show that early inspiratory neurons with small whole cell capacitance ( CM) are 36% TMR-SP+ and 64% TMR-SP−. Also, late inspiratory neurons with large CM are 67% TMR-SP+ and 33% are TMR-SP−. Thus NKR+ and NKR− neurons exhibit the same phenotypic properties, which suggests that they may share functional roles also. Substance P (SP) alone evoked a voltage-insensitive inward current ( ISP) that reversed at –19 mV and was associated with an increase in membrane conductance in both NKR+ and NKR− neurons. Gap junctions may be needed to confer SP sensitivity to neurons that appear to lack NKR expression. We propose that cell death in NKR+ preBötC neurons, by targeted lesion or neurodegeneration, may impair breathing behavior by killing less than one half of the rhythmogenic preBötC neurons and a large number of respiratory premotoneurons.
Panulirus interruptus Ih-Channel Gene PIIH: Modification of Channel Properties by Alternative Splicing and Role in Rhythmic Activity
Authors: Ouyang Qing , Goeritz Marie , Harris-Warrick Ronald M.
Published: April 2007

We cloned 10 full-length variants of PIIH, the gene for Ih from the spiny lobster, Panulirus interruptus, using reverse transcription-PCR (RT-PCR) and rapid amplification of cDNA ends (RACE). This gene shows a significant amount of alternative splicing in the S3–S4 and S4–S5 linkers, in the P-loop and the entire S6 transmembrane domain, in the cyclic nucleotide binding domain (CNBD), and near the 3′ end of the gene. Functional expression of seven splice variants in Xenopus oocytes generated slowly activating hyperpolarization-activated inward currents, which were blocked by the Ih channel blockers CsCl and ZD7288. The different splice variants had markedly varying activation kinetics and voltage dependence of activation. Bath application of 8-Br-cAMP shifted the V1/2 to more positive potentials and accelerated the activation kinetics in an isoform-specific manner. Two variants containing a segment with an ER-retention motif in the S4-S5 loop did not produce currents in oocytes. Overexpression of one splice variant, PIIH ABS -I, in pyloric dilator (PD) neurons in the lobster stomatogastric ganglion produced an average threefold increase in Ih without evoking a compensatory increase in IA. The voltage for half-maximal activation of Ih in PIIH ABS-I-expressing PDs was shifted in the depolarizing direction by 9 mV, whereas the slope factor decreased by 3.8 mV. Moreover, its activation kinetics were significantly faster than in control PDs. PIIH ABS-I overexpression enhanced PD neuron rhythmic firing in an amplitude-dependent manner above a minimal threshold two- to threefold increase in amplitude.
Phosphoprotein Pathway Mapping: Akt/Mammalian Target of Rapamycin Activation Is Negatively Associated with Childhood Rhabdomyosarcoma Survival
Authors: Petricoin Emanuel F. , Espina Virginia , Araujo Robyn P. , Midura Brieanne , Yeung Choh , Wan Xiaolin , Eichler Gabriel S. , Johann Donald J. , Qualman Stephen , Tsokos Maria , Krishnan Kartik , Helman Lee J. , Liotta Lance A.
Published: April 2007

Mapping of protein signaling networks within tumors can identify new targets for therapy and provide a means to stratify patients for individualized therapy. Despite advances in combination chemotherapy, the overall survival for childhood rhabdomyosarcoma remains ∼60%. A critical goal is to identify functionally important protein signaling defects associated with treatment failure for the 40% nonresponder cohort. Here, we show, by phosphoproteomic network analysis of microdissected tumor cells, that interlinked components of the Akt/mammalian target of rapamycin (mTOR) pathway exhibited increased levels of phosphorylation for tumors of patients with short-term survival. Specimens (n = 59) were obtained from the Children's Oncology Group Intergroup Rhabdomyosarcoma Study (IRS) IV, D9502 and D9803, with 12-year follow-up. High phosphorylation levels were associated with poor overall and poor disease-free survival: Akt Ser473 (overall survival P < 0.001, recurrence-free survival P < 0.0009), 4EBP1 Thr37/46 (overall survival P < 0.0110, recurrence-free survival P < 0.0106), eIF4G Ser1108 (overall survival P < 0.0017, recurrence-free survival P < 0.0072), and p70S6 Thr389 (overall survival P < 0.0085, recurrence-free survival P < 0.0296). Moreover, the findings support an altered interrelationship between the insulin receptor substrate (IRS-1) and Akt/mTOR pathway proteins (P < 0.0027) for tumors from patients with poor survival. The functional significance of this pathway was tested using CCI-779 in a mouse xenograft model. CCI-779 suppressed phosphorylation of mTOR downstream proteins and greatly reduced the growth of two different rhabdomyosarcoma (RD embryonal P = 0.00008; Rh30 alveolar P = 0.0002) cell lines compared with controls. These results suggest that phosphoprotein mapping of the Akt/mTOR pathway should be studied further as a means to select patients to receive mTOR/IRS pathway inhibitors before administration of chemotherapy. [Cancer Res 2007;67(7):3431–40]
CXCL10 Promotes Invasion-Related Properties in Human Colorectal Carcinoma Cells
Authors: Zipin-Roitman Adi , Meshel Tsipi , Sagi-Assif Orit , Shalmon Bruria , Avivi Camila , Pfeffer Raphael M. , Witz Isaac P. , Ben-Baruch Adit
Published: April 2007

CXCL10 was recently shown to exert antimalignancy functions by influencing the tumor microenvironment. Here, we have taken a different approach, investigating the effects of CXCL10 directly on tumor-promoting functions in colorectal carcinoma (CRC) cells. CXCL10 expression was detected in preferred metastatic sites of CRC (liver, lungs, and lymph nodes), and its CXCR3 receptor was expressed by eight CRC cell lines (detected: reverse transcription-PCR and/or flow cytometry). Detailed analysis was done on two cell lines derived from primary CRC tumors (SW480, KM12C) and their metastatic descendents (SW620 and KM12SM). The three known variants of CXCR3 (CXCR3-A, CXCR3-B, and CXCR3-alt) were detected in all four cell lines. CXCR3 expression was also observed on colorectal tumor cells in biopsies of CRC patients (immunohistochemistry). CXCL10 and CXCR3 expression were potently induced in CRC cells by Interferon γ and all four CRC cell lines responded to CXCL10 by extracellular signal-regulated kinase 1/2 dephosphorylation. The chemokine did not affect tumor cell growth or angiogenesis-related functions in the tumor cells, such as CXCL8 and vascular endothelial growth factor secretion. Importantly, CXCL10 significantly up-regulated invasion-related properties in CRC cells: It promoted matrix metalloproteinase 9 expression and induced CRC cell migration. Of note, CXCL10-induced migration was detected only in the two metastatic cells and not in their primary counterparts. Also, CXCL10 promoted the adhesion of metastatic cells to laminin. These results suggest that CXCL10 can be exploited by CRC cells toward their progression, thus possibly antagonizing the antimalignancy effects of the chemokine on the tumor microenvironment. Therefore, care should be taken when considering CXCL10 as a therapeutic antitumor modality for CRC treatment. [Cancer Res 2007;67(7):3396–405]
Aurora A and B Overexpression and Centrosome Amplification in Early Estrogen-Induced Tumor Foci in the Syrian Hamster Kidney: Implications for Chromosomal Instability, Aneuploidy, and Neoplasia
Authors: Hontz Adrianne E. , Li Sara Antonia , Lingle Wilma L. , Negron Vivian , Bruzek Amy , Salisbury Jeffrey L. , Li Jonathan J.
Published: April 2007

Estrogen-induced Syrian hamster tumors in the kidney represent a useful model to gain insight into the role of estrogens in oncogenic processes. We provided evidence that early tumor foci in the kidney arise from interstitial ectopic uterine-like germinal stem cells, and that early tumor foci and well-established tumors are highly aneuploid (92–94%). The molecular mechanisms whereby estrogens mediate this process are unclear. Here, we report that estrogen treatment induced significant increases in Aurora A protein expression (8.7-fold), activity (2.6-fold), mRNA (6.0-fold), and Aurora B protein expression (4.6-fold) in tumors, compared with age-matched cholesterol-treated kidneys. Immunohistochemistry revealed that this increase in Aurora A and B protein expression was essentially confined to cells within early and large tumor foci at 3.5 and 6 months of estrogen treatment, respectively. Upon estrogen withdrawal or coadministration of tamoxifen for 10 days, a 78% to 79% and 81% to 64% reduction in Aurora A and B expression, respectively, were observed in primary tumors compared with tumors continuously exposed to estrogens. These data indicate that overexpressed Aurora A and B in these tumors are under estrogen control via estrogen receptor α. Aurora A coenriched with the centrosome fraction isolated from tumors in the kidney. Centrosome amplification (number and area/cell) was detected in early tumor foci and large tumors but not in adjacent uninvolved or age-matched control kidneys. Taken together, these data indicate that persistent overexpression of Aurora A and B is under estrogen control, and is coincident with centrosome amplification, chromosomal instability, and aneuploidy, and represent an important mechanism driving tumorigenesis. [Cancer Res 2007;67(7):2957–63]
Dual Role of Inactivating Lef1 Mutations in Epidermis: Tumor Promotion and Specification of Tumor Type
Authors: Niemann Catherin , Owens David M. , Schettina Peter , Watt Fiona M.
Published: April 2007

The NH2 terminus of LEF1 is frequently mutated in human sebaceous tumors. To investigate how this contributes to cancer, we did two-stage chemical carcinogenesis on K14ΔNLef1 transgenic mice, which express NH2-terminally truncated Lef1 in the epidermal basal layer. Transgenic mice developed more tumors, more rapidly than littermate controls, even without exposure to tumor promoter. They developed sebaceous tumors, whereas controls developed squamous cell carcinomas. K14ΔNLef1 epidermis failed to up-regulate p53 and p21 proteins during tumorigenesis or in response to UV irradiation, and this correlated with impaired p14ARF induction. We propose that LEF1 NH2-terminal mutations play a dual role in skin cancer, specifying tumor type by inhibiting Wnt signaling and acting as a tumor promoter by preventing induction of p53. [Cancer Res 2007;67(7):2916–21]
A High-Throughput Study in Melanoma Identifies Epithelial-Mesenchymal Transition as a Major Determinant of Metastasis
Authors: Alonso Soledad R. , Tracey Lorraine , Ortiz Pablo , Pérez-Gómez Beatriz , Palacios José , Pollán Marina , Linares Juan , Serrano Salvio , Sáez-Castillo Ana I. , Sánchez Lydia , Pajares Raquel , Sánchez-Aguilera Abel , Artiga Maria J. , Piris Miguel A. , Rodríguez-Peralto José L.
Published: April 2007

Metastatic disease is the primary cause of death in cutaneous malignant melanoma (CMM) patients. To understand the mechanisms of CMM metastasis and identify potential predictive markers, we analyzed gene-expression profiles of 34 vertical growth phase melanoma cases using cDNA microarrays. All patients had a minimum follow-up of 36 months. Twenty-one cases developed nodal metastatic disease and 13 did not. Comparison of gene expression profiling of metastatic and nonmetastatic melanoma cases identified 243 genes with a >2-fold differential expression ratio and a false discovery rate of <0.2 (206 up-regulated and 37 down-regulated). This set of genes included molecules involved in cell cycle and apoptosis regulation, epithelial-mesenchymal transition (EMT), signal transduction, nucleic acid binding and transcription, protein synthesis and degradation, metabolism, and a specific group of melanoma- and neural-related proteins. Validation of these expression data in an independent series of melanomas using tissue microarrays confirmed that the expression of a set of proteins included in the EMT group (N-cadherin, osteopontin, and SPARC/osteonectin) were significantly associated with metastasis development. Our results suggest that EMT-related genes contribute to the promotion of the metastatic phenotype in primary CMM by supporting specific adhesive, invasive, and migratory properties. These data give a better understanding of the biology of this aggressive tumor and may provide new prognostic and patient stratification markers in addition to potential therapeutic targets. [Cancer Res 2007;67(7):3450–60]
Systemic Targeting Inhibitor of κB Kinase Inhibits Melanoma Tumor Growth
Authors: Yang Jinming , Pan Wei-Hua , Clawson Gary A. , Richmond Ann
Published: April 2007

Constitutive activation of nuclear factor-κB (NF-κB) has been directly implicated in tumorigenesis of various cancer types, including melanoma. Inhibitor of κB kinase (IKK) functions as a major mediator of NF-κB activation. Thus, development of an IKK-specific inhibitor has been a high priority, although it remains unclear whether systemic inhibition of IKK will provide therapeutic benefit. In this study, we show that inhibition of NF-κB activity in melanocytes that are persistently expressing an active H-RasV12 gene and are deficient in the tumor suppressors inhibitor A of cyclin-dependent kinase 4/alternative reading frame results in reduction of melanoma tumor growth in vivo. This effect is, at least in part, via regulation of NF-κB nuclear activation and RelA phosphorylation. Based on this result, we developed a double hammerhead ribozyme long-term expression system to silence either IKKα or IKKβ. The ribozymes were placed in an EBV construct and delivered i.v. to nude mice bearing melanoma lesions, which developed after i.v. injection of H-Ras–transformed melanoma cells. Our in vivo data show that knockdown of endogenous IKKβ significantly reduces the growth of the melanoma lesions and knockdown of either IKKα or IKKβ prolongs the life span of immunocompetent mice. [Cancer Res 2007;67(7):3127–34]
Tumor Suppression by IFN Regulatory Factor-1 Is Mediated by Transcriptional Down-regulation of Cyclin D1
Authors: Kröger Andrea , Stirnweiss Anja , Pulverer Julia Elisabeth , Klages Katjana , Grashoff Martina , Reimann Jörg , Hauser Hansjörg
Published: April 2007

IFNs have been ascribed to mediate antitumor effects. IFN regulatory factor-1 (IRF-1) is a major target gene of IFNs. It inhibits cell proliferation and oncogenic transformation. Here, we show that 60% of all mRNAs deregulated by oncogenic transformation mediated by c-myc and H-ras are reverted to the expression levels of nontransformed cells by IRF-1. These include cell cycle–regulating genes. An indirect target is cyclin D1. Activation of IRF-1 decreased cyclin D1 expression and cyclin-dependent kinase 4 kinase activity concomitant with change in the levels of hyperphosphorylated retinoblastoma protein. These effects are mediated by inhibition of the mitogen-activated protein kinase kinase/extracellular signal-regulated kinase pathway and a transcriptional repression of cyclin D1. As shown by in vitro assays and tumor growth in nude mice, IRF-1–mediated effects on cell cycle progression were found to be overridden by ectopic expression of cyclin D1. Conversely, decrease of cyclin D1 by RNA interference experiments prevents transformation and tumor growth. The data show that cyclin D1 is a key target for IRF-1–mediated tumor-suppressive effects. [Cancer Res 2007;67(7):2972–81]
The Aryl Propionic AcidR-Flurbiprofen Selectively Induces p75NTR-Dependent Decreased Survival of Prostate Tumor Cells
Authors: Quann Emily J. , Khwaja Fatima , Zavitz Kenton H. , Djakiew Daniel
Published: April 2007

Epidemiologic studies show that patients chronically consuming nonsteroidal anti-inflammatory drugs (NSAID) for arthritis exhibit a reduced incidence of prostate cancer. In addition, some NSAIDs show anticancer activity in vitro. NSAIDs exert their anti-inflammatory effects by inhibiting cyclooxygenase (COX) activity; however, evidence suggests that COX-independent mechanisms mediate decreased prostate cancer cell survival. Hence, we examined the effect of selected aryl propionic acid NSAIDs and structurally related compounds on the decreased survival of prostate cancer cell lines PC-3, DU-145, and LNCaP by induction of the p75NTR protein. p75NTR has been shown to function as a tumor suppressor in the prostate by virtue of its intracellular death domain that can initiate apoptosis and inhibit growth. The most efficacious compounds for induction of p75NTR and decreased survival, in rank-order, were R-flurbiprofen, ibuprofen, oxaprozin, fenoprofen, naproxen, and ketoprofen. Because R-flurbiprofen and ibuprofen exhibited the greatest efficacy, we examined their dose-dependent specificity of induction for p75NTR relative to other members of the death receptor family. Whereas treatment with R-flurbiprofen or ibuprofen resulted in a massive induction of p75NTR protein levels, the expression of Fas, p55TNFR, DR3, DR4, DR5, and DR6 remained largely unchanged. Moreover, transfection of either cell line before R-flurbiprofen or ibuprofen treatment with a dominant negative form of p75NTR to antagonize p75NTR activity or p75NTR small interfering RNA to prevent p75NTR protein expression rescued both cell lines from decreased survival. Hence, R-flurbiprofen and ibuprofen selectively induce p75NTR-dependent decreased survival of prostate cancer cells independently of COX inhibition. [Cancer Res 2007;67(7):3254–62]
Regulation of Vascular Endothelial Growth Factor Receptor-1 Expression by Specificity Proteins 1, 3, and 4 in Pancreatic Cancer Cells
Authors: Abdelrahim Maen , Baker Cheryl H. , Abbruzzese James L. , Sheikh-Hamad David , Liu Shengxi , Cho Sung Dae , Yoon Kyungsil , Safe Stephen
Published: April 2007

Vascular endothelial growth factor receptor-1 (VEGFR1) is expressed in cancer cell lines and tumors and, in pancreatic and colon cancer cells, activation of VEGFR1 is linked to increased tumor migration and invasiveness. Tolfenamic acid, a nonsteroidal anti-inflammatory drug, decreases Sp protein expression in Panc-1 and L3.6pl pancreatic cancer cells, and this was accompanied by decreased VEGFR1 protein and mRNA and decreased luciferase activity on cells transfected with constructs (pVEGFR1) containing VEGFR1 promoter inserts. Comparable results were obtained in pancreatic cancer cells transfected with small inhibitory RNAs for Sp1, Sp3, and Sp4 and all three proteins bound to GC-rich elements in the VEGFR1 promoter. These results show that VEGFR1 is regulated by Sp proteins and that treatment with tolfenamic acid decreases expression of this critical angiogenic factor. Moreover, in vitro studies in Panc-1 cells show that activation of VEGFR1 by VEGFB to increase mitogen-activated protein kinase 1/2 phosphorylation and cell migration on collagen-coated plates is also inhibited by tolfenamic acid. Thus, targeted degradation of Sp proteins is highly effective for inhibiting VEGFR1 and associated angiogenic responses in pancreatic cancer. [Cancer Res 2007;67(7):3286–94]
Isogenic Normal Basal and Luminal Mammary Epithelial Isolated by a Novel Method Show a Differential Response to Ionizing Radiation
Authors: Huper Gudrun , Marks Jeffrey R.
Published: April 2007

Epithelial cells within the normal breast duct seem to be the primary target for neoplastic transformation events that eventually produce breast cancer. Normal epithelial cells are easily isolated and propagated using standard techniques. However, these techniques almost invariably result in populations of cells that are largely basal in character. Because only ∼20% of human breast cancers exhibit a basal phenotype, our understanding of the disease may be skewed by using these cells as the primary comparator to cancer. Further, because germ line mutations in BRCA1 yield breast cancers that are most often of the basal type, a comparison of normal basal and luminal cells could yield insight into the tissue and cell type specificity of this hereditary cancer susceptibility gene. In this report, we describe a simplified and efficient method for isolating basal and luminal cells from normal human breast tissue. These isogenic cells can be independently propagated and maintain phenotypic markers consistent with their respective lineages. Using these cultured cells, we show that basal and luminal cells exhibit distinct responses to ionizing radiation. Basal cells undergo a rapid but labile cell cycle arrest, whereas luminal cells show a much more durable arrest, primarily at the G2-M boundary. Molecular markers, including p53 protein accumulation, p53-activated genes, and BRCA1 nuclear focus formation all correlate with the respective cell cycle responses. Further, we show that short-term cultures of human breast tissue fragments treated with ionizing radiation show a similar phenomenon as indicated by the biphasic accumulation of p53 protein in the basal versus luminal layer. Together, these results indicate that normal basal cells have a transitory cell cycle arrest after DNA damage that may underlie their increased susceptibility to transformation after the loss of functional BRCA1. [Cancer Res 2007;67(7):2990–3001]
Tumor-Specific Cooperation of Retinoblastoma Protein Family and Snf5 Inactivation
Authors: Chai Jingjing , Lu Xiangdong , Godfrey Virginia , Fletcher Christopher , Roberts Charles W.M. , Van Dyke Terry , Weissman Bernard E.
Published: April 2007

Malignant rhabdoid tumors (MRT) are rare aggressive cancers that occur in young children. Seventy-five percent of sporadic MRTs harbor inactivating SNF5 mutations, and mice heterozygous for an Snf5-null allele develop MRTs with partial penetrance. The diagnosis of choroid plexus carcinomas (CPC) in addition to MRTs in families with a single mutant SNF5 allele prompted us to assess the role of SNF5 loss in CPC in genetically engineered mice. With high frequency, TgT121 mice develop CPCs that are initiated by inactivation of retinoblastoma protein (pRb) and related proteins p107 and p130. However, CPC penetrance and latency were not significantly affected by Snf5 heterozygosity, consistent with recent evidence that CPCs in SNF5 families were, in many cases, misdiagnosed MRTs. Surprisingly, although the CPC phenotype was unaffected, TgT121;Snf5+/− mice developed MRTs with increased penetrance and decreased latency compared with TgT121;Snf5+/+ littermates. MRTs expressed the T121 protein with a concomitant increase in mitotic activity. The predominant appearance of TgT121;Snf5+/− MRTs in the spinal cord led to the discovery that these tumors likely arose from a subset of spinal cord neural progenitor cells expressing T121 rather than from transdifferentiation of CPC. Significantly, the target cell type(s) for MRT is unknown. Hence, this study not only shows that pRbf and SNF5 inactivation cooperate to induce MRTs but also provides new insight into the MRT target population. [Cancer Res 2007;67(7):3002–9]
Improvement of Antitumor Activity by Gene Amplification with a Replicating but Nondisseminating Adenovirus
Authors: Bourbeau Denis , Lau Cara Jean , Jaime Jairo , Koty Zafiro , Zehntner Simone P. , Lavoie Geneviève , Mes-Masson Anne-Marie , Nalbantoglu Josephine , Massie Bernard
Published: April 2007

Gene therapy is a promising approach for cancer treatment; however, efficacy of current vectors remains insufficient. To improve the success of suicide gene therapy, we constructed a replication-competent adenoviral vector that has its protease gene deleted and expresses bacterial cytosine deaminase fused with bacterial uracil phosphoribosyltransferase (CU). The prodrug, 5-fluorocytosine, is transformed into the highly toxic and tissue-diffusible 5-fluorouracil by CU in infected cells. This vector is incapable of producing infectious particles but is able to undergo a single round of replication, thereby increasing transgene copy number and expression. In the presence of 5-FC, compared with the first-generation vector (AdCU), the replication-competent vector, Ad(dPS)CU-IRES-E1A, was significantly more efficacious for in vitro tumor cell killing and in bystander assays, whereas 25-fold fewer viral particles were required in a three-dimensional spheroid model. For in vivo experiments, in which virus was injected into preestablished intracranial glioma xenografts, followed by 5-FC treatment, mice receiving Ad(dPS)CU-IRES-E1A had significantly smaller tumors at 35 days postinjection as well as significantly longer median survival than mice treated with the replication-deficient, protease-deleted vector [Ad(dPS)CU]. In an immunocompetent syngeneic model, Ad(dPS)CU + 5-FC–treated mice had a median survival of only 23 days, whereas Ad(dPS)CU-IRES-E1A + 5-FC–treated animals had a survival of 57.1% at 365 days. In conclusion, Ad(dPS)CU-IRES-E1A in the presence of 5-FC produces more potent tumoricidal effects than its replication-deficient counterparts. [Cancer Res 2007;67(7):3387–95]
Identification of Putative Stem Cell Markers, CD133 and CXCR4, in hTERT–Immortalized Primary Nonmalignant and Malignant Tumor-Derived Human Prostate Epithelial Cell Lines and in Prostate Cancer Specimens
Authors: Miki Jun , Furusato Bungo , Li Hongzhen , Gu Yongpeng , Takahashi Hiroyuki , Egawa Shin , Sesterhenn Isabell A. , McLeod David G. , Srivastava Shiv , Rhim Johng S.
Published: April 2007

Understanding normal and cancer stem cells may provide insight into the origin of and new therapeutics for prostate cancer. Normal and cancer stem cells in prostate have recently been identified with a CD44+/α2β1high/CD133+ phenotype. Stromal cell–derived factor-1 (SDF-1) and its receptor, CXCR4, have multiple essential functions, including homing of stem cells and metastasis of cancer cells. We show here that human telomerase reverse transcriptase (hTERT)-immortalized primary nonmalignant (RC-165N/hTERT) and malignant (RC-92a/hTERT) tumor-derived human prostate epithelial cell lines retain stem cell properties with a CD133+/CD44+/α2β1+/34βE12+/CK18+/p63−/androgen receptor (AR)−/PSA− phenotype. Higher CD133 expression was detected in the hTERT-immortalized cells than in primary prostate cells. These immortalized cells exhibited “prostaspheres” in nonadherent culture systems and also maintained higher CD133 expression. The CD133+ cells from these immortalized cell lines had high proliferative potential and were able to differentiate into AR+ phenotype. In three-dimensional culture, the CD133+ cells from RC-165N/hTERT cells produced branched structures, whereas the CD133+ cells from RC-92a/hTERT cells produced large irregular spheroids with less branched structures. SDF-1 induced, but anti-CXCR4 antibody inhibited, migration of CD133+ cells from RC-92a/hTERT cells, which coexpressed CXCR4. CXCR4/SDF-1 may sustain tumor chemotaxis in cancer stem cells. Furthermore, immunostaining of clinical prostate specimens showed that CD133 expression was detected in a subpopulation of prostate cancer cells and corresponded to the loss of AR. Expression of CXCR4 was also detected in CD133+ cancer cells. These novel in vitro models may offer useful tools for the study of the biological features and functional integration of normal and cancer stem cells in prostate. [Cancer Res 2007;67(7):3153–61]
Item Selection Strategy for Reducing the Number of Items Rated in an Angoff Standard Setting Study
Authors: Ferdous Abdullah A. , Plake Barbara S.
Published: April 2007

In an Angoff standard setting procedure, judges estimate the probability that a hypothetical randomly selected minimally competent candidate will answer correctly each item in the test. In many cases, these item performance estimates are made twice, with information shared with the panelists between estimates. Especially for long tests, this estimation process can be time-consuming and fatiguing for the judges. This study extended an item selection strategy earlier proposed to form subsets of test items. The results of this study suggest that 40% to 50% of test items may be sufficient to estimate an equivalent passing score in an Angoff setting study when those items are selected to represent the full test in content, discrimination, and difficulty.
Assessing Fit of Cognitive Diagnostic Models A Case Study
Authors: Sinharay Sandip , Almond Russell G.
Published: April 2007

A cognitive diagnostic model uses information from educational experts to describe the relationships between item performances and posited proficiencies. When the cognitive relationships can be described using a fully Bayesian model, Bayesian model checking procedures become available. Checking models tied to cognitive theory of the domains provides feedback to educators about the underlying cognitive theory. This article suggests a number of graphics and statistics for diagnosing problems with cognitive diagnostic models expressed as Bayesian networks. The suggested diagnostics allow the authors to identify the inadequacy of an earlier cognitive diagnostic model and to hypothesize an improved model that provides better fit to the data.