bluuebunny/crossref_metadata_2025 huggingface dataset:
Colorimetric characterization of a computer‐controlled liquid crystal display
Authors: Day Ellen A. , Taplin Lawrence , Berns Roy S.
Published: July 2004

A new method was used to characterize computer‐controlled liquid crystal displays (LCDs). The characterization, which was performed to enable colorimetric image display, included channel independence, spatial independence, screen uniformity, and colorimetry. The colorimetric model consisted of three one‐dimensional look‐up tables (LUTs) describing each channel's optoelectronic transfer function and a 3 × 4 matrix transformation that included black‐level flare. The matrix coefficients were estimated statistically by minimizing the average CIEDE2000 color difference for a data set sampling the display's colorimetric gamut. The LUTs were recreated dynamically throughout the optimization of the matrix coefficients. The characterization was implemented with three different instruments to evaluate the robustness of the method with respect to measurement uncertainty. The average performance ranged between 0.1 and 0.4 ΔE00 and was well correlated with instrument precision. The optimization approach improved performance by a factor of two compared with direct measurements. Despite differences in instrument design, the chromaticities of each primary following optimization and black‐level flare compensation were very similar. This excellent performance was a result of the display's optoelectronic properties well matching the model assumptions. The technique was also used to characterize three additional LCD displays ranging in their matching of the model assumptions. In this case, performance worsened. For one display, more complex models would be required for colorimetric characterization. Finally, a colorimetric characterization based on measurements at the center of the display and perpendicular to the face was used to predict measurements at the edges and at different angles. The results indicated that characterizations would be required at multiple positions and angles in order to achieve sufficient accuracy. © 2004 Wiley Periodicals, Inc. Col Res Appl, 29, 365–373, 2004; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/col.20046
A study of colour emotion and colour preference. Part III: Colour preference modeling
Authors: Ou Li‐Chen , Luo M. Ronnier , Woodcock Andrée , Wright Angela
Published: July 2004

In this study three colour preference models for single colours were developed. The first model was developed on the basis of the colour emotions, clean–dirty, tense–relaxed, and heavy–light. In this model colour preference was found affected most by the emotional feeling “clean.” The second model was developed on the basis of the three colour‐emotion factors identified in Part I, colour activity, colour weight, and colour heat. By combining this model with the colour‐science‐based formulae of these three factors, which have been developed in Part I, one can predict colour preference of a test colour from its colour‐appearance attributes. The third colour preference model was directly developed from colour‐appearance attributes. In this model colour preference is determined by the colour difference between a test colour and the reference colour (L*, a*, b*) = (50, −8, 30). The above approaches to modeling single‐colour preference were also adopted in modeling colour preference for colour combinations. The results show that it was difficult to predict colour‐combination preference by colour emotions only. This study also clarifies the relationship between colour preference and colour harmony. The results show that although colour preference is strongly correlated with colour harmony, there are still colours of which the two scales disagree with each other. © 2004 Wiley Periodicals, Inc. Col Res Appl, 29, 381–389, 2004; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/col.20047
Prediction of optical efficacy of vital tooth bleaching using regression analysis
Authors: Ishikawa‐Nagai Shigemi , Terui Takayuki , Ishibashi Kanji , Weber Hans‐Peter , Ferguson Michael
Published: July 2004

Establishing a colorimetric guideline to predict the effectiveness of tooth bleaching could produce a more reliable dental treatment. The purpose of this study was to evaluate the effectiveness of tooth bleaching and to test the predictability of tooth color changes. A 10% carbamide peroxide bleaching system was used in studies at Harvard University and at Iwate Medical University in Japan. L*, a*, and b* values (CIELAB) for pre‐ and postbleaching were obtained and color differences (ΔE) were calculated. The b* and L* values of the original tooth color indicated a relatively strong to moderate correlation with ΔE values, whereas a* showed a weak correlation. The multiple‐regression equation obtained from the color data of Harvard subjects performed better than the predictive model. The predicted ΔE correlated strongly with the observed ΔE (r = 0.78). The validation of this equation on data collected from Iwate confirmed the strong correlation (r = 0.74). © 2004 Wiley Periodicals, Inc. Col Res Appl, 29, 390–394, 2004; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/col.20048
Bezolde‐Brucke effect exists in related and unrelated colors and resembles the Abney effect
Authors: Pridmore R. W.
Published: July 2004

The original article to which this Erratum refers was published in Color Res Appl 2004;29:241–246

No abstract.
Elliptic equations with BMO coefficients in Reifenberg domains
Authors: Byun Sun‐Sig , Wang Lihe
Published: July 2004

The inhomogeneous Dirichlet problems concerning divergence form elliptic equations are studied. Optimal regularity requirements on the coefficients and domains for the W1,p theory, 1 < p < ∞, are obtained. The principal coefficients are supposed to be in the John‐Nirenberg space with small BMO seminorms. The domain is a Reifenberg domain. These conditions for the W1,p theory not only weaken the requirements on the coefficients but also lead to a more general geometric condition on the domains. In fact, these domains might have fractal dimensions. © 2004 Wiley Periodicals, Inc.
Existence of two‐dimensional Skyrmions via the concentration‐compactness method
Authors: Lin Fanghua , Yang Yisong
Published: July 2004

In this paper, we prove the existence of a unit‐charge energy minimizer in the two‐dimensional Skyrme model by the method of concentration‐compactness. © 2004 Wiley Periodicals, Inc.
Global existence, singular solutions, and ill‐posedness for the Muskat problem
Authors: Siegel Michael , Caflisch Russel E. , Howison Sam
Published: July 2004

The Muskat, or Muskat‐Leibenzon, problem describes the evolution of the interface between two immiscible fluids in a porous medium or Hele‐Shaw cell under applied pressure gradients or fluid injection/extraction. In contrast to the Hele‐Shaw problem (the one‐phase version of the Muskat problem), there are few nontrivial exact solutions or analytic results for the Muskat problem. For the stable, forward Muskat problem, in which the higher‐viscosity fluid expands into the lower‐viscosity fluid, we show global‐in‐time existence for initial data that is a small perturbation of a flat interface. The initial data in this result may contain weak (e.g., curvature) singularities. For the unstable, backward problem, in which the higher‐viscosity fluid contracts, we construct singular solutions that start off with smooth initial data but develop a point of infinite curvature at finite time. © 2004 Wiley Periodicals, Inc.
Infinite Prandtl number limit of Rayleigh‐Bénard convection
Authors: Wang Xiaoming
Published: July 2004

We rigorously justify the infinite Prandtl number model of convection as the limit of the Boussinesq approximation to the Rayleigh‐Bénard convection as the Prandtl number approaches infinity. This is a singular limit problem involving an initial layer. © 2003 Wiley Periodicals, Inc.
Effect of inducible costimulator blockade on the pathological and protective immune responses induced by the gastrointestinal helminth Trichinella spiralis
Authors: Scales Hannah E. , Ierna Michelle X. , Gutierrez‐Ramos Jose‐Carlos , Coyle Anthony J. , Garside Paul , Lawrence Catherine E.
Published: August 2004

Infections with gastrointestinal helminths elicit potent Th2 responses, which ultimately result in their expulsion. However, during expulsion of Trichinella spiralis this Th2 response also induces a severe enteropathy characterized by villus atrophy and crypt hyperplasia. Inducible costimulator (ICOS), a homologue of CD28, interacts with B7‐related protein 1, and has been shown to be important in T‐B cell interactions and antibody class switching. Significantly, ICOS appears to be involved in the induction of both Th1 and Th2 responses, but may be of heightened importance in Th2 responses. Here we employed a blocking antibody against ICOS to investigate the contribution of ICOS costimulation to the development of the protective and pathological immune responses induced during infection with T. spiralis. We show that, although blocking ICOS resulted in a decrease in TNF‐α and the Th2 cytokines IL‐4 and IL‐5 and serum levels of total IgE, it did not affect the expulsion of the adult parasites. Surprisingly, levels of IL‐9, IL‐13 and IL‐10 were elevated and protection against the larval muscle stage of the parasite was enhanced. Importantly, these findings may relate to the fact that ICOS blockade significantly ameliorated the enteropathy that usually accompanies expulsion of the adult parasite.
Primary induction of CD4 T cell responses in nasal associated lymphoid tissue during group A streptococcal infection
Authors: Park Hae‐Sun , Costalonga Massimo , Reinhardt R. Lee , Dombek Priscilla E. , Jenkins Marc K. , Cleary P. Patrick
Published: August 2004

CD4 T cells are important for development of long‐term immunity to bacterial infections. Here we describe construction of a group A streptococcus (GAS) strain that expresses the model ovalbumin epitope (OVA) on its surface, and the use of this strain in adoptive transfer experiments to study CD4 T cell response to bacterial infection in nasal‐associated lymphoid tissue (NALT), which was previously shown to be a specific target for GAS colonization. The OVA+ GAS, but not the wild‐type strain was shown to activate CD4 T cells in an antigen‐specific manner both in vitro and in vivo. After intranasal infection of mice with this strain, OVA‐specific CD4 T cells were first activated in NALT, which is functionally equivalent to human tonsils, rather than in the cervical lymph nodes. During localized infection, OVA+ GAS induced rapid and prolonged activation of CD4 T cells at higher magnitudes in the NALT than in draining lymph nodes and spleen, where CD4 T cells underwent little or no activation. In contrast, systemic infection induced significantly higher activation of CD4 T cells in both lymph nodes and spleens, compared to when the infection was localized in NALT. Further investigation of cellular immune responses in NALT during GAS infection using adoptive T cell transfer, combined with the model antigen on the pathogen may ultimately shed light on mechanisms for failure of children to develop protective immune responses following streptococcal tonsillitis.
Transgene‐mediated hyper‐expression of IL‐5 inhibits autoimmune disease but increases the risk of B cell chronic lymphocytic leukemia in a model of murine lupus
Authors: Wen Xiangshu , Zhang Danqing , Kikuchi Yuji , Jiang Yi , Nakamura Kazuhiro , Xiu Yan , Tsurui Hiromichi , Takahashi Kazuko , Abe Masaaki , Ohtsuji Mareki , Nishimura Hiroyuki , Takatsu Kiyoshi , Shirai Toshikazu , Hirose Sachiko
Published: August 2004

IL‐5 preferentially activates B1 cells to produce natural antibodies cross‐reactive to self antigens. To determine the role of IL‐5 in antibody‐mediated autoimmune disease, we generated systemic lupus erythematosus (SLE)‐prone (NZB×NZW)F1 mice congenic for IL‐5 transgene (TG‐F1). The transgene unexpectedly reduced the incidence of lupus nephritis. Anti‐DNA antibodies in sera and those produced by splenic B cells in vitro were markedly decreased in TG‐F1 mice, while total polyclonal Ig levels were comparable to those in IL‐5 transgene‐negative (NZB×NZW)F1 (non‐TG‐F1) littermates. Flow cytometry‐sorted splenic B1 cells showed a significant reduction of anti‐DNA antibody synthesis in response to IL‐5, while proliferative responses to IL‐5 did not significantly differ between TG‐F1 and non‐TG‐F1 mice. As TG‐F1 mice aged, frequencies of peripheral B1 cells progressively increased, and the mice frequently developed B cell chronic lymphocytic leukemia (B‐CLL). Our results suggest that dysregulated, continuous high expression of IL‐5 in SLE‐prone mice may directly or indirectly mediate a skewed signaling of proliferation/differentiation of self‐antigen‐activated B1 cells, leading to suppression of autoimmune disease, but instead to aberrant expansion of B1 cells, giving rise to B‐CLL. Thus, this model may provide a clue to the pathogenesis of both SLE and B‐CLL.
The acute effects of L‐theanine in comparison with alprazolam on anticipatory anxiety in humans
Authors: Lu Kristy , Gray Marcus A. , Oliver Chris , Liley David T. , Harrison Ben J. , Bartholomeusz Cali F. , Phan K. Luan , Nathan Pradeep J.
Published: July 2004

L‐Theanine (δ‐glutamylethylamide) is one of the predominant amino acids ordinarily found in green tea, and historically has been used as a relaxing agent. The current study examined the acute effects of L‐theanine in comparison with a standard benzodiazepine anxiolytic, alprazolam and placebo on behavioural measures of anxiety in healthy human subjects using the model of anticipatory anxiety (AA). Sixteen healthy volunteers received alprazolam (1 mg), L‐theanine (200 mg) or placebo in a double‐blind placebo‐controlled repeated measures design. The acute effects of alprazolam and L‐theanine were assessed under a relaxed and experimentally induced anxiety condition. Subjective self‐reports of anxiety including BAI, VAMS, STAI state anxiety, were obtained during both task conditions at pre‐ and post‐drug administrations. The results showed some evidence for relaxing effects of L‐theanine during the baseline condition on the tranquil–troubled subscale of the VAMS. Alprazolam did not exert any anxiolytic effects in comparison with the placebo on any of the measures during the relaxed state. Neither L‐theanine nor alprazalam had any significant anxiolytic effects during the experimentally induced anxiety state. The findings suggest that while L‐theanine may have some relaxing effects under resting conditions, neither L‐theanine not alprazolam demonstrate any acute anxiolytic effects under conditions of increased anxiety in the AA model. Copyright © 2004 John Wiley & Sons, Ltd.
Evidence for synergistic modulation of early information processing by nicotinic and muscarinic receptors in humans
Authors: Erskine F. F. , Ellis J. R. , Ellis K. A. , Stuber E. , Hogan K. , Miller V. , Moore E. , Bartholomeusz C. , Harrison B. J. , Lee B. , Phan K. L. , Liley D. , Nathan P. J.
Published: July 2004

Impairments in early information processing are a hallmark feature of diverse neuropsychiatric disorders including schizophrenia and Alzheimer's disease (AD). Several lines of evidence implicate a dysfunction of the cholinergic system in these disorders, particularly in AD where there is known degeneration in major cholinergic pathways. Inspection time (IT), a measure of early visual information processing speed, has been shown to be sensitive to cholinergic manipulation. The current study employed the IT task to (1) examine the independent roles of nicotinic and muscarinic receptors in modulating information processing and (2) investigate the interaction of nicotinic and muscarinic receptor systems in modulating information processing. Twelve healthy participants completed a randomized, double‐blind, placebo‐controlled study under four drug conditions; (1) placebo, (2) mecamylamine (15 mg; oral), (3) scopolamine (0.4 mg, s.c.), (4) mecamylamine (15 mg)+scopolamine (0.4 mg). IT measures were examined at baseline and 2.5 h post drug administration. Selective blockade of nicotinic receptors with mecamylamine did not significantly impair IT, whereas selective blockade of muscarinic receptors with scopolamine produced a significant but small impairment in IT. Combined blockade of both receptor types with scopolamine and mecamylamine produced a large impairment in IT performance. The results indicate that both nicotinic and muscarinic receptors are involved in modulating IT, and that the two systems may function synergistically to modulate early visual information processing. These findings suggest that functional abnormalities in both nicotinic and muscarinic systems may underlie deficits in early visual information processing seen in disorders such as Alzheimer's disease and schizophrenia. Copyright © 2004 John Wiley & Sons, Ltd.
Lubrication and wear properties of grafted polyelectrolytes, hyaluronan and hylan, measured in the surface forces apparatus
Authors: Benz Marcel , Chen Nianhuan , Israelachvili Jacob
Published: August 2004

Hyaluronan is believed to have an important function in the boundary biolubrication of articular cartilage. Using a Surface Forces Apparatus, we tested the tribological properties of surface bound, rather than “free” hyaluronan. The grafting process of the polyelectrolyte included either a biological route via an HA‐binding protein or a chemical reaction to covalently bind the polymer to a lipid bilayer coated surface. In another reaction, we constructed a surface with covalently grafted hylan (crosslinked hyaluronan). We studied the normal and shear forces between these surfaces. None of the systems demonstrated comparable lubrication to that found between cartilage surfaces except at very low loads. Both grafted hyaluronan and hylan generated coefficients of friction between 0.15 and 0.3. Thus, the polysaccharide, which is a constituent of the lamina splendens (outermost cartilage layer), is not expected to be the responsible molecule for the great lubricity of cartilage; however, it may contribute to the load bearing and wear protection of these surfaces. This was concluded from the results with hylan, where a thin gel layer was sufficient to shield the underlying surfaces from damage even at applied pressures of over 200 atmospheres during shear. Our study shows that a low coefficient of friction is not a requirement for, or necessarily a measure of, wear protection. © 2004 Wiley Periodicals, Inc. J Biomed Mater Res 71A: 6–15, 2004
Chondrogenic differentiation of murine embryonic stem cells: Effects of culture conditions and dexamethasone
Authors: Tanaka Hiroshi , Murphy Christopher L. , Murphy Chiho , Kimura Mitsuhiro , Kawai Shinya , Polak Julia M.
Published: July 2004

Pluripotent embryonic stem (ES) cells have the capability to differentiate to various cell types and may represent an alternative cell source for the treatment of cartilage defects. Here, we show that differentiation of ES cells toward the chondrogenic lineage can be enhanced by altering the culture conditions. Chondrogenesis was observed in intact embryoid body (EB) cultures, as detected by an increase in mRNA levels for aggrecan and Sox9 genes. Collagen IIB mRNA, the mature chondrocyte‐specific splice variant, was absent at day 5, but appeared at later time points. Dexamethasone treatment of alginate‐encapsulated EB cultures did not have a strong chondrogenic effect. Nor was chondrogenesis enhanced by alginate encapsulation compared to simple plating of EBs. However, disruption of day 5 EBs and culture as a micromass or pelleted mass, significantly enhanced the expression of the cartilage marker gene collagen type II and the transcription factor Sox9 compared to all other treatments. Histological and immunohistochemical analysis of pellet cultures revealed cartilage‐like tissue characterized by metachromatically stained extracellular matrix and type II collagen immunoreactivity, indicative of chondrogenesis. These findings have potentially important implications for cartilage tissue engineering, since they may enable the increase in differentiated cell numbers needed for the in vitro development of functional cartilaginous tissue suitable for implantation. © 2004 Wiley‐Liss, Inc.
Differential transcriptional effects of PTH and estrogen during anabolic bone formation
Authors: von Stechow D. , Zurakowski D. , Pettit A.R. , Müller R. , Gronowicz G. , Chorev M. , Otu H. , Libermann T. , Alexander Joseph M.
Published: July 2004

The aim of this study was to compare transcriptional regulation in vivo during anabolic bone formation induced by either estradiol (E2) treatment or intermittent parathyroid hormone[1‐34] (PTH) therapy. We utilized an ovariectomized (OVX) mouse model of osteoporosis and transcriptional profiling to identify genes upregulated by either high‐dose E2 or PTH. Five weeks post‐OVX, the mice were administered either E2 and/or PTH, or vehicle for 4 weeks. Femoral bones were analyzed by microCT and histomorphometry to confirm the anabolic effect of each treatment. OVX vehicle‐treated control mice lost metaphyseal trabecular bone, with significant decrease in trabecular number, thickness, and connectivity. Both E2 and PTH treatments increased trabecular and cortical bone indices above the level of the sham operated controls, fully restoring both bone volume and bone mineral density (BMD). Moreover, PTH/E2 combination treatment led to significantly greater increase in cancellous bone and BMD than would be expected from the additive effects of the separate treatments. To determine whether PTH and E2 treatments were stimulating similar bone anabolic mechanisms, or were activating distinct signaling pathways, we compared patterns of gene expression using transcriptional profiling after either E2 or PTH treatment. After 4, 11, and 24 days of treatment, total RNA was collected from both the distal femoral metaphysis and diaphysis. Transcriptional profiling was performed using Affymetrix GeneChip probe arrays, comprised of approximately 36,000 full‐length mouse genes and EST clusters from the UniGene database. Several markers of osteoblast activity, including c‐fos, RANKL, PHEX, and PTHR1, were consistently upregulated by PTH in both skeletal sites. PTH treatment also increased expression of Cathespin K, consistent with the predicted increase in osteoclast activity. E2 treatment upregulated a largely distinct set of genes, including TGFβ3, and BMP1, as well as several genes critical for cell cycle control, including Cyclin D1 and CDK inhibitor 1A. Overall, comparison of transcriptional profiles suggest that anabolic responses in bone to PTH and high‐dose E2 treatment after OVX‐induced osteoporosis involve largely distinct patterns of gene regulation, each resulting in restoration of bone mass. © 2004 Wiley‐Liss, Inc.
Autoinhibited proteins as promising drug targets
Authors: Peterson Jeffrey R. , Golemis Erica A.
Published: July 2004

Current drug discovery efforts generally focus on a limited number of protein classes, typically including proteins with well‐defined catalytic active sites (e.g., kinases) or ligand binding sites (e.g., G protein‐coupled receptors). Nevertheless, many clinically important pathways are mediated by proteins with no such obvious targets for small molecule inhibitors. Allosteric inhibitors offer an alternative approach to inhibition of protein activities, particularly for proteins that undergo conformational changes as part of their activity cycle. Proteins regulated by autoinhibitory domains represent one broad class of proteins that meets this criterion. In this article, we discuss the potential of autoinhibited proteins as targets for allosteric inhibitors and describe two examples of small molecules that act by stabilizing native autoinhibited conformations of their targets. We propose that proteins regulated by autoinhibition may be generally amenable to allosteric inhibition by small molecules that stabilize the native, autoinhibited fold. © 2004 Wiley‐Liss, Inc.
Changes in adipocyte hormones leptin, resistin, and adiponectin in thyroid dysfunction
Authors: Yaturu Subhashini , Prado Susan , Grimes Sidney R.
Published: July 2004

Thyroid hormones as well as the recently discovered secretory products of adipose tissue adiponectin and resistin take part in energy metabolism. To study the changes in the adipocyte hormones with changes in the thyroid functional status, we measured adiponectin, resistin, and leptin in 69 subjects with Graves' disease before and 32 patients at follow up after treatment for hyperthyroidism at hypothyroid state. Concentrations of serum adiponectin and resistin were higher in hyperthyroid state than in hypothyroid state (adiponectin: 5.73 ± 1.1 vs. 3.0 ± 0.5 ng/ml, P = 0.03) (resistin: 6.378 ± 0.6 vs. 5.81 ± 0.57 ng/ml, P < 0.0001). Resistin levels correlate positively with free t4(r = 0.37, P < 0.01), free t3 levels(r = 0.33, P < 0.01) and negatively with TSH(r = −0.22, P < 0.05). Adiponectin levels correlate with free t4(r = 0.33, P < 0.01) and free t3 (r = 0.44, P < 0.01). Though the adiponectin levels did not correlate with leptin or resistin levels, strong positive correlation of both resistin and adiponectin with thyroid hormones is noted. Serum levels of leptin did not change with change in the thyroid functional status (leptin: 53.38 ± 2.47 vs. 55.10 ± 2.58 NS). Leptin levels did not correlate with resistin and adiponectin. We conclude that thyroid function has effect on adipocyte hormones adiponectin and resistin but not leptin. © 2004 Wiley‐Liss, Inc.
Transforming growth factor beta stimulation of biglycan gene expression is potentially mediated by sp1 binding factors
Authors: Heegaard Anne‐Marie , Xie Zhongjian , Young Marian Frances , Nielsen Karina Lishmann
Published: July 2004

Biglycan is a small leucine‐rich proteoglycan which is localized in the extracellular matrix of bone and other specialized connective tissues. Both biglycan mRNA and protein are up‐regulated by transforming growth factor‐β1 (TGF‐β1) and biglycan appears to influence TGF‐β1 activity. In this study, we have investigated the mechanism by which TGF‐β1, TGF‐β2 and TGF‐β3 stimulate biglycan mRNA expression in the osteoblastic cell line MG‐63. The cells were transfected with a series of deletional human biglycan promoter constructs and a region in the biglycan 5′ DNA was found to respond to TGF‐β1 with increased transcriptional activity in a dose‐dependent manner. Also TGF‐β2 and TGF‐β3, two structurally highly related TGF‐β isoforms stimulated biglycan transcription. A TGF‐β responsive region was identified within the first 218 bp of the human biglycan promoter upstream from the transcriptional start site, which contained several binding sites for the transcription factor Sp1. Electrophoretic mobility shift assays with nuclear extracts from MG‐63 cells showed binding of both Sp1 and Sp3 to a site at −216 to −208. When the biglycan promoter construct was co‐transfected with Sp1 and Sp3 expression vectors in Sp1‐deficient Drosophila Schneider‐2 cells, Sp1 induced the transcriptional activity of biglycan. Addition of Sp3 augmented the effect of Sp1 on biglycan gene expression. Induction of biglycan mRNA expression in response to TGF‐β in MG‐63 cells was abrogated by mithramycin, an inhibitor of Sp1 binding to GC‐rich DNA sequences. A mutation in the Sp1 site at −216 to −208 within the −218 biglycan promoter construct substantially diminished the transcriptional up‐regulation by TGF‐β1. Taken together this data shows for the first time that TGF‐β1 stimulation of human biglycan mRNA expression relies on increased transcription of the biglycan gene, and is mediated by members of the Sp1 family of transcription factors. © 2004 Wiley‐Liss, Inc.
Long‐term in vitro analysis of limb cartilage development: Involvement of Wnt signaling
Authors: Daumer Kathleen M. , Tufan A. Cevik , Tuan Rocky S.
Published: July 2004

Endochondral skeletal development involves the condensation of mesenchymal cells, their differentiation into chondrocytes, followed by chondrocyte maturation, hypertrophy, and matrix mineralization, and replacement by osteoblasts. The Wnt family of secreted proteins have been shown to play important roles in vertebrate limb formation. To examine the role(s) of Wnt members and their transmembrane‐spanning receptor(s), Frizzled (fz), we retrovirally misexpressed Wnt‐5a, Wnt‐7a, chicken frizzled‐1 (Chfz‐1), and frizzled‐7 (Chfz‐7) in long‐term (21 day) high density, micromass cultures of stage 23/24 chick embryonic limb mesenchyme. This culture system recapitulates in vitro the entire differentiation (days 1–10), growth (days 5–12), and maturation and hypertrophy (from day 12 on) program of cartilage development. Wnt‐7a misexpression severely inhibited chondrogenesis from day 7 onward. Wnt‐5a misexpression resulted in a poor hypertrophic phenotype by day 14. Chfz‐7 misexpression caused a slight delay of chondrocyte maturation based on histology, whereas Chfz‐1 misexpression did not affect the chondrogenic phenotype. Misexpression of all Wnt members decreased collagen type X expression and alkaline phosphatase activity at day 21. Our findings implicate functional role(s) for Wnt signaling throughout embryonic cartilage development, and show the utility of the long‐term in vitro limb mesenchyme culture system for such studies. © 2004 Wiley‐Liss, Inc.
Myocardin/MKL family of SRF coactivators: Key regulators of immediate early and muscle specific gene expression
Authors: Cen Bo , Selvaraj Ahalya , Prywes Ron
Published: July 2004

Myocardin, megakaryoblastic leukemia‐1 (MKL1), and MKL2 belong to a newly defined family of transcriptional coactivators. All three family members bind to serum response factor (SRF) and strongly activate transcription from promoters with SRF binding sites. SRF is required for the serum induction of immediate early genes such as c‐fos and for the expression of many muscle specific genes. Consistent with a role in muscle specific gene expression, myocardin is specifically expressed in cardiac and smooth muscle cells while MKL1 and 2 are broadly expressed. Myocardin has particularly been shown to be required for smooth muscle development while MKL1/2 are required for the RhoA signaling pathway for induction of immediate early genes. SRF can be activated by at least two families of coactivators, p62TCF and myocardin/MKL. These factors bind to the same region of SRF such that their binding is mutually exclusive. This provides one mechanism of regulation of SRF target genes by pathways that differentially activate the coactivators. The RhoA pathway appears to activate MKL1 by altering MKL1's binding to actin and causing MKL1's translocation from the cytoplasm to the nucleus. However, this mechanism of activation of the myocardin/MKL family has not been observed in all cell types such that other regulatory mechanism(s) likely exist. In particular, rapid serum inducible phosphorylation of MKL1 was observed. The regulation of this coactivator family is key to understanding how SRF target genes are activated during muscle cell differentiation or growth factor induced cell proliferation. © 2004 Wiley‐Liss, Inc.
Effect of overexpression and nuclear translocation of constitutively active PKB‐α on cellular survival and proliferation in HepG2 cells
Authors: Gupta Dhananjay , Syed Noor A. , Roesler William J. , Khandelwal Ramji L.
Published: July 2004

Protein kinase B (Akt/PKB) is a key component in the PI 3‐kinase mediated cell survival pathway and has oncogenic transformation potential. Although the over‐expression of PKB‐α can prevent cell death following growth factor withdrawal, the long‐term effects of stable over‐expression of PKB‐α on cell survival in the absence of growth factors remain to be resolved. In the present study, we generated HepG2 cells with stable expression of active PKB‐α and compared its characteristics with HepG2 cells. Basal as well as insulin‐stimulated levels of Ser473 and Thr308 phosphorylation in PKB‐α transfected HepG2 cells were much higher than HepG2 cells. Constitutive expression of active PKB‐α enabled HepG2 cells to survive up to 96 h without serum in growth media while HepG2 cells fail to survive after 48 h of serum withdrawal. A strong positive correlation (R2 = 0.71) between cell proliferation and phosphorylated form of PKB‐α at Thr308 was observed along with higher levels of phosphorylated 3′‐phosphoinositide‐dependent kinase‐1 (PDK‐1). HepG2 cells with constitutive expression of active PKB‐α also showed higher levels of phosphorylated p65 subunit of nuclear factor‐κB (NFκB) in comparison with HepG2 cells. Predominant nuclear localization of phosphorylated PKB‐α was observed in stably transfected HepG2 cells. These results indicate that constitutive expression of active PKB‐α renders HepG2 cells independent of serum based growth factors for survival and proliferation. © 2004 Wiley‐Liss, Inc.
Changes in receptor activator of nuclear factor‐kappaB, and its ligand, osteoprotegerin, bone‐type alkaline phosphatase, and tartrate‐resistant acid phosphatase in ovariectomized rats
Authors: Miyazaki Takashi , Matsunaga Toshiyuki , Miyazaki Shuichi , Hokari Shigeru , Komoda Tsugikazu
Published: July 2004

We investigated time‐course changes in the expression of receptor activator of nuclear factor‐kappaB (RANK), its ligand (RANKL), osteoprotegerin (OPG), bone‐type alkaline phosphatase (BAP), and tartrate‐resistant acid phosphatase (TRAP) in ovariectomized (OVX) rats. Samples of sera and coccyges were used for analysis of the enzyme activities and expression levels of proteins and mRNAs, and an immunohistochemical analysis was also performed. Serum BAP activity increased to 158.6% of the pre‐operation value at 1 week after OVX, and then decreased to 38.7% at 8 weeks after OVX. On the other hand, the serum TRAP activity increased to 130.9% of the pre‐operation level at 1 week after OVX, and was maintained at a high level, compared with the pre‐operation level. The patterns of BAP and TRAP activity in the coccyges specimens were similar to those seen in the sera. The expression profiles of TRAP, RANK, and RANKL proteins in the coccyx specimens were similar to the pattern of serum TRAP activity, while the profiles of the BAP and OPG proteins were similar to the pattern of serum BAP activity in OVX rats. The changes in the mRNA expression levels of the osteogenic proteins were similar to those for protein expression. These biochemical changes in OVX rats were confirmed by immunohistochemical studies. Our results suggest that not only osteoclastogenesis accelerated but also osteoblastogenesis transiently increased during the early phase of osteoporosis. © 2004 Wiley‐Liss, Inc.
Thiol oxidation of cell signaling proteins: Controlling an apoptotic equilibrium
Authors: Cross Janet V. , Templeton Dennis J.
Published: July 2004

Studies of cell signal transduction have predominantly focused on regulation of protein function by phosphorylation. However, recent efforts have begun to uncover another layer of regulation mediated by direct oxidation of cysteine residues in signaling proteins. Typically induced during signaling responses accompanied by generation of reactive oxygen species, these thiol modifications have a variety of functional consequences for target proteins. Using specific signaling protein targets as examples, we discuss how thiol oxidation generally activates pro‐apoptotic signaling pathways while inhibiting pathways that promote cell survival. We propose a model in which thiol oxidation acts to control the equilibrium between survival and apoptosis, fine tuning cellular responses that play a central role in the apoptotic decision‐making process. We identify areas of focus for future work, including a better understanding of specificity in thiol oxidation events, and a critical need for approaches to examine these modifications under physiologically relevant signaling conditions. © 2004 Wiley‐Liss, Inc.
Simultaneous detection of DsRed2‐tagged and EGFP‐tagged human β‐interferons in the same single cells
Authors: Maruyama Masato , Nishio Teruko , Yoshida Toyokazu , Ishida Chisaki , Ishida Kayo , Watanabe Yoshihiko , Nishikawa Makiya , Takakura Yoshinobu
Published: July 2004

The red fluorescent protein DsRed2 is a useful fusion tag for various proteins, together with the enhanced green fluorescent protein (EGFP). These chromoproteins have spectral properties that allow simultaneous distinctive detection of tagged proteins in the same single cells by dual color imaging. We used them for tagging a secretory protein, human interferon‐β (IFN‐β). Expression plasmids for human IFN‐β tagged with DsRed2 or with EGFP at the carboxyl terminal were constructed and their coexpression was examined in Mardin–Darby canine kidney epithelial cells. Although maturation of DsRed2 for coloration was slow and the color intensity was weak compared with EGFP, low temperature treatment (20°C) allowed DsRed2‐tagged human IFN‐β to be detected in the cells using color imaging. Consequently, the two chimeric proteins were shown to be colocalized in the same single cells by dual color confocal microscopy. This approach will be useful for investigating subcellular localization of not only cell resident proteins but also secretory proteins. © 2004 Wiley‐Liss, Inc.
Effects of phosphorylation by protein kinase CK2 on the human basal components of the RNA polymerase II transcription machinery
Authors: Cabrejos María Eugenia , Allende Catherine C. , Maldonado Edio
Published: July 2004

We have investigated the role of phosphorylation by vertebrate protein kinase CK2 on the activity of the General Transcription Factors TFIIA, TFIIE, TFIIF, and RNAPII. The largest subunits of TFIIA, TFIIE, and TFIIF were phosphorylated by CK2 holoenzyme. Also, RNA polymerase II was phosphorylated by CK2 in the 214,000 and 20,500 daltons subunits. Our results show that phosphorylation of TFIIA, TFIIF, and RNAPII increase the formation of complexes on the TATA box of the Ad‐MLP promoter. Also, phosphorylation of TFIIF increases the formation of transcripts, where as phosphorylation of RNA polymerase II dramatically inhibits transcript formation. Furthermore, we demonstrate that CK2β directly interacts with RNA polymerase II, TFIIA, TFIIF, and TBP. These results strongly suggest that CK2 may play a role in regulating transcription of protein coding genes. © 2004 Wiley‐Liss, Inc.
Multiple functions of BMPs in chondrogenesis
Authors: Yoon Byeong S. , Lyons Karen M.
Published: July 2004

The ability of bone morphogenetic proteins (BMPs) to promote chondrogenesis has been investigated extensively over the past two decades. Although BMPs promote almost every aspect of chondrogenesis, from commitment to terminal differentiation is well known, the mechanisms of BMP action in discrete aspects of endochondral bone formation have only recently begun to be investigated. In this review, we focus on in vivo studies that have identified interactions between BMP signaling pathways and key downstream targets of BMP action in chondrogenesis. We also discuss evidence regarding the potential roles of BMP receptors in mediating distinct aspects of chondrogenesis, and studies investigating the intersection of BMP pathways with other pathways known to coordinate the progression of chondrocytes through the growth plate. These studies indicate that both Smad‐dependent and ‐independent BMP pathways are required for chondrogenesis, and that BMPs exert essential roles via regulation of the Indian hedgehog (IHH)/parathyroid hormone‐related protein (PTHrP) and fibroblast growth factor (FGF) pathways in the growth plate. © 2004 Wiley‐Liss, Inc.
Down‐regulation of p21WAF1 promotes apoptosis in senescent human fibroblasts: Involvement of retinoblastoma protein phosphorylation and delay of cellular aging
Authors: Huang Ying , Corbley Michael J. , Tang Zuoqin , Yang Long , Peng Yong , Zhang Zong Yu , Tong Tan Jun
Published: July 2004

It has been suggested that genes which exercise checkpoint control during cell cycle traverse are equally important to the process of apoptotic cell death. In this study, we show that the key cell cycle regulatory gene p21WAF1 is also involved in the execution of apoptosis. p21WAF1 expression was down‐regulated during NaBu‐induced apoptosis of senescent normal diploid human 2BS fibroblasts. Conversely, when p21WAF1 expression was actively suppressed in 2BS cells by a stably transfected antisense p21WAF1 construct, apoptosis was accelerated and senescence was delayed, as shown by several markers of cell aging. Down‐regulation of p21WAF1 by antisense caused an increase in the phosphorylation and inactivation of pRb. Phosphorylation of pRb was further enhanced upon induction of apoptosis by NaBu. Our results suggest that p21WAF1, acting through the phosphorylation of pRb, regulates whether 2BS cells cease to proliferate and become senescent but resistant to apoptosis, or whether they accelerate proliferation while becoming more susceptible to apoptotic stimuli. © 2004 Wiley‐Liss, Inc.
Bone morphogenetic protein‐2 suppresses collagenase‐3 promoter activity in osteoblasts through aruntdomain factor 2 binding site
Authors: Varghese Samuel , Rydziel Sheila , Canalis Ernesto
Published: July 2004

Transforming growth factor‐β (TGFβ) superfamily of growth factors, which include bone morphogenetic proteins (BMPs), have multiple effects in osteoblasts. In this study, we examined the regulation of collagenase‐3 promoter activity by BMP‐2 in osteoblast‐enriched (Ob) cells from fetal rat calvariae. BMP‐2 suppressed the activity of a −2 kb collagenase‐3 promoter/luciferase recombinant in a time‐ and dose‐dependent manner. The BMP‐2 effect on the collagenase‐3 promoter was further tested in several collagenase‐3 promoter deletion constructs and it was narrowed down to a −148 to −94 nucleotide segment of the promoter containing aruntdomain factor 2 (Runx2) site at nucleotide −132 to −126. The effect of BMP‐2 was obliterated in a collagenase‐3 promoter/luciferase construct containing a mutated Runx2 (mRunx2) sequence indicating that the Runx2 site mediates the BMP‐2 response. Electrophoretic mobility shift assays, using nuclear extracts from control and BMP‐2‐treated Ob cells, indicated that the Runx2 protein is a component of the specific DNA–protein complex formed on the Runx2 site and that the BMP‐2 effect may be associated with minor protein modifications rather than major changes in the composition of specific proteins interacting with the Runx2 site. We confirmed that other members of the TGFβ family can down‐regulate the collagenase‐3 promoter by showing that TGFβ1 also suppresses the promoter activity in a time‐ and dose‐dependent manner. In conclusion, this study demonstrates that BMP‐2 and TGFβ1 suppress collagenase‐3 promoter activity in osteoblasts and establishes a link between BMP‐2 action and collagenase‐3 expression via Runx2, a major regulator of osteoblast formation and function. © 2004 Wiley‐Liss, Inc.
KCNQ1/KCNE1 channels during germ‐cell differentiation in the rat: Expression associated with testis pathologies
Authors: Tsevi Irini , Vicente Rubén , Grande Maribel , López‐Iglesias Carmen , Figueras Agnès , Capellà Gabriel , Condom Enric , Felipe Antonio
Published: August 2004

KCNQ1/KCNE1 channels are responsible for the Jervell–Lange–Nielsen cardiac syndrome, which is also characterized by congenital deafness. KCNQ1/KCNE1 is crucial for K+ transport in the inner ear. We show that KCNQ1 and KCNE1 are associated in testis and that their expression is closely regulated during development. Both genes were expressed in undifferentiated germ cells in 21‐day‐old rats and mostly confined to basal immature germ cells in adulthood. Leydig and Sertoli cells were negative. KCNQ1 and KCNE1 were also studied in various germ‐cell pathologies. First, in spontaneous unilateral rat testis atrophy, hematoxylin–eosin analysis revealed massive germ‐cell aplasia with only Sertoli cells and groups of interstitial Leydig cells. In these samples, KCNQ1 and KCNE1 were not expressed. In human seminoma samples characterized by a proliferation of undifferentiated germ cells, KCNQ1/KCNE1 protein levels were higher than in healthy samples. Our results demonstrate that the expression of KCNQ1 and KCNE1 is associated with early stages of spermatogenesis and with the presence of undifferentiated healthy or neoplastic germ cells. The presence of a K+ rich‐fluid in the seminiferous tubule suggests that KCNQ1/KCNE1 is involved in K+ transport, probably during germ‐cell development. © 2004 Wiley‐Liss, Inc.
Key role of the achievement of an appropriate ribosomal RNA complement for G1‐S phase transition in H4‐II‐E‐C3 rat hepatoma cells
Authors: Derenzini Massimo , Montanaro Lorenzo , Chillà Alessandra , Tosti Elena , Vici Manuela , Barbieri Stefania , Govoni Marzia , Mazzini Giuliano , Treré Davide
Published: August 2004

Cell growth is closely related to cell proliferation and an adequate ribosome biogenesis appears to be necessary for cell duplication. In the present study, we have investigated the relationship between rRNA synthesis and cell cycle progression. For this purpose, in a first set of experiments, we evaluated the effect of rRNA synthesis variation on cycle duration in asynchronously growing H4‐II‐E‐C3 rat hepatoma cells. Cells were either treated with insulin or insulin plus actinomycin D (AMD). The hormone stimulated ribosome biogenesis, which was later followed by an increased synthesis of DNA and a shortening of cell doubling time (DT). Bivariate flow cytometry indicated that the reduced length of the cell cycle was mainly due to the shorter G1‐phase. AMD, at the concentration of 0.04 μg/ml, hindered ribosome biogenesis without affecting heterogeneous RNA production. A 12‐h reduction in ribosome biogenesis level by AMD caused a lowering of DNA synthesis and a lengthening of cell DT with a longer G1‐phase. In a second set of experiments, we analyzed the cell content variations of 28S and 18S rRNA transcripts during G1 phase in H4‐II‐E‐C3 cells, synchronized by serum deprivation, and then stimulated by serum, serum plus insulin, and serum plus insulin and AMD. In control cells, a progressive increase in rRNA content occurred until the highest value of rRNA content was reached 21 h after serum stimulation. In insulin‐treated cells, the highest rRNA value was reached at 12 h whereas in AMD‐treated cells, the rRNA quantity was constantly low until 18 h and then sharply increased at 21 h. In the three experimental conditions, the highest values of rRNA amount were reached at the end of G1 phase and were quite similar to one another. We also evaluated, by real‐time RT‐PCR, cyclin E mRNA expression, which appeared to sharply increase at those times in which the maximum increase in the rRNA content was observed. Our results indicated that the achievement of an appropriate amount of rRNA allows G1/S phase transition, probably by modulating the expression of cyclin E mRNA. © 2004 Wiley‐Liss, Inc.
Differences in X‐ray absorption due to cadmium treatment in Saponaria officinalis leaves
Authors: Reale Lucia , Lai Antonia , Tucci Adele , Poma Anna , Faenov Anatoly , Pikuz Tania , Flora Francesco , Spano Laura , Limongi Tania , Palladino Libero , Ritucci Antonio , Tomassetti Giuseppe , Petrocelli Giovanni , Francucci Massimo , Martellucci Sergio
Published: July 2004

A method for detecting cadmium uptake in leaves of Saponaria officinalis doped with a solution of cadmium acetate is described. The technique based on the exposure of dried leaves to X‐rays of a wavelength close to that of the metal K‐edge could be useful for phytoremediation studies as it could reveal the bioaccumulation in plants due to the treatment either in vivo or in vitro with heavy metals. X‐ray microradiography measurements are in agreement with those from peroxidase enzyme assay utilized to follow the oxidative damage induced by heavy metals. At present, as we will see in this report, microradiography has still poorer sensitivity in comparison with enzyme assay, but it has the advantage of being faster, not destructive, and usable even at very high doping levels, where the enzyme assay technique results are fully saturated. Further analysis of the optical density values could lead to a quantitative measurement of the heavy metal in the sample. Thus, the technology developed in this article could be useful for tracing the intake in phytoremediation studies. Microsc. Res. Tech. 64:21–29, 2004. © 2004 Wiley‐Liss, Inc.