bluuebunny/crossref_metadata_2025 huggingface dataset:
Behavior of rat periodontal ligament cells on fibroblast growth factor‐2‐immobilized titanium surfaces treated by plasma modification
Authors: Kokubu Eitoyo , Yoshinari Masao , Matsuzaka Kenichi , Inoue Takashi
Published: September 2008

The aim of this study was to investigate the behavior of rat periodontal ligament (PDL) cells cultured on fibroblast growth factor‐2 (FGF‐2)‐immobilized titanium surfaces treated by oxygen (O2) plasma. We used cell disks (15 mm in diameter), and 35‐mm culture dishes sputter‐coated with titanium. These were treated with oxygen plasma and dipped in FGF‐2 solution. Immobilized FGF‐2 was visualized with a confocal laser‐scanning microscope, and its weight was calculated to be ˜22.6 ng/cm2 using a quartz crystal microbalance‐dissipation apparatus. The PDL cells were obtained from rat incisors. Cells from fourth subculture were seeded onto the FGF‐2‐immobilized titanium surface. Proliferation ratio, alkaline phosphatase (ALP) activity, and expressions of type I collagen and vascular endothelial growth factor (VEGF) mRNAs were evaluated. Proliferation ratio and expressions of type I collagen and VEGF mRNAs were significantly higher, whereas ALP activity was significantly lower in FGF‐2‐immobilized cells than in control group (p < 0.05). These findings suggest that oxygen plasma modification can immobilize FGF‐2 onto a titanium surface. Immobilized FGF‐2, although inferior to culture medium with FGF‐2, influenced the proliferation of PDL cells and might have promoted collagen and vascular synthesis. © 2008 Wiley Periodicals, Inc. J Biomed Mater Res 2009
Effect of carbon nanotubes on cellular functions in vitro
Authors: Li Xiaoming , Gao Hong , Uo Motohiro , Sato Yoshinori , Akasaka Tsukasa , Feng Qingling , Cui Fuzhai , Liu Xinhui , Watari Fumio
Published: September 2008

Carbon nanotubes (CNTs) have been shown to affect cell behavior. But how and why the CNTs affect potential differentiation of the attached cells has not been largely known. In this study, multiwalled carbon nanotubes (MWNTs) and graphite (GP) were pressed as compacts. Higher ability of CNTs to adsorb proteins, compared with GP, was shown. Myoblastic mouse cells (C2C12) were cultured and the cell responses to the two kinds of compacts were compared in vitro. Meanwhile, we used cell culture on the culture plate as a control. During the conventional culture, significantly better cell attachment, proliferation, and differentiation of cells on the MWNTs were found. To confirm the hypothesis that the larger amount of protein adsorbed on the CNTs was crucial for this, we made the compacts adsorb more proteins in culture medium with 50% fetal bovine serum (FBS) before cell culture. With the adsorption of the proteins in advance, the increments of the total‐protein/DNA and alkaline phosphatase (ALP)/DNA for the MWNTs was respectively as about 11 times and 18 times as the increments of those for GP and the control at both day 4 and day 7. Therefore, the CNTs might induce cellular functions by adsorbing more proteins, which indicated that the CNTs might be a candidate for scaffold material for tissue engineering. © 2008 Wiley Periodicals, Inc. J Biomed Mater Res 2009
Bioactive glass sol‐gel foam scaffolds: Evolution of nanoporosity during processing and in situ monitoring of apatite layer formation using small‐ and wide‐angle X‐ray scattering
Authors: FitzGerald V. , Martin R. A. , Jones J. R. , Qiu D. , Wetherall K. M. , Moss R. M. , Newport R. J.
Published: September 2008

Recent work has highlighted the potential of sol‐gel–derived calcium silicate glasses for the regeneration or replacement of damaged bone tissue. The work presented herein provides new insight into the processing of bioactive calcia‐silica sol‐gel foams, and the reaction mechanisms associated with them when immersed in vitro in a simulated body fluid (SBF). Small‐angle X‐ray scattering and wide‐angle X‐ray scattering (diffraction) have been used to study the stabilization of these foams via heat treatment, with analogous in situ time‐resolved data being gathered for a foam immersed in SBF. During thermal processing, pore sizes have been identified in the range of 16.5–62.0 nm and are only present once foams have been heated to 400°C and above. Calcium nitrate crystallites were present until foams were heated to 600°C; the crystallite size varied from 75 to 145 nm and increased in size with heat treatment up to 300°C, then decreased in size down to 95nm at 400°C. The in situ time‐resolved data show that the average pore diameter decreases as a function of immersion time in SBF, as calcium phosphates grow on the glass surfaces. Over the same time, Bragg peaks indicative of tricalcium phosphate were evident after only 1‐h immersion time, and later, hydroxycarbonate apatite was also seen. The hydroxycarbonate apatite appears to have preferred orientation in the (h,k,0) direction. © 2008 Wiley Periodicals, Inc. J Biomed Mater Res, 2009
Osteoblast activity on collagen‐GAG scaffolds is affected by collagen and GAG concentrations
Authors: Tierney Claire M. , Jaasma Michael J. , O'Brien Fergal J.
Published: September 2008

Optimization of a tissue engineering scaffold for use in bone tissue engineering requires control of many factors such as pore size, porosity, permeability and, as this study shows, the composition of the matrix. The collagen‐glycosaminoglycan (GAG) scaffold variants were fabricated by varying the collagen and GAG content of the scaffold. Scaffolds were seeded with MC3T3 osteoblasts and cultured for up to 7 days. During the culture period, osteoblastic activity was evaluated by measuring metabolic activity, cell number, and spatial distribution. Collagen and GAG concentrations both affected osteoblast viability, proliferation, and spatial distribution within the scaffold. Scaffolds containing 1% collagen (w/v) and 0.088% GAG (w/v) were found to have a porosity of ∼99%, high cell metabolic activity and cell number, and good cell infiltration over the 7 days in culture. Taken together, these results indicate the need to tailor the parameters of a biological substrate for use in a specific tissue application, in this case bone tissue engineering. © 2008 Wiley Periodicals, Inc. J Biomed Mater Res, 2009
Mammary epithelial cell transformation is associated with deregulation of the vitamin D pathway
Authors: Kemmis Carly M. , Welsh JoEllen
Published: September 2008

The vitamin D endocrine system mediates anti‐proliferative and pro‐differentiating signaling in multiple epithelial tissues, including mammary gland and breast tumors. The vitamin D metabolite 1α,25(OH)2D3 mediates growth inhibitory signaling via activation of the vitamin D receptor (VDR), a ligand dependent transcription factor. 1α,25(OH)2D3 is synthesized from 25(OH)D3 (the major circulating form of the vitamin) by the mitochondrial enzyme CYP27b1 in renal and other tissues. Human mammary epithelial (HME) cells express VDR and CYP27b1 and undergo growth inhibition when exposed to physiological concentrations of 25(OH)D3, suggesting that autocrine or paracrine vitamin D signaling contributes to maintenance of differentiation and quiescence in the mammary epithelium. In the current studies we tested the hypothesis that cancer cells would exhibit reduced sensitivity to vitamin D mediated negative growth regulation. We used a series of progressively transformed HME cell lines expressing known oncogenic manipulations to study the effects of transformation per se on the vitamin D pathway. We report that mRNA and protein levels of VDR and CYP27b1 were reduced greater than 70% upon stable introduction of known oncogenes (SV40 T antigens and H‐rasV12) into HME cells. Oncogenic transformation was also associated with reduced 1α,25(OH)2D3 synthesis, and cellular sensitivity to growth inhibition by 1α,25(OH)2D3 and 25(OH)D3 was decreased approximately 100‐fold in transformed cells. These studies provide evidence that disruption of the vitamin D signaling pathway occurs early in the cancer development process. J. Cell. Biochem. 105: 980–988, 2008. © 2008 Wiley‐Liss, Inc.
E‐cadherin dis‐engagement activates the Rap1 GTPase
Authors: Asuri Sirisha , Yan Jingliang , Paranavitana Nivanka C. , Quilliam Lawrence A.
Published: September 2008

E‐cadherin based adherens junctions are finely regulated by multiple cellular signaling events. Here we show that the Ras‐related Rap1 GTPase is enriched in regions of nascent cell–cell contacts and strengthens E‐cadherin junctions: constitutively active Rap1 expressing MDCK cells exhibit increased junctional contact and resisted calcium depletion‐induced cell–cell junction disruption. E‐cadherin disengagement activated Rap1 and this correlated with E‐cadherin association with the Rap GEFs, C3G and PDZ‐GEF I. PDZ‐GEF I associated with E‐cadherin and β‐catenin whereas C3G interaction with E‐cadherin did not involve β‐catenin. Knockdown of PDZ‐GEF I in MDCK cells decreased Rap1 activity following E‐cadherin junction disruption. We hereby show that Rap1 plays a role in the maintenance and repair of E‐cadherin junctions and is activated via an “outside‐in” signaling pathway initiated by E‐cadherin and mediated at least in part by PDZ‐GEF I. J. Cell. Biochem. 105: 1027–1037, 2008. © 2008 Wiley‐Liss, Inc.
Negative effects of the amino acids Lys, His, and Thr on S6K1 phosphorylation in mammary epithelial cells
Authors: Prizant Rotem Ladovsky , Barash Itamar
Published: September 2008

The role of essential amino acids (AA) on protein synthesis via the mTOR pathway was studied in murine mammary epithelial cells cultured under lactogenic conditions. Leu, Ile, and Val increased S6K1 phosphorylation compared to that measured in AA‐deprived cells. Trp, Phe, and Met had no effect. Surprisingly, Lys, His, and Thr inhibited S6K1 phosphorylation in both murine and bovine mammary cells. Thr exhibited the most potent inhibition, being the only amino acid that competed with Leu's positive role. In non‐deprived cells, there was no observable effect of Lys, His, or Thr on S6K1 phosphorylation at concentrations up to five times those in the medium. However, their addition as a mix revealed a synergistic negative effect. Supplementation of Lys, His, and Thr abrogated mTOR Ser 2448 phosphorylation, with no effect on Akt Ser 473—an mTORC2 target. This confirms specific mTORC1 regulation of S6K1 phosphorylation. The individual supplementation of Lys, His, and Thr maintained a low level of IRS‐1 phosphorylation, which was dose‐dependently increased by their combined addition. Thus, in parallel to inhibiting S6K1 activity, these AA may act synergistically to activate an additional kinase, phosphorylating IRS‐1 via an S6K1‐independent pathway. In cultures supplemented by Lys, His, and Thr, cellular protein synthesis decreased by up to 65%. A more pronounced effect was observed on β‐casein synthesis. These findings indicate that positive and negative signaling from AA to the mTOR pathway, combined with modulation of insulin sensitization, mediate the synthesis rates of total and specific milk proteins in mammary epithelial cells. J. Cell. Biochem. 105: 1038–1047, 2008. © 2008 Wiley‐Liss, Inc.
Overexpression of cFLIPs inhibits oxaliplatin‐mediated apoptosis through enhanced XIAP stability and Akt activation in human renal cancer cells
Authors: Kim Shin , Lee Tae‐Jin , Park Jong‐Wook , Kwon Taeg Kyu
Published: September 2008

cFLIP inhibits caspase 8 recruitment and processing at the death‐inducing signaling complex (DISC), which is known to inhibits apoptosis mediated by death receptors such as Fas and death receptor 5 (DR5) as well as apoptosis mediated by anticancer therapeutic drugs. We observed that oxaliplatin induced apoptosis, the activation of DEVDase activity, DNA fragmentation, and cleavage of PLC‐γ1 and degradation of XIAP protein in dose‐dependent manners, which was prevented by pretreatment with z‐VAD or NAC, suggesting that oxaliplatin‐induced apoptosis was mediated by caspase‐ or reactive oxygen species (ROS)‐dependent pathways. Furthermore, ectopic expression of cFLIPs potently attenuated oxaliplatin‐induced apoptosis, whereas cFLIPL had less effect. Interestingly, we found that the protein level of XIAP was sustained in oxaliplatin‐treated cFLIPs overexpressing cell, which was caused by the increased XIAP protein stability and that the phospho‐Akt level was high compared to vector‐transfected cell. The increased XIAP protein stability was lessened by PI3K inhibitor LY294002 treatment in cFLIPs overexpressing cells. Thus, our findings imply that the anti‐apoptotic functions of cFLIPs may be attributed to inhibit oxaliplatin‐induced apoptosis through the sustained XIAP protein level and Akt activation. J. Cell. Biochem. 105: 971–979, 2008. © 2008 Wiley‐Liss, Inc.
Pim‐1 kinase phosphorylates and stabilizes RUNX3 and alters its subcellular localization
Authors: Kim Hye‐Ryun , Oh Byung‐Chul , Choi Joong‐Kook , Bae Suk‐Chul
Published: September 2008

The loci of the Pim and Runx gene families have been identified as targets for viral insertions in CD2‐myc mice. Synergistic cooperation between Pim and RUNX was also found in the CD2‐Runx2 transgenic mouse lymphoma model. RUNX genes have come to prominence recently because of their roles as essential regulators of cell fate in development. Paradoxically, they appear to function either as tumor‐suppressor genes or dominant oncogenes according to the cellular context. However, the molecular mechanism of the ambiguous roles played by this family of transcription factors in cancer has remained largely uninvestigated. Here we demonstrate that Pim‐1 phosphorylates four Ser/Thr residues within the Runt domain and stabilizes RUNX3 protein. In addition, Pim‐1 markedly altered the cellular localization of RUNX3 from the nucleus to the cytoplasm. Our results demonstrate that the subcellular localization of RUNX3 is altered by phosphorylation. We propose that RUNX family members may behave as oncogenes if mislocalized to a cellular micro‐compartment. J. Cell. Biochem. 105: 1048–1058, 2008. © 2008 Wiley‐Liss, Inc.
Butyrate metabolism in human colon carcinoma cells: Implications concerning its growth‐inhibitory effect
Authors: Andriamihaja Mireille , Chaumontet Catherine , Tome Daniel , Blachier François
Published: September 2008

Butyrate and acetate are bacterial metabolites present in the large intestine lumen. Although butyrate is well known to inhibit the in vitro proliferation of human colon carcinoma cells in a process involving the hyperacetylation of specific nuclear histones, little is known about the possible link between butyrate metabolism and its growth‐inhibitory effect. In a previous study (Leschelle et al., 2000, Eur J Biochem 267: 6435–6442), we showed that butyrate accumulates and is metabolized in HT‐29 Glc−/+ cells without increasing oxygen consumption. In the present study, using the same cell line incubated with 14C‐labeled butyrate, we determined that a minor part of 14C from butyrate was recovered in nuclear histones. Unlike butyrate, acetate exerted no effect on cell growth but was a precursor for overall net histone acetylation. Although butyrate was able to increase the cellular AMP/ADP ratio, it did not affect the ATP cell content or the adenylate charge or the oxidation of endogenous L‐glutamine. Butyrate oxidation was found to be markedly sensitive to the presence of other substrates with D‐glucose decreasing this oxidation and L‐malate stimulating it. Furthermore, in the presence of L‐malate, the growth‐inhibitory effect of butyrate was significantly weaker than in its absence. From these data, we conclude that the metabolism of butyrate downstream acetyl‐CoA synthesis is not involved in the butyrate antiproliferative effect. The suggestion that butyrate metabolism in mitochondria is not used in these cells as a fuel but acts as a regulator of butyrate free concentrations (thus limiting its action upon cellular targets), is discussed. J. Cell. Physiol. 218: 58–65, 2009. © 2008 Wiley‐Liss, Inc.
Inhibition of retinal neovascularization by gene transfer of small interfering RNA targeting HIF‐1α and VEGF
Authors: Jiang Jian , Xia Xiao‐Bo , Xu Hui‐Zhuo , Xiong Yu , Song Wei‐Tao , Xiong Si‐Qi , Li Yan
Published: September 2008

Retinal neovascularization (NV) occurs in various ocular disorders including proliferative diabetic retinopathy, retinopathy of prematurity and secondary neovascular glaucoma, which often result in blindness. Vascular endothelial growth factor (VEGF) is an essential growth factor for angiogenesis, and is particularly regulated by hypoxia inducible factor‐1α (HIF‐1α) under hypoxic conditions. Therefore, HIF‐1α and VEGF could provide targets for therapeutic intervention on retinal NV. In this study, we investigated the inhibitory effects of small interfering RNA (siRNA) targeting HIF‐1α and VEGF on the expression of HIF‐1α and VEGF in human umbilical vein endothelial cells (HUVEC) in vitro and on retinal NV in vivo. siRNA‐expressing plasmids targeting human HIF‐1α (HIF‐1α siRNA) and human VEGF165 (VEGF siRNA) were constructed. They were transfected and co‐transfected to HUVEC and C57BL/6J mice of ischemic retinopathy model. HIF‐1α siRNA and VEGF siRNA specifically downregulated HIF‐1α and VEGF at both mRNA and protein levels in vitro and in vivo. Neovascular tufts and neovascular nuclei were decreased in gene therapy group compared to control hypoxia group. Co‐transfection of HIF‐1α siRNA and VEGF siRNA resulted in maximal effects on VEGF suppression in vitro and in vivo. It also manifested the maximal inhibitory effect on retinal NV. These results indicate that the application of HIF‐1α siRNA and VEGF siRNA technology holds great potential as a novel therapeutic for retinal NV. J. Cell. Physiol. 218: 66–74, 2009. © 2008 Wiley‐Liss, Inc.
Non‐peptidic small molecule inhibitors against Bcl‐2 for cancer therapy
Authors: Azmi Asfar S. , Mohammad Ramzi M.
Published: September 2008

A critical regulator of the apoptotic machinery is the Bcl‐2 family proteins whose over expression confers a protective effect on malignant cells against death signals of apoptosis. Cancer cells that are resistant to various anti‐cancer drugs and treatment regimen are found to over express these Bcl‐2 proteins such as Bcl‐2, Bcl‐XL, Mcl‐1, Bcl‐w, and A1/Bfl1. In recent years there has been an exponential growth in the identification as well as synthesis of non‐peptidic cell permeable small‐molecule inhibitors (SMIs) of protein–protein interaction. The focus of this article is on inhibitors of anti‐apoptotic protein Bcl‐2. This review summarizes an up to date knowledge of the available SMIs, their mode of action as well as their current status in preclinical as well as clinical development. J. Cell. Physiol. 218: 13–21, 2009. © 2008 Wiley‐Liss, Inc.
Strategies for profiling MicroRNA expression
Authors: Kong William , Zhao Jian‐Jun , He Lili , Cheng Jin Q.
Published: September 2008

MicroRNAs (miRNAs) are a class of small RNAs (∼22‐nt) that play an important role in the control of different cell processes by negative regulation of protein‐coding genes. In the last several years, a number of miRNA profiling strategies have been used to document the miRNA expression changes during physiological and pathological processes. Aberrant expression of miRNAs has been linked to developmental defects, cancer, neurological disorders, and heart diseases. Over 540 human miRNAs have been validated to date; however, computer models suggest there may be thousands more. As bench work continue to verify in silico predictions, miRNA profiling will remain a prominent tool for identification of differential expression miRNAs in normal cellular courses and human disorders. This review focuses on current strategies for miRNA expression profiling and discusses their sensitivity and specificity, as well as advantage and disadvantage. J. Cell. Physiol. 218: 22–25, 2009. © 2008 Wiley‐Liss, Inc.
Mechanisms of induction of adenosine receptor genes and its functional significance
Authors: St. Hilaire Cynthia , Carroll Shannon H. , Chen Hongjie , Ravid Katya
Published: September 2008

Adenosine is a metabolite generated and released from cells, particularly under injury or stress. It elicits protective or damaging responses via signaling through the adenosine receptors, including the adenylyl cyclase inhibitory A1and A3, and the adenylyl cyclase stimulatory A2Aand A2B. Multiple adenosine receptor types, including stimulatory and inhibitory, can be found in the same cell, suggesting that a careful balance of adenosine receptor expression in a particular cell is necessary for a specific adenosine‐induced response. This balance could be controlled by differential expression of the adenosine receptor genes under different stimuli. Here, we have reviewed an array of studies that have characterized basal or induced expression of the adenosine receptors and common as well as distinct mechanisms of effect, in hopes that ongoing studies on this topic will further elucidate detailed mechanisms of adenosine receptor regulation, leading to potential therapeutic applications. J. Cell. Physiol. 218: 35–44, 2009. © 2008 Wiley‐Liss, Inc.
Biomonitoring of biosurfactant production by green fluorescent protein‐marked Bacillus subtilis W1012
Authors: das Neves Luiz Carlos Martins , Kobayashi Márcio Junji , Rodrigues Thaís Miranda , Converti Attilio , Penna Thereza Christina Vessoni
Published: September 2008

BACKGROUND: Biosurfactant production was investigated using two strains of Bacillus subtilis, one being a reference strain (B. subtilis 1012) and the other a recombinant of this (B. subtilis W1012) made able to produce the green fluorescent protein (GFP).

RESULTS: Batch cultivations carried out at different initial levels of glucose (G0) in the presence of 10 g L−1 casein demonstrated that the reference strain was able to release higher levels of biosurfactants in the medium at 5.0≤G0≤10 g L−1 (Bmax = 104–110 mg L−1). The recombinant strain exhibited slightly lower levels of biosurfactants (Bmax = 90–104 mg L−1) but only at higher glucose concentrations (G0 ≥ 20 g L−1). Under these nutritional conditions, the fluorescence intensity linked to the production of GFP was shown to be associated with the cell concentration even after achievement of the stationary phase.

CONCLUSION: The ability of the genetically‐modified strain to simultaneously overproduce biosurfactant and GFP even at low biomass concentration makes it an interesting candidate for use as a biological indicator to monitor indirectly the biosurfactant production in bioremediation treatments. Copyright © 2008 Society of Chemical Industry
Ultrastructural observations on tegumental surface of Raillietina echinobothrida and its alterations caused by root‐peel extract of Millettia pachycarpa
Authors: Roy Bishnupada , Dasgupta Shyamashree , Tandon Veena
Published: September 2008

Millettia pachycarpa Benth (Leguminosae) has a usage in traditional medicine system practiced among the Lushai tribes of Mizoram, a state in North East India, who customarily consume the aqueous extract of the root peel of the plant to get rid of intestinal worm infections. The crude ethanol, methanol, and acetone fractions of the plant were assayed against Raillietina echinobothrida, the intestinal cestode parasite of domestic fowl, to authenticate the putative anthelmintic efficacy and cestocidal potential in particular of the plants. In vitro exposure of the worm to the extract at a concentration of 25 mg/mL phosphate buffered saline (at 37°C ± 1°C) revealed distortion and disruption of mitochondria, nucleus, nucleolus, nuclear membrane, basal lamina, and tegumental vacuolization in the distal cytoplasm leading to scar formation in the surface. The possible use of the plant as a potential anthelmintic against cestode parasite is discussed. Microsc. Res. Tech., 2008. © 2008 Wiley‐Liss, Inc.
Laser scanning confocal microscopy characterization of water repellent distribution in a sandstone pore network
Authors: Zoghlami Karima , Gómez‐Gras David , Corbella Mercè , Darragi Fadila
Published: September 2008

In the present work, we propose the use of the Laser Scanning Confocal Microscopy (LSCM) to determine the effect of water repellents on rock's pore‐network configuration and interconnection. The rocks studied are sandstones of Miocene age, a building material that is commonly found in the architectural heritage of Tunisia. The porosity quantitative data of treated and untreated samples, obtained by mercury porosimetry tests, were compared. The results show a slight decrease in total porosity with the water repellent treatment, which reduced both microporosity and macroporosity. This reduction produced a modification in pore size distribution and a shift of the pore access size mode interval toward smaller pore diameters (from the 30–40 μm to the 20–30 μm intervals). The water repellent was observed in SEM images as a continuous film coating grain surfaces; moreover, it was easily visualized in LSCM, by staining the water repellent with Epodye fluorochrome, and the coating thickness was straightforwardly measured (1.5–2 μm). In fact, the combination of mercury intrusion porosimetry data and LSCM observations suggests that the porosity reduction and the shift of the pore diameter mode were mainly due to the general reduction of pore diameters, but also to the plugging of the smallest pores (less than 3–4 μm in diameter) by the water repellent film. Finally, the LSCM technique enabled the reconstruction of 3D views of the water repellent coating film in the pore network, indicating that its distribution was uniform and continuous over the 100 μm thick sample. The LSCM imaging facilitates the integration and interpretation of mercury porosimetry and SEM data. Microsc. Res. Tech., 2008. © 2008 Wiley‐Liss, Inc.
A qualitative and quantitative study of the hepatic pigmented macrophage aggregates during the breeding cycle of ohrid trout, Salmo letnica Kar. (Teloestei, Salmonidae)
Authors: Jordanova Maja , Miteva Nada , Rocha Eduardo
Published: September 2008

It is well known that factors such as aging and water quality may influence pigmented macrophages (PMacs) in fishes, but it is not established yet if PMacs undergo seasonal and breeding dependent variations. This study explored this caveat and reports qualitative histological and stereological data on liver PMacs from wild female Ohrid trout, Salmo letnica, during the annual breeding cycle. Data showed that a minority of PMacs contained melanin and that the vast majority of them contained only hemosiderin or hemosiderin and lipofuscin/ceroid pigment. It was suggested that this is the normal scenario for the species. One remarkable result was the demonstration of a striking increase, after spawning, of the relative and total volumes of the hepatic macrophages, both parenchyma and stroma located. Because the melano PMacs did not vary, those changes were due to fluctuations in the hemosiderin‐laden PMacs. We concluded that Ohrid trout presented a pigment composition in liver macrophages that differed from other fish, including salmonids, where most liver phagocytes essentially display melanin. Our quantitative data support interspecies differences in the amount of liver macrophages and also that after spawning expansion of the macrophage pool is crucial and most likely connected with the needs of liver remodeling (leading to a decrease in hepatic mass). So, we suggested that the hormonal (sex steroidal) constellation influenced the liver macrophage pool. Additionally, we proved that the use of fish liver macrophages for biomonitoring should take into account the considerable natural breeding/seasonal dependent variations that are expected to occur. Microsc. Res. Tech., 2008. © 2008 Wiley‐Liss, Inc.
Ultimobranchial and parathyroid glands of the pigeon Columba livia in response to 1,25(OH)2D3 administration
Authors: Yadav Seema , Srivastav Ajai K.
Published: September 2008

Columba livia were given daily intraperitoneal injections of 100 pmol of 1,25(OH)2D3/100 g body wt. for 15 days. Ultimobranchial and parathyroid glands were fixed on 1st, 3rd, 5th, 10th and 15th day of the experiment. Following 1,25(OH)2D3 treatment, the plasma calcium levels of pigeon remain unchanged on day 1. The levels increase significantly after day 3 which progresses up to day 10. The plasma calcium level becomes normal at day 15. The plasma inorganic phosphate levels of Columba livia injected with 1,25(OH)2D3 remain unaffected up to day 3. The levels exhibit a progressive increase from day 5 to day 10. The levels become normophosphatemic at day 15. Up to day 3 following 1,25(OH)2D3 treatment, there is no change in the ultimobranchial gland of Columba livia. The gland exhibits an increased activity after 5 days 1,25(OH)2D3 treatment which is evident by the increased nuclear volume and weak staining response of the cytoplasm of ultimobranchial cells. After day 10, the nuclear volume depicts a further increase and a few completely exhausted cells are discerned. Following 15‐day 1,25(OH)2D3 treatment the nuclear volume records an increase and degenerating cells have been observed at certain places. The parathyroid glands of 1,25(OH)2D3‐treated Columba livia remain unaffected up to day 5. After day 10 and day 15, there is a progressive decrease in the nuclear volume of parathyroidal cells and reduced chromaticity of nuclei has been noticed. Moreover, after 15 days few degenerating cells are observed. Microsc. Res. Tech., 2008. © 2008 Wiley‐Liss, Inc.
Minicircle‐DNA production by site specific recombination and protein–DNA interaction chromatography
Authors: Mayrhofer Peter , Blaesen Markus , Schleef Martin , Jechlinger Wolfgang
Published: September 2008

Background

Conventional plasmid‐DNA (pDNA) used in gene therapy and vaccination can be subdivided into a bacterial backbone and a transcription unit. Bacterial backbone sequences are needed for pDNA production in bacteria. However, for gene transfer application, these sequences are dispensable, reduce the overall efficiency of the DNA agent and, most importantly, represent a biological safety risk. For example, the dissemination of antibiotic resistance genes, as well as the uncontrolled expression of backbone sequences, may have profound detrimental effects and unmethylated CpG motifs have been shown to contribute to silencing of episomal transgene expression. Therefore, an important goal in nonviral vector development is to produce supercoiled pDNA lacking bacterial backbone sequences.

Methods

A method is described to provide circular, supercoiled minimal expression cassettes (minicircle‐DNA) based on two processes: (i) an inducible, sequence specific, in vivo recombination process that is almost 100% efficient and (2) a novel affinity‐based chromatographic purification approach for the isolation of the minicircle‐DNA.

Results

Quantitative real‐time polymerase chain reaction analysis, capillary gel electrophoresis and restriction analysis of the recombination products, and the minicircle‐DNA revealed a recombination efficiency greater than 99.5% and a purity of the isolated minicircle‐DNA of more than 98.5%.

Conclusions

The results obtained in the present study demonstrate that the described technology facilitates the production of highly pure minicircle‐DNA for direct application in gene therapy and vaccination. The process described is efficient, stable and suitable for further scale‐up in industrial large‐scale manufacturing. Copyright © 2008 John Wiley & Sons, Ltd.
Insulin‐like growth factor‐1 gene therapy and cell transplantation in diabetic wounds
Authors: Hirsch Tobias , Spielmann Malte , Velander Patrik , Zuhaili Baraa , Bleiziffer Oliver , Fossum Magdalena , Steinstraesser Lars , Yao Feng , Eriksson Elof
Published: September 2008

Background

Impaired wound healing is a frequent phenomenon in diabetes mellitus. However, little is known of the fundamental cause of this pathology. The present study examined the effect of human insulin‐like growth factor (hIGF)‐1 overexpression in combination with autologous cell transplantation to diabetic wounds in a preclinical large‐animal model.

Methods

Diabetes was induced in Yorkshire pigs with streptozotocin. Keratinocytes were cultured and transfected with hIGF‐1 or LacZ transgene. Plasmids were lipoplexed with either Lipofectin or Lipofectamin 2000. Transgene expression was assessed by enzyme‐linked immunosorbent assay or X‐gal staining. For in vivo studies, full‐thickness wounds were created and dressed with a sealed chamber. Transfected cells were transplanted into the wounds. Wound contraction was monitored and biopsies were obtained for measurement of re‐epithelialization. Wound fluid was collected and analysed for IGF‐1 concentrations.

Results

Quantification showed up to 740 ng/ml IGF‐1 in vitro and significantly higher concentrations over 14 days compared to controls for the Lipofectamin 2000 group. Lipofectin‐mediated gene transfer showed peak expression on day 2 with 68.5 ng/ml. In vivo, transfected cells showed peak expression of 457 ng/ml at day 1, followed by subsequent decline to 5 ng/ml on day 12 with Lipofectamin 2000. For Lipofectin, no significant IGF‐1 expression could be detected. Gene therapy caused significantly faster wound closure (83%) than both controls (native‐cell therapy = 57%; control wounds = 32%).

Conclusions

The present study demonstrates that optimized nonviral gene transfer increased IGF‐1 expression in diabetic wounds by up to 900‐fold. This high IGF‐1 concentration in combination with cell therapy improved diabetic wound healing significantly. Copyright © 2008 John Wiley & Sons, Ltd.
Molecular dynamics study of the vibrational pattern of ring structures in the Raman spectra of vitreous silica
Authors: To Thi Thao , Bougeard Daniel , Smirnov Konstantin S.
Published: September 2008

Raman scattering spectra of silica glass models were studied by means of molecular dynamics computer simulation. The study principally aimed at the inspection of the assignment of the defect bands of the Raman spectra of a‐SiO2 proposed by Galeener and co‐workers (Galeener FL, Barrio RA, Martinez E, Elliott RJ. Phys. Rev. Lett. 1984; 53: 2429). The results of the study have shown that the D2 peak at 606 cm−1 can probably be related to the presence of three‐membered rings in a‐SiO2, whereas the D1 feature at 495 cm−1 correlates with the presence of both three‐ and four‐membered rings, and can therefore, be used for structural characterisation of silica glasses only with care. Copyright © 2008 John Wiley & Sons, Ltd.
An analytical form for the Raman shift dependence on size of nanocrystals
Authors: Salis M. , Ricci P. C. , Anedda A.
Published: September 2008

An analytical form of the Raman shift dependence on the size of nanocrystals is presented. On the basis of the hard confinement model, this form describes the deviations from Raman shifts in infinite crystals as Δω = π2A[1 − exp(−η)]/12x2η2, where η = L/12ax and x = (A/Γ0)1/2, L standing for the crystal size, Γ0 for the intrinsic band linewidth, a for the lattice parameter and A for a suitable phonon curve parameter. It works in those cases where the average phonon curve shows a quadratic dependence on the phonon quasi‐momentum in the range of interest. Copyright © 2008 John Wiley & Sons, Ltd.
Variations in morphology and composition of barley endosperm cell walls
Authors: Lazaridou Athina , Chornick Tricia , Izydorczyk Marta S
Published: September 2008

BACKGROUND: The endosperm cell walls (CWs) impact upon barley grain utilization as well as influence animal and human nutrition. The objective of this study was to examine the morphological and compositional differences in the endosperm CWs derived from barley grain grown in different (uncontrolled) environments in Canada, and differing in grain hardness, protein, and total β‐glucan contents.

RESULTS: The endosperm CWs were isolated from barley, cv. Metcalfe, grown in Davidson, SK (sample A), Hythe, AB (sample B), and Hamiota, MB (sample C). The inner wall surfaces of sample A exhibited deep indentations made by large and small starch granules, whereas the CWs of sample B were thicker, had smooth surface, and exhibited a greater degree of binding between the adjoining cells. The CWs of sample A contained the lowest amount of β‐glucans but the highest amount of arabinoxylans and the mannose‐containing (glucomannans) polysaccharides. The CWs of sample B contained the highest ratio of ferulic and dehydrodiferulate residues to Xyl residues among the samples, indicating the greatest potential and degree of cross‐linking of arabinoxylans in these preparations which might be related to the strength and rigidity of the grain endosperm. The amino acid composition of the proteins detected in the isolated CW indicated the presence of 37–47 g kg−1 of structural proteins.

CONCLUSIONS: Substantial differences in the morphological appearance and chemical composition of the CW preparations were observed and some attempts were made to relate these differences to the variable composition and physical properties of barley grain as affected by the environmental factors. Copyright © 2008 Crown in the Right of Canada, Canadian Grain Commission. Published by John Wiley and Sons, Ltd
The impact of margin status on local recurrence following breast conserving therapy for invasive carcinoma in Manitoba
Authors: Hardy K. , Fradette K. , Gheorghe R. , Lucman L. , Latosinsky S.
Published: September 2008

Background

Histologically positive margins are generally considered unacceptable with breast conserving therapy (BCT) given the increased risk of local recurrence (LR). What constitutes an adequate negative margin remains controversial. Margin status was explored as a predictor of LR post‐BCT.

Methods

Manitoba women with loco‐regional progression and/or mastectomy >6 months following BCT for Stage I/II invasive cancer (1995–2004) were identified from the Manitoba Cancer Registry; LR cases were confirmed by chart review. Three controls per case were matched by age, grade, stage, and adjuvant chemotherapy use. Margin status was categorized as histologically positive, ≤1 mm, ≤2 mm or >2 mm. Conditional logistic regression determined the odds ratio of LR by margin category.

Results

There were 50 LR cases in 3,017 patients who underwent BCT, with a median follow‐up of 60 months. Wider margins were associated with a non‐significant reduction in LR: >1 mm versus ≤1 mm (OR 0.69; 95% CI 0.28–1.69) and >2 mm versus ≤2 mm (OR 0.90; 95% CI 0.44–1.84).

Conclusions

No clear benefit to wider histologically negative margins is demonstrated. J. Surg. Oncol. © 2008 Wiley‐Liss, Inc.
Recurrence in patients following curative resection of early gastric carcinoma
Authors: Wu Benyan , Wu Daohong , Wang Mengwei , Wang Gangshi
Published: September 2008

Objective

Post‐gastrectomy recurrences of early gastric cancer occur in a few cases. We investigated the outcome of early gastric cancer patients treated surgically, with special respect to the risk factor(s) for tumor recurrence.

Patients and Methods

A total of 308 patients with mono‐foci early gastric cancer underwent curative surgical resection. Clinicopathological variables and tumor recurrence patterns were analyzed retrospectively.

Results

Recurrence was observed in 30 out of 245 patients (12.24%) who had completed follow‐up. The median interval from surgery to diagnosis of recurrence was 28 (range 3–188) months. Hematogenous recurrence (3.27%) was twice as frequent as peritoneal recurrence (1.63%). Gastric stump cancer occurred in 9 patients (3.67%), anastomotic recurrence in 7 patients (2.86%) and lymphatic recurrence in 4 patients (1.63%). Older patients (≥60 years old) had a more frequent recurrence rate than younger patients (P < 0.05). Multivariate analyses identified lymph node metastasis and depth of invasion as risk factors of recurrence. All the patients with a positive family history of cancer got recurrence within 3 years after surgery.

Conclusions

Early gastric cancer patients, who were elderly, had lymphatic and submucosal involvement, and patients with a positive family history of cancer, tended to have a greater risk of recurrence. J. Surg. Oncol. © 2008 Wiley‐Liss, Inc.
Single sample extraction protocol for the quantification of NAD and NADH redox states in Saccharomyces cerevisiae
Authors: Sporty Jennifer L. , Kabir Md. Mohiuddin , Turteltaub Kenneth W. , Ognibene Ted , Lin Su‐Ju , Bench Graham
Published: September 2008

A robust redox extraction protocol for quantitative and reproducible metabolite isolation and recovery has been developed for simultaneous measurement of nicotinamide adenine dinucleotide (NAD) and its reduced form, NADH, from Saccharomyces cerevisiae. Following culture in liquid media, yeast cells were harvested by centrifugation and then lysed under nonoxidizing conditions by bead blasting in ice‐cold, nitrogen‐saturated 50 mM ammonium acetate. To enable protein denaturation, ice cold nitrogen‐saturated CH3CN/50 mM ammonium acetate (3:1 v/v) was added to the cell lysates. Chloroform extractions were performed on supernatants to remove organic solvent. Samples were lyophilized and resuspended in 50 mM ammonium acetate. NAD and NADH were separated by HPLC and quantified using UV–Vis absorbance detection. NAD and NADH levels were evaluated in yeast grown under normal (2% glucose) and calorie restricted (0.5% glucose) conditions. Results demonstrate that it is possible to perform a single preparation to reliably and robustly quantitate both NAD and NADH contents in the same sample. Robustness of the protocol suggests it will be (i) applicable to quantification of these metabolites in other cell cultures; and (ii) amenable to isotope labeling strategies to determine the relative contribution of specific metabolic pathways to total NAD and NADH levels in cell cultures.
Rapid and sensitive analysis of multiple bioactive constituents in Compound Danshen preparations using LC‐ESI‐TOF‐MS
Authors: Xia Li , Liu Hai‐Ling , Li Ping , Zhou Jian‐Liang , Qi Lian‐Wen , Yi Ling , Chen Jun
Published: September 2008

By fully optimizing the separation and analytical conditions, a rapid‐resolution LC (RRLC, 1.8 μm particle size) coupled with TOF‐MS method was developed and validated for multiple compounds analysis of traditional Chinese medicinal compound preparations (TCMCPs). As a typical example, determination of Compound Danshen preparations (CDPs, mainly comprising Salvia miltiorrhiza and Panax notoginseng), was improved on three groups of bioactive constituents (phenolic acids, diterpenoids, and saponins). LODs down to 1.2 pg, permitting very good separation of 20 analytes and 3 internal standards (ISs) in 17 min, represented an approximate five‐fold reduction of time compared to conventional HPLC. Unequivocal identification of target compounds in CDPs was based on accurate mass measurement of ions, and comparison of retention performance with available standards. Quantitation was carried out using the narrow window extracted ion chromatograms (XICs) of each compound with an interference‐free baseline (using a ±0.02 Da window) yielding good linearity of response (r2 >0.9940), and excellent precision of retention time (tR) both interday (RSD, 0.01–0.19%) and intraday (RSD, 0.02–1.08%). The acceptable recoveries obtained were in the range of 77.69–113.8%. The results demonstrated the robustness and applicability of RRLC‐TOF‐MS for multicomponents analysis in TCMCPs with diverse chemical compositions and properties.
Fast field analysis of short‐chain aliphatic amines in water using solid‐phase microextraction and a portable gas chromatograph
Authors: Lin Huaqing , Deng Chunhui , Zhang Xiangmin
Published: September 2008

A novel method is firstly presented for field and rapid analysis of short‐chain aliphatic amines in water as their pentafluorobenzaldehyde (PFBAY) derivative using solid‐phase microextraction (SPME) and portable GC. In the proposed method, short‐chain aliphatic amines in water rapidly reacted with PFBAY, and then were headspace extracted and concentrated by SPME. The formed amines derivatives were analyzed by portable GC. The SPME parameters of fiber selection, extraction temperature, extraction time, and stirring rate were studied. The method validations including LOD, recovery, precision, and linearity were studied. It was found that the proposed method required the whole analysis time 22 min, and provided low LOD of 1.2–4.6 ng/mL, good recovery of 91–106%, good precision of RSD value 3.5–9.3%, and linear range 20.0–500 ng/mL (r2 >0.99). The obtained results demonstrated that the SPME‐portable GC is a simple, rapid, and efficient method for the field analysis of short‐chain aliphatic amines. Finally, the proposed method was further applied to the quantification of ethylamine, propylamine, and butylamine in environmental water.
To go or stay home and watch: exploring the balance between motives and perceived constraints for major events: a case study of the 2008 Beijing Olympic Games
Authors: Funk Daniel C. , Alexandris Konstantinos , Ping Yang
Published: September 2008

The current study offers insight into the role of motivation and constraints on behavioural intentions prior to a mega event. The Psychological Continuum Model framework was used to integrate motivation and leisure constraints theory to examine attraction towards the 2008 Olympic Games. Semi‐structured interviews (N = 47) and a questionnaire (N = 235) distributed in Australia and the USA were used to identify and measure three motivational themes: cultural learning, cultural experience and olympic event interest, and three perceived constraints: structural, interpersonal and intrapersonal. Structural Equation Modelling revealed that motives were positively related to behavioural intentions while constraints were negatively related. Analysis further revealed the interaction between motives and perceived constraints led to two different forms of behavioural intentions; intentions to travel and attend the event vs. stay home to watch the event on TV. Policy and marketing implications are discussed that illustrate the benefits of understanding both motives and perceived constraints for tourism travel. Copyright © 2008 John Wiley and Sons, Ltd.
Type of diversification and firm resources: new empirical evidence from the Spanish tourism industry
Authors: Andreu Rosario , Claver Enrique , Quer Diego
Published: September 2008

This paper analyses the diversification strategies followed by tourism firms from the perspective of the resource‐based view. More specifically, it seeks to verify whether the composition of the firms' resources has a bearing on the decision to undertake one type of diversification or another (related or unrelated). From a mail survey to 80 Spanish tourism firms, we identified 94 entries into new business areas. We applied an ordered logit regression and have statistically confirmed that intangible resources lead to a more related type of diversification. Copyright © 2008 John Wiley & Sons, Ltd.
Tourism and the state in Cuba: from the past to the future
Authors: Sharpley Richard , Knight Martin
Published: September 2008

It has long been recognised that nature and extent of state intervention in tourism development closely reflects the prevailing political‐economy and ideology within the destination state. This is certainly the case with Cuba which, since the 1959 revolution and despite the collapse of communism elsewhere, remains the world's only centrally‐planned economy that boasts a significant international tourism sector. Tracing the development of tourism since 1959, this paper explores the relationship between the evolution of Cuba's political‐economic structures and processes and their subsequent influence on the planning, control, development and ownership of tourism on the island. In particular, it considers the potential future of tourism in Cuba, challenging the widespread belief that, in a post‐Castro era, the island's tourism sector faces a bright future. It concludes that, even with a potential move towards market reform, significant improvements will be required with respect to the quality, value and diversity of the island's tourism product. Copyright © 2008 John Wiley & Sons, Ltd.