bluuebunny/crossref_metadata_2025 huggingface dataset:
Determinants of access to physician services in Italy: a latent class seemingly unrelated probit approach
Authors: Atella Vincenzo , Brindisi Francesco , Deb Partha , Rosati Furio C.
Published: March 2004

We examine access to general practitioners and specialists who work in the public and private sectors in Italy using a seemingly unrelated system of probits. We use a latent class formulation that provides a rich and flexible functional form and can accommodate non‐normality of response probabilities. The empirical analysis shows that patient behavior can be clustered in two latent classes. We find that income strongly influences the mix of services. Richer individuals are less likely to seek care from GP's and more likely to seek care from specialists, and especially private specialists. Health status and societal vulnerability are the most important indicators of class membership. Copyright © 2004 John Wiley & Sons, Ltd.
Two New Insecticidal Amide Dimers from Fruits of Piper nigrum Linn.
Authors: Siddiqui Bina S. , Gulzar Tahsin , Begum Sabira , Afshan Farhana , Sattar Fouzia A.
Published: March 2004

The methanolic extract of dried ground seeds of Piper nigrum Linn. afforded fourteen compounds, of which thirteen were amides, including two new isomeric insecticidal amides, pipsaeedine (1) and pipbinine (2), along with eleven known amides and piptaline; (this is the first report of isolation of these compounds from this plant). The structures of 1 and 2 have been elucidated as (E,E)‐1‐[(E)‐5‐(7‐{6‐[5‐(piperidin‐1‐yl)‐5‐oxopent‐3‐enyl]‐1,3‐benzodioxol‐4‐yl}‐1,3‐benzodioxol‐5‐yl)‐1‐oxopenta‐2,4‐dienyl]piperidine and (E,E)‐1‐[(E)‐5‐(4‐{6‐[5(piperidin‐1‐yl)‐5‐oxopent‐3‐enyl]‐1,3‐benzodioxol‐4‐yl}‐1,3‐benzodioxol‐5‐yl)‐1‐oxopenta‐2,4‐dienyl]piperidine, respectively, through extensive 1D‐ and 2D‐NMR spectral studies, while the known constituents have been identified through comparison of their spectral data with those reported in the literature. Compounds 1 and 2 exhibited toxicities of 45.0 and 40.0 ppm, respectively, against fourth instar larvae of Aedes aegypti Liston.
Studies Directed to the Total Synthesis of Cepacin A. Preliminary Communication
Authors: Tang Chao‐Jun , Wu Yikang
Published: March 2004

The synthesis of the bromoallenyl‐substituted epoxide derivative 34 related to cepacin A (1) starting from D‐arabinose as a chiral pool is reported.
Prognostic factors in children requiring admission to an intensive care unit after hematopoietic stem cell transplant
Authors: Cheuk Daniel Ka Leung , Ha Shau Yin , Lee So Lun , Chan Godfrey Chi Fung , Tsoi Nai Shun , Lau Yu Lung
Published: March 2004

The objectives of this study are to identify prognostic factors of survival to discharge in pediatric hematopoietic stem cell transplant (HSCT) recipients requiring intensive care unit (ICU) admission, and to determine the utility of the Oncological Pediatric Risk of Mortality (O‐PRISM) in predicting death of these patients. A retrospective cohort of 125 pediatric HSCT recipients from October 1992 to September 2002 was analysed to evaluate risk factors of mortality in those admitted to ICU after HSCT. Nineteen patients (median age 7.8 years, 14 boys) required 24 ICU admissions post‐HSCT. The most frequent underlying diseases were acute myeloid leukemia (n=5). The survival rate on discharge from ICU was 54%. In univariate analysis, risk factors of mortality included earlier requirement of ICU admission post‐HSCT (median 34 versus 166 days, p=0.002), a longer delay before ICU admission (median 12 versus 5 h, p=0.02), lack of neutrophil (p=0.011) or platelet engraftment (p=0.008), macroscopic hemorrhage (p<0.001), tachypnoea (p=0.033), hypoxemia (p=0.031), renal impairment (p=0.011), coagulopathy (p=0.012), mechanical ventilation (p<0.001), and an increasing number of organ failures (p=0.003). Macroscopic hemorrhage and mechanical ventilation remained significant in multivariate analysis. Both PRISM and O‐PRISM scores were significant composite prognosticators. It was concluded that mortality of post‐HSCT children requiring ICU admission is high, especially in those with poor prognosticators. Copyright © 2004 John Wiley & Sons, Ltd.
Boundary and border considerations in hydrology
Authors: Woo Ming‐ko
Published: March 2004

This paper examines several issues related to hydrological boundaries and their border zones. In a two‐dimensional space, a boundary is a line that separates two domains possessing different hydrological properties or dominated by different hydrological processes, and a border is an area that experiences an edge effect owing to transitions or mixing of processes. Hydrological boundaries may be static, such as drainage divides, or dynamic, such as the edges of a seasonal snow cover. They may be open or closed to the transfer of matter and energy, although most boundaries tend to be perforated, permitting different rates of movement across different segments. Borders may be narrow or the edge effect can affect large areas, as happens to the sensible heat flux over a highly fragmented melting snowfield. The introduction of artificial boundaries, notably the grid patterns of remote sensing pixels, digital elevation models and land surface schemes, gives rise to problems of mismatch with the natural hydrological boundaries. Incorrect demarcation, omission and generalization of boundaries can produce errors that are hard to rectify. Serious biases are involved when point observations are used to calibrate parameters or to validate model outputs integrated over a bounded area. Examples are drawn mainly from cold climate hydrology to illustrate the boundary issues but the questions transcend disciplinary areas. The intent of this presentation is to stimulate discussions that could be a prelude to finding solutions to many boundary problems which have thus far eluded hydrological investigations. Copyright © 2004 John Wiley & Sons, Ltd.
A simulation of large‐scale groundwater flow and travel time in a fractured rock environment for waste disposal purposes
Authors: Ophori Duke U.
Published: March 2004

Two‐dimensional regional groundwater flow was simulated based on a conceptual model of low‐permeability crystalline rocks of the Whiteshell Research Area (WRA) in south‐eastern Manitoba. The conceptual model consists of fracture zones that strike in different directions and dip at various angles in the background rock mass. The thickness and hydraulic properties of the fracture zones in the conceptual model were varied as were the fluid properties and the boundary conditions of the groundwater flow system. The effects of these variations on the groundwater flow pattern and on the convective travel time along pathways from a hypothetical disposal vault at 500 m depth to discharge locations at the ground surface were evaluated. The vault was located in the regional discharge area of the groundwater system. A homogeneous conceptual model of the WRA, having only freshwater flow, formed a groundwater flow pattern with a regional flow system. Local flow systems developed increasingly with the introduction of fracture zones 20 m and 3 m thick, and depth‐dependent fluid density. This indicates a reduction in groundwater residence time by fracture zones and fluid density. Flow pathways were analysed using both a stream‐function and a particle‐tracking technique. The pathways and their lengths from the location of the vault to the surface varied spatially according to the flow patterns. The minimum travel time along these pathways was less than 150 000 and greater than 4 000 000 years in models with and without fracture zones, respectively, indicating that the presence of fracture zones was the major controlling factor. A precise knowledge and refinement of conceptual model parameters is necessary during site selection for waste disposal purposes. Copyright © 2004 John Wiley & Sons, Ltd.
Modelling the effects of land‐use change on water and salt delivery from a catchment affected by dryland salinity in south‐east Australia
Authors: Vaze Jai , Barnett Paul , Beale Geoffrey , Dawes Warrick , Evans Ray , Tuteja Narendra Kumar , Murphy Brian , Geeves Guy , Miller Michelle
Published: March 2004

A comprehensive framework for the assessment of water and salt balance for large catchments affected by dryland salinity is applied to the Boorowa River catchment (1550 km2), located in south‐eastern Australia. The framework comprised two models, each focusing on a different aspect and operating on a different scale. A quasi‐physical semi‐distributed model CATSALT was used to estimate runoff and salt fluxes from different source areas within the catchment. The effects of land use, climate, topography, soils and geology are included. A groundwater model FLOWTUBE was used to estimate the long‐term effects of land‐use change on groundwater discharge. Unlike conventional salinity studies that focus on groundwater alone, this study makes use of a new approach to explore surface and groundwater interactions with salt stores and the stream.

Land‐use change scenarios based on increased perennial pasture and tree‐cover content of the vegetation, aimed at high leakage and saline discharge areas, are investigated. Likely downstream impacts of the reduction in flow and salt export are estimated.

The water balance model was able to simulate both the daily observed stream flow and salt load at the catchment outlet for high and low flow conditions satisfactorily. Mean leakage rate of about 23·2 mm year−1 under current land use for the Boorowa catchment was estimated. The corresponding mean runoff and salt export from the catchment were 89 382 ML year−1 and 38 938 t year−1, respectively.

Investigation of various land‐use change scenarios indicates that changing annual pastures and cropping areas to perennial pastures is not likely to result in substantial improvement of water quality in the Boorowa River. A land‐use change of about 20% tree‐cover, specifically targeting high recharge and the saline discharge areas, would be needed to decrease stream salinity by 150 µS cm−1 from its current level. Stream salinity reductions of about 20 µS cm−1 in the main Lachlan River downstream of the confluence of the Boorowa River is predicted.

The FLOWTUBE modelling within the Boorowa River catchment indicated that discharge areas under increased recharge conditions could re‐equilibrate in around 20 years for the catchment, and around 15 years for individual hillslopes. Copyright © 2004 John Wiley & Sons, Ltd.
A fast method of flood discharge estimation
Authors: Chen Yen‐Chang , Chiu Chao‐Lin
Published: March 2004

Discharge, especially during flood periods, is among the most important information necessary for flood control, water resources planning and management. Owing to the high flood velocities, flood discharge usually cannot be measured efficiently by conventional methods, which explains why records of flood discharge are scarce or do not exist for the watersheds in Taiwan. A fast method of flood discharge estimation is presented. The greatest advantage of the proposed method is its application to estimate flood discharge that cannot be measured by conventional methods. It has as its basis the regularity of open‐channel flows, i.e. that nature maintains a constant ratio of mean to maximum velocities at a given channel section by adjusting the velocity distribution and the channel geometry. The maximum velocity at a given section can be determined easily over a single vertical profile, which tends to remain invariant with time and discharge, and can be converted to the mean velocity of the entire cross‐section by multying by the constant ratio. Therefore the mean velocity is a common multiple of maximum velocity and the mean/maximum velocity ratio. The channel cross‐sectional area can be determined from the gauge height, the water depth at the y‐axis or the product of the channel width multiplied by the water depth at the y‐axis. Then the most commonly used method, i.e. the velocity–area method, which determines discharge as the product of the cross‐sectional area multiplied by mean velocity, is applied to estimate the flood discharge. Only a few velocity measurements on the y‐axis are necessary to estimate flood discharge. Moreover the location of the y‐axis will not vary with time and water stage. Once the relationship of mean and maximum velocities is established, the flood estimation can be determined efficiently. This method avoids exposure to hazardous environments and sharply reduces the measurement time and cost. The method can be applied in both high and low flows in rivers. Available laboratory flume and stream‐flow data are used to illustrate accuracy and reliability, and results show that this method can quickly and accurately estimate flood discharges. Copyright © 2004 John Wiley & Sons, Ltd.
Ser‐249TP53 mutation in tumour and plasma DNA of hepatocellular carcinoma patients from a high incidence area in the Gambia, West Africa
Authors: Szymańska Katarzyna , Lesi Olufunmilayo A. , Kirk Gregory D. , Sam Omar , Taniere Philippe , Scoazec Jean‐Yves , Mendy Maimuna , Friesen Marlin D. , Whittle Hilton , Montesano Ruggero , Hainaut Pierre
Published: March 2004

Hepatocellular carcinoma (HCC) is frequent in areas of high exposure to aflatoxin and high prevalence of HBV infection, such as western Africa and south‐east China. A selective mutation in TP53 (AGG→AGT at codon 249, Arg→Ser) has been identified as a hotspot in HCCs from such areas, reflecting DNA damage caused by aflatoxin metabolites. Recent studies have shown that circulating free DNA can be retrieved from human plasma, and it is hypothesised that plasma DNA may serve as a source for biomarkers of tumorigenic processes. In our study, we have determined the prevalence of Ser‐249 mutation, using a PCR‐restriction digestion method, with selective use of short oligonucleotide mass spectrometry analysis (SOMA), in a series of 29 biopsy specimens of HCC from The Gambia in West Africa. Overall, we identified the Ser‐249 mutation in 35% (10/29) of the tumours. In parallel, we tested 17 plasma samples from HCC patients with matching tumour tissue. The 249 status concordance between tumour tissues and matched plasma was 88.5%. These results indicate that the Ser‐249 mutation is common in HCC in The Gambia (35%), although a higher prevalence has been reported in other regions with high population exposure to aflatoxin (e.g., eastern China: >50%). Moreover, our studies indicate that plasma is a convenient source of liver tumour‐derived DNA, thus holding promise for earlier detection and diagnosis of cancer. © 2004 Wiley‐Liss, Inc.
Expression of the metastasis‐associated MTA1 protein and its relationship to deacetylation of the histone H4 in esophageal squamous cell carcinomas
Authors: Toh Yasushi , Ohga Takefumi , Endo Kazuya , Adachi Eisuke , Kusumoto Hiroki , Haraguchi Masaru , Okamura Takeshi , Nicolson Garth L.
Published: March 2004

Metastasis‐associated protein MTA1 and histone deacetylase form a protein complex with histone deacetylase activity that plays an important role in histone deacetylation, alteration of chromatin structure and transcriptional control. The precise role of the MTA1 protein in the malignant progression of human cancers remains unknown, however, especially its overexpression and relationship with histone acetylation/deacetylation in experimental and clinical tumors. The expression levels of MTA1 protein and the acetylation levels of histone H4 were examined in 70 cases of surgically resected esophageal squamous cell carcinomas, using immunohistochemistry. The intensities of immunostaining of MTA1 protein and acetylated histone H4 in carcinoma tissues (Ca) were compared to normal epithelium (N) contained in the same section. Thirty of 70 cases (42.9%) displayed overexpression of MTA1 protein (N < Ca). Cancers overexpressing MTA1 protein invaded deeper into the esophageal wall (p < 0.005) and showed significantly higher degrees of lymph node metastasis (p < 0.01), higher pathological stage, more lymphatic involvement and poorer prognosis (p < 0.05) than the remaining cases. The acetylation levels of histone H4 inversely correlated to the depth of cancer invasion and pathological stage (p < 0.05), and the patients with higher level of histone H4 acetylation had a better prognosis (p < 0.05). Furthermore, immunostaining patterns of MTA1 and acetylated histone H4 were inversely correlated (p < 0.001), demonstrating the relationship of deacetylation of histone H4 in MTA1‐overexpressing carcinomas. In conclusion, the data suggest that the overexpression of MTA1 protein and acetylation level of histone H4 protein, both of which are closely related, might be useful predictors of malignant potential of esophageal squamous cell carcinomas. Thus, strategies involving inhibition of MTA1 function as well as inhibition of histone deacetylation could be novel approaches for the treatment of esophageal squamous cell carcinomas. © 2004 Wiley‐Liss, Inc.
Succimer treatment and calcium supplementation reduce tissue lead in suckling rats
Authors: Marija Varnai Veda , Piasek Martina , Blanuša Maja , Matek Sarić Marijana , Jureša Dijana , Kostial Krista
Published: March 2004

The effect of combined treatment with meso‐2,3‐dimercaptosuccinic acid (DMSA) and calcium supplementation in reducing lead absorption and enhancing lead elimination was evaluated in suckling rats under two experimental conditions: during ongoing oral lead exposure (lead acetate, 2 mg Pb kg−1 day−1, total dose 16 mg Pb kg−1) or after lead exposure (72 h after a 2‐day lead exposure, total dose 12 mg Pb kg−1 s.c.). The artificial feeding method was used for calcium supplementation, with 6% Ca (as CaHPO4) suspension in cow's milk to increase the daily calcium intake about three times above control values. Artificial feeding lasted for 7 h a day over eight consecutive days. During this period DMSA was administered on 6 days twice a day (0.5 mmol kg−1 day−1 p.o.). At the end of the experiments, Pb, Ca and Zn in the carcass and Pb, Fe and Cu in the liver, kidneys and brain were analysed by atomic absorption spectrometry.

Calcium supplementation during lead exposure reduced tissue lead but had no effect when applied after lead exposure, and DMSA administered either during or after lead exposure lowered the tissue lead. Combined treatment during ongoing lead exposure caused a greater reduction in tissue lead than either DMSA or calcium treatment alone. When administered after lead exposure, it had no advantage over DMSA treatment alone but did not impair its efficacy. Combined treatment had no influence on growth and did not seriously disturb essential element status. It is concluded that calcium supplementation could be applied during DMSA therapy, when indicated. Copyright © 2004 John Wiley & Sons, Ltd.
Tissue regeneration patterns in acellular bovine pericardia implanted in a canine model as a vascular patch
Authors: Chang Yen , Liang Huang‐Chien , Wei Hao‐Ji , Chu Chih‐Ping , Sung Hsing‐Wen
Published: March 2004

It was noted in our previous study that acellular tissues can provide a natural microenvironment for host cell migration and proliferation to accelerate tissue regeneration. The purpose of this study was to further investigate the tissue regeneration patterns in acellular bovine pericardia fixed with glutaraldehyde or genipin as a biological patch to repair a defect in the pulmonary trunk in a canine model. The implanted samples were retrieved at distinct durations postoperatively. The structural remodeling of retrieved samples was then examined. It was found that the degree of inflammatory reaction observed for the genipin‐fixed acellular patch was significantly less than its glutaraldehyde‐fixed counterpart. At 1 month postoperatively, intimal thickening was found on the inner surfaces of both studied groups. The intimal thickening observed on the glutaraldehyde‐fixed acellular patch was significantly thicker than its genipin‐fixed counterpart. An intact layer of endothelial cells was found on the intimal thickening of the genipin‐fixed acellular patch, whereas endothelial cells did not universally and totally cover the entire surface of the glutaraldehyde‐fixed acellular patch. Additionally, fibroblasts with neocollagen fibrils and myofibroblasts were observed in the acellular patches for both studied groups, an indication of tissue regeneration. This phenomenon was more prominent for the genipin‐fixed acellular patch than its glutaraldehyde‐fixed counterpart. At 6 months postoperatively, foci of chondroid and/or bony metaplasia were found in each retrieved sample for both studied groups. The observed adverse response of chondroid metaplasia may be attributed to a compliance mismatch at the implanted site of the canine pulmonary trunk after implantation or a lack of angiogenesis in the regenerated tissue observed at 1 month postoperatively. Bony metaplasia may then develop as in other chondroid tissues. It was reported that ischemia is a usual cause of metaplasia. © 2004 Wiley Periodicals, Inc. J Biomed Mater Res 69A: 323–333, 2004
Hierarchically biomimetic bone scaffold materials: Nano‐HA/collagen/PLA composite
Authors: Liao S. S. , Cui F. Z. , Zhang W. , Feng Q. L.
Published: March 2004

A bone scaffold material (nano‐HA/ collagen/PLA composite) was developed by biomimetic synthesis. It shows some features of natural bone both in main composition and hierarchical microstructure. Nano‐hydroxyapatite and collagen assembled into mineralized fibril. The three‐dimensional porous scaffold materials mimic the microstructure of cancellous bone. Cell culture and animal model tests showed that the composite material is bioactive. The osteoblasts were separated from the neonatal rat calvaria. Osteoblasts adhered, spread, and proliferated throughout the pores of the scaffold material within a week. A 15‐mm segmental defect model in the radius of the rabbit was used to evaluate the bone‐remodeling ability of the composite. Combined with 0.5 mg rhBMP‐2, the material block was implanted into the defect. The segmental defect was integrated 12 weeks after surgery, and the implanted composite was partially substituted by new bone tissue. This scaffold composite has promise for the clinical repair of large bony defects according to the principles of bone tissue engineering. © 2004 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 69B: 158–165, 2004
The effect of cesium‐containing leucite additions on the thermal and mechanical properties of two leucite‐based porcelains
Authors: Rasmussen Stephen T. , McLaren Charles I. , O'Brien William J.
Published: March 2004

It has been demonstrated that a Cs2O‐stabilized leucite porcelain was susceptible to a stress‐induced phase transformation, but toughening was not observed.1 It was hypothesized that if Cs2O‐stabilized leucite core particles were added to a cesium‐free matrix porcelain, selected or designed to enhance the toughening mechanism, toughening would occur. A commercial porcelain (VP) and a synthesized leucite‐based porcelain (NP) were the matrix materials. Core particles of Cs2O (0.0–2.0 mol%) containing synthetic leucite were mixed with the two cesium‐free matrix porcelains and vacuum fired into specimens for testing. The toughness of both types of matrix‐based materials was dependent upon cesium content of the added core particles with a maximum toughness reached for those containing 0.75 mol% Cs2O. The toughness of the 0.75 mol% specimens (1.42 MNm−3/2 for VP based and 2.15 MNm−3/2 for NP based) was statistically (p < .02) higher than either of the matrix materials alone or the matrix materials containing added core particles of synthetic cesium‐free leucite. However, the toughest materials (0.75 mol% Cs2O) were not the strongest materials, most likely because of large internal flaws. The results suggested that transformation toughening was possible. © 2004 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 69B: 195–204, 2004
A new resin matrix for dental composite having low volumetric shrinkage
Authors: Kim Y. , Kim C. K. , Cho B. H. , Son H. H. , Um C. M. , Kim O. Y.
Published: March 2004

The applications of dental restorative composite resins containing 2,2 bis [4‐(2‐hydroxy‐3‐methacryloyloxy propoxy) phenyl] propane (Bis‐GMA), as a base resin, and triethylene glycol dimethacrylate (TEGDMA), as a diluent, are often limited in dentistry due to the relatively large amount of volumetric shrinkage that occurs during the curing reaction. In this study, various new resin matrices were examined for use as dental composites in order to reduce the amount of volumetric shrinkage that occurs in dental composites as a result of curing. Bis‐GMA derivatives were synthesized by substituting methyl groups for hydrogen on the phenyl ring. The derivatives of TEGDMA with different chain lengths or reactive groups were also examined. The molecular structural changes in the TEGDMA derivatives were not effective in reducing the level of volumetric shrinkage. The resin matrix containing a Bis‐GMA derivative and TEGDMA showed a reduced amount of volumetric shrinkage in proportion to the number of methyl groups on the phenyl rings. Polymerization with a mixture of Bis‐GMA, its derivatives and a diluent is a promising strategy for obtaining a polymer with a low amount of volumetric shrinkage. A comparison of the volumetric shrinkage of dental composites containing Bis‐GMA, TMBis‐GMA (2,2‐bis[3,5‐dimethyl, 4‐(2‐hydroxy‐3‐methacryloyloxy propoxy) phenyl] propane)), and TEGDMA with that prepared from a Bis‐GMA and TEGDMA mixture showed that the volumetric shrinkage reduction in the new resin was approximately 50%. Furthermore, the mechanical strength of the former was higher than that of the latter. © 2004 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 70B: 82–90, 2004
Quantitative analysis of biologic specimens by X‐ray scanning analytic microscopy
Authors: Uo Motohiro , Tanaka Masaya , Watari Fumio
Published: March 2004

X‐ray scanning analytic microscopy (XSAM) can be used to visualize the elemental distribution in biologic specimens. In this article, the authors prepared standard specimens for XSAM and performed quantitative analysis of various elements dissolved in soft tissues. Two different types of standard specimens were prepared. Methylmethacrylate (MMA) resin‐based standard specimens were prepared with organic compounds of elements for low‐concentration standards and lithium borate glass‐based standard specimens were prepared with oxides of elements for higher concentration standards. Using these standard specimens, the P and Ca concentrations in normal rat tissue and dissolved Ni, Fe, and Ni concentrations around metal‐implanted tissues were quantitatively analyzed. The estimated concentrations of dissolved Fe, Cu, and Ni from the implants were 1000, 40, and 20 mM, respectively. From the concentration levels causing inflammation around these implants, the high toxicity for soft tissue of Ni and Cu at low concentrations, for example, 10 mM, was confirmed. The toxicity of Cu was estimated as next to that of Ni. In contrast, Fe had low toxicity despite high concentrations of dissolved Fe of as much as 1000 mM. In this article, it was possible to estimate the nonmetallic elements and low‐concentration metallic elements dispersed in soft tissue by XSAM. © 2004 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 70B: 146–151, 2004
Proliferation of human melanoma cells is under tight control of protein kinase C alpha
Authors: Krasagakis Konstantin , Lindschau Carsten , Fimmel Sabine , Eberle Jürgen , Quass Petra , Haller Hermann , Orfanos Constantin E.
Published: March 2004

Exponential proliferation of human melanoma cells has been associated with low levels of protein kinase C (PKC)‐α. The aim of the present study was to investigate the functional relationship between PKC‐α and melanoma cell proliferation. Treatment of human melanoma cells with the selective PKC inhibitor Ro‐31‐8220 resulted in a significant increase of cell proliferation as measured by 3H‐thymidine incorporation and a fluorometric microassay. In addition, phosphorothioate antisense‐oligodeoxynucleotides (ODNs) to PKC‐α enhanced DNA‐synthesis of human melanoma cells. Furthermore, microinjection and transient transfection of melanoma cells with PKC‐α decreased their proliferation, as shown by the reduction of nuclear staining with the proliferation marker Ki‐67. The presented data demonstrate a cause–effect relationship between PKC‐α and melanoma cell growth, whereby PKC‐α reversely influences the rate of cell proliferation. © 2004 Wiley‐Liss, Inc.
Different roles of ERK and p38 MAP kinases during tube formation from endothelial cells cultured in 3‐dimensional collagen matrices
Authors: Yang Baohua , Cao Dian J. , Sainz Irma , Colman Robert W. , Guo Yan‐Lin
Published: March 2004

In a two‐dimensional (2D) culture dish, the major activity of endothelial cells is proliferation with limited morphological change. When cultured in a three‐dimensional (3D) collagen gel matrix, endothelial cells undergo a series of morphological changes starting with development of intracellular vacuoles and followed by cell elongation. Adjacent cells then coalesce to form tube‐like structures. This process mimics the steps of capillary formation during angiogenesis. Using this model, we investigated the roles of extracellular signal‐regulated kinase (ERK) and p38 MAP kinase (p38) in the tube formation from human umbilical vein endothelial cells (HUVEC). Proliferating HUVEC gradually lost their ability to divide after being transferred to 3D collagen matrices, where differentiation became the dominant cellular activity. The transition from proliferation to the differentiation state was accompanied by a drastic reduction of cyclin‐dependent kinases CDC2, CDK4, and retinoblastoma (Rb) protein, but the expression of cyclin‐dependent kinase inhibitor, p27kip1, was increased. Inhibition of p38 by SB203580 partially prevented these changes and increased the proliferation rate of HUVEC. However, cells under this condition exhibited unusually elongated cell bodies, and they were unable to coalesce to form tube structures. Inhibition of ERK neither affected the cell proliferation rate nor the expression levels of cell cycle regulators, but it completely blocked tube formation by inducing apoptosis, a finding different from the best‐known role of ERK in cell proliferation in the 2D cell culture systems. We conclude that the major function of ERK is to maintain cell viability while p38 plays multiple roles in controlling cell proliferation, viability, and morphogenesis during tube formation. © 2004 Wiley‐Liss, Inc.
Subcellular trafficking of exogenously expressed interferon‐β in Madin–Darby canine kidney cells
Authors: Maruyama Masato , Nishio Teruko , Kato Takako , Yoshida Toyokazu , Ishida Chisaki , Watanabe Yoshihiko , Nishikawa Makiya , Kaneda Yasufumi , Takakura Yoshinobu
Published: March 2004

We have recently demonstrated that when IFN‐β was exogenously expressed in epithelial cells, transiently expressed IFN‐β was predominantly secreted from the cell side to which the transfection was performed, while stably expressed one was almost equally secreted to the apical and basolateral sides. In the present study, we analyzed the subcellular transport of IFN‐β using confocal imaging with green fluorescent protein (GFP)‐tagged IFN‐β in Madin–Darby canine kidney (MDCK) cells. Stably expressed and transiently expressed human IFN‐β (HuIFN‐β)–GFPs were seen in upper regions of the nucleus. In stable HuIFN β–GFP‐producing transformants, transiently expressed mouse IFN‐β (MuIFN‐β) was apparently co‐localized with the bulk of the constitutive HuIFN β–GFP proteins at TGN, and a significant quantity of them then appeared to pass into distinct post‐TGN vesicles, accepting either type of IFN. Meanwhile, when cells were co‐transfected with both expression vectors, transiently expressed both IFNs tended to co‐localize not only at TGN but in post‐TGN vesicles. These results suggest that stably and transiently expressed IFN‐βs, albeit co‐localized at TGN, were transported through apparently discriminated post‐TGN routes. J. Cell. Physiol. 201: 117–125, 2004. © 2004 Wiley‐Liss, Inc.
MAP kinases differentially regulate the expression of macrophage hyperactivity after thermal injury
Authors: Alexander Michelle , Daniel Tanjanika , Chaudry Irshad H. , Schwacha Martin G.
Published: March 2004

Thermal injury increases the capacity of macrophages (Mϕ) to produce various inflammatory mediators, (i.e., Mϕ hyperactivity), which is believed to be involved in the development of subsequent immunosuppression, sepsis, and multiple organ failure. The signal transduction pathways involved in the expression of Mϕ hyperactivity post‐burn, however, remain to be clearly elucidated. To study this C57BL/6 female mice were subjected to a 25% TBSA burn and splenic Mϕs were isolated 7 days later. LPS‐stimulated inflammatory mediator production and MAPK expression (P38 ERK 1/2 and JNK) were determined. Burn injury increased LPS‐induced P38 MAPK, suppressed JNK activation and ERK 1/2 activation was unaltered. These changes in MAPK activation were paralleled by the increased production of PGE2, TNF‐α, IL‐1β, IL‐6, and IL‐10. Differential sensitivity to the inhibition of the MAPK pathways was observed with regard to the mediator evaluated and the presence or absence of burn injury. In general cytokine production in the burn group was in part resistant to the inhibition of a single MAPK pathway as compared with shams. Thus, burn injury increases cross‐talk between the MAPKs pathways, suggesting that alterations MAPK activation and signal transduction contribute to the development Mϕ hyperactivity post‐injury. J. Cell. Physiol. 201: 35–44, 2004. © 2004 Wiley‐Liss, Inc.
TRPC5 activation kinetics are modulated by the scaffolding protein ezrin/radixin/moesin‐binding phosphoprotein‐50 (EBP50)
Authors: Obukhov Alexander G. , Nowycky Martha C.
Published: March 2004

TRPC1‐7 proteins are members of a family of mammalian non‐specific cation channels that mediate receptor‐operated, phospholipase Cβ/Cγ dependent Ca2+ influx in various cell types. TRPC4 and TRPC5 form a subfamily within TRPCs. Uniquely in the TRPC family, these channels possess a C‐terminal “VTTRL” motif that binds to PDZ‐domains of the scaffolding protein, EBP50 (NHERF1; Tang et al., J Biol Chem 275:37559–37564). The functional effects of EBP50 on TRPC4/5 activity have not been investigated. We have cloned rat TRPC5 (rTRPC5), functionally expressed it in HEK293 cell, and studied channel regulation with patch‐clamp techniques. Both rTRPC5 and its VTTRL deletion mutant (r5dV) were localized to the plasma membrane. rTRPC5 did not display any significant basal activity in unstimulated HEK293 cells. In cells co‐expressing rTRPC5 and H1 histamine receptor, rTRPC5 current evoked by GTPγS or histamine developed in two phases: a slowly developing, small inward current was followed by a rapidly developing, transient, large inward current. Each phase had a characteristic non‐linear current–voltage (I–V) relationship. Deletion of the VTTRL motif had no detectable effect on the biophysical properties of the channel. Co‐expression of EBP50 with rTRPC5 caused a significant delay in the time‐to‐peak of the histamine‐evoked, transient large inward current. EBP50 did not modify the activation kinetics of the VTTRL‐deletion mutant. We conclude that the VTTRL motif is not necessary for activation of TRPC5, but may mediate the modulatory effect of EBP50 on TRPC5 activation kinetics. © 2004 Wiley‐Liss, Inc.
Synergistic anti‐tumor effect of glycosylphosphatidylinositol‐anchored IL‐2 and IL‐12
Authors: Ji Jianfei , Li Jinhua , Holmes Lillia M. , Burgin Kelly E. , Yu Xianzhong , Wagner Thomas E. , Wei Yanzhang
Published: March 2004

Background

Preclinical and clinical studies have demonstrated that interleukin 2 (IL‐2), interleukin 12 (IL‐12), and some other cytokines, play important roles in activating host immune responses against tumor growth. However, severe side effects caused by systemic high‐dose administration of these cytokines limit their clinical application. In our previous study, local high doses of IL‐2 were achieved by a GPI‐anchoring technology; therefore, it will be interesting to know if this technology works for other cytokines.

Methods

A fusion gene containing murine IL‐12 and the glycosylphosphatidylinositol (GPI) anchor signal sequence was generated and transfected into the murine melanoma tumor cell line B16F0 either alone or together with a vector encoding GPI‐anchored IL‐2. The GPI‐anchored cytokine expression of the selected stable clones was assayed in vitro by ELISA and their anti‐tumor effects were analyzed in vivo by tumor lymphocyte infiltration and tumor growth studies.

Results

GPI‐anchored IL‐12 was successfully expressed on the cell surface as indicated by FACS analysis and IL‐12 ELISA assay. The GPI‐anchored IL‐12 enhanced lymphocyte infiltration and significantly inhibited tumor growth. More importantly, when GPI‐anchored IL‐12 and GPI‐anchored IL‐2 were co‐delivered, a synergistic anti‐tumor effect was observed in both subcutaneous and intravenous tumor models.

Conclusions

GPI anchorage of cytokines represents a new approach to locally deliver high doses of cytokines without the severe adverse effects normally accompanied with systematic high‐dose administration of these cytokines. Copyright © 2004 John Wiley & Sons, Ltd.
Trans‐complementing adenoviral vectors for oncolytic therapy of malignant melanoma
Authors: Wolkersdörfer G. W. , Morris J. C. , Ehninger G. , Ramsey W. J.
Published: March 2004

Background

Despite attempts to develop efficient viral‐based gene transfer therapies for the treatment of malignant tumors, only limited progress has been made to improve the efficacy of this approach. As an alternative, the use of replicating oncolytic adenoviruses with and without the expression of therapeutic transgenes is an area of active investigation.

Methods

We used a human melanoma xenograft tumor nude mouse model to test the efficacy of a bivalent vector approach consisting of two trans‐complementing replication‐incompetent adenoviral vectors that resulted in tumor‐restricted oncolysis. We combined an E1‐deleted non‐replicating adenoviral vector expressing the herpes simplex virus thymidine kinase gene (AV.C2.TK) and Ad5.dl1014, an E4‐deleted/E4orf4‐only expressing adenovirus, to allow full replication competence when tumor cells were co‐infected with both vectors.

Results

A375 tumors showed apoptosis at the ultrastructural level after transduction with the trans‐complementing vector system that was not seen with injection of either vector alone. Apoptotic DNA fragments could be co‐localized to sites of infection with the adenoviral vectors. A significant survival benefit was achieved for the trans‐complementing vector treated animals compared to animals treated with either vector alone. Interestingly, the administration of GCV did not further increase animal survival over treatment with the trans‐complementing system of viruses alone, and long‐term survival was only seen in the trans‐complementing vector treatment group. Intraperitoneal administration of a pseudo‐wild‐type vector Ad.dl327 resulted in significant hepatotoxicity, while intraperitoneal administration of the trans‐complementing vectors resulted in only mild liver abnormalities.

Conclusions

The trans‐complementing vector approach using a combination of E1‐ and E4‐deleted adenoviral vectors showed similar antitumor efficacy as reported for monovalent replicating vector systems, but may offer additional safety by reducing the risk of dissemination of the replication‐competent vectors by requiring the presence of both vectors in a cell to achieve replication competence. Copyright © 2004 John Wiley & Sons, Ltd.
Bitter taste in cassava roots correlates with cyanogenic glucoside levels
Authors: Chiwona‐Karltun Linley , Brimer Leon , Kalenga Saka John D , Mhone Albert R , Mkumbira Jonathan , Johansson Lisbeth , Bokanga Mpoko , Mahungu Nzola Meso , Rosling Hans
Published: March 2004

Cassava roots contain cyanogenic glucosides. Malawian farmers classify cultivars into two groups based on the perceived danger of eating raw roots that they associate with bitterness. In the vernacular, cultivars that produce roots with bitter taste are called vyakubaba (bitter), whereas those yielding non‐bitter roots are called vyakuzizra (cool). In the scientific literature they are distinguished as ‘bitter’ or ‘sweet’. Roots from ‘bitter’ cultivars are processed prior to consumption. We studied the ability of farmers to predict the cyanogenic glucoside levels of 492 roots from the 10 most commonly grown cultivars. Twenty‐eight farmers predicted the taste of each of the cultivars that they grew, and scored bitterness on a five‐point scale by tasting the root tip. Thereafter cyanogenic glucosides were determined on half of the root, while a taste panel scored the taste of the other half. The mean cyanogenic glucoside level in 132 roots from ‘cool’ cultivars was 29 mg HCN eq kg−1 fresh weight (CI 25–33, range 1–123) and in 360 roots from ‘bitter’ cultivars was 153 mg HCN eq kg−1 fresh weight (CI 143–163, range 22–661). Farmers' distinction of ‘cool’ and ‘bitter’ cultivars predicts glucoside levels. The tasting of the tip of the root improved the farmers' prediction of toxicity. Scoring of bitterness by a trained taste panel showed a stronger correlation with glucoside levels (r2 = 0.67). This suggests that cyanogenic glucosides confer the bitter taste, notwithstanding the probability of additional modifying intrinsic factors. Copyright © 2004 Society of Chemical Industry
Effects of various coatings and antioxidants on peel browning of ‘Bartlett’ pears
Authors: Feng Xuqiao , Biasi Bill , Mitcham Elizabeth J
Published: March 2004

Peel browning of ‘Bartlett’ pears (Pyrus communis L) caused by various mechanical injuries during postharvest handling results in a reduction in visual quality. Various commercial and experimental coatings and antioxidants with potential to reduce peel browning of pears have been evaluated. Treatment with 0.2% diphenylamine (DPA) or 0.3% ethoxyquin (Eth) reduced peel browning on ‘Bartlett’ pears induced by vibration, rolling or scuffing. However, this effect only lasted for about 5 days when treated pears were stored at 0 °C. For the consideration of practical use, pears would have to be treated and packed shortly before transport to achieve a reduction in peel browning. DPA and Eth did not reduce peel browning on pears induced by handling at 20 °C after treatment. It is suggested that tight‐fill packaging and use of DPA or Eth would best reduce peel browning. Copyright © 2004 Society of Chemical Industry
A systematic analysis of a flow‐switching modulator for comprehensive two‐dimensional gas chromatography
Authors: LaClair Russell W. , Bueno Pedro A. , Seeley John V.
Published: March 2004

A simple flow‐switching device has been designed for use as a comprehensive two‐dimensional gas chromatography modulator. The device is constructed from fused silica tubing, t‐unions, and a solenoid valve. A series of experiments were conducted to determine the influence of primary flow, secondary flow, modulation time, and device dimensions on the performance of the modulator. The flow‐switching device was found to produce pulses with widths near the theoretical minimum. High‐performance was maintained over a wide range of modulation times. The flow‐switching device did not introduce extra broadening along the primary retention axis. However, the modulator performance was optimal only over a narrow range of primary to secondary flow ratios. The ideal flow ratio is determined by the dimensions of the tubes that connect the t‐unions. A simple flow resistance model has been developed that can predict the dimensions that will produce optimal results for a specified primary to secondary flow ratio. Thus, it is possible to construct a device that operates near the theoretical limit without numerous alterations. Under optimal conditions, the flow switching modulator generates peaks that are narrower than those produced by a diaphragm valve.
Separation, identification, and quantification of selected withanolides in plant extracts of Withania somnifera by HPLC‐UV(DAD) – Positive ion electrospray ionisation–mass spectrometry
Authors: Khajuria Ravi K. , Suri Krishan A. , Gupta Rajinder K. , Satti Naresh K. , Amina Musarat , Suri Om P. , Qazi Ghulam N.
Published: March 2004

This paper describes a method for separation, identification, and quantification of selected withanolides in Withania somnifera plant extracts by HPLC‐UV(DAD)–Mass Spectrometry (HPLC‐MS). Withaferin‐A (WS‐3), 12‐deoxywithastramonolide (WS‐12DS), Withanolide A (WS‐1), and Withanone (WS‐2) were used as external standards. The compounds were isolated from Withania somnifera by repeated column chromatography of the root extract and their identity was established by 1H‐ and 13C‐NMR and mass spectral data. The compounds were chromatographed on a Merck (250×4.6 mm ID, 5 μm) column and analyzed by Electrospray Ionization on a mass spectrometer in Selected Ion Mode (SIM). For quantification, [M + Na]+ ions were monitored. Linear calibration curves were obtained in the concentration range of 1.50 μg/mL to 6.5 μg/mL. The method was applied successfully to the detection and quantification of the said withanolides in a number of samples.
Evolution of Polyhydroxyalkanoate (PHA) Production System by “Enzyme Evolution”: Successful Case Studies of Directed Evolution
Authors: Taguchi Seiichi , Doi Yoshiharu
Published: March 2004

Summary: Biotechnological studies towards the biosynthesis of polyhydroxyalkanoates (PHAs) biopolyesters have extensively progressed through the development of various metabolic engineering strategies. Historically, efficient PHA production has been achieved using the fermentation technology of naturally occurring PHA‐producing bacteria based on external substrate manipulation (1st generation), and subsequent reinforcement with recombinant gene technology (2nd generation). More recently, “enzyme evolution” is becoming the 3rd generation approach for PHA production. A break‐through in the chemical synthesis of macromolecules with desirable properties was achieved by the development of prominent chemical catalysts via “catalyst evolution”, as represented by a series of Ziegler‐Natta catalysts. Thus, one can easily accept the concept that the molecular evolution of the biocatalysts (enzymes) relevant to PHA synthesis will provide us with a chance to create novel PHA materials with high performance. The first trial of an in vitro enzyme evolution in PHA biosynthesis was reported by our group in 2001. The following literature data, as well as our own experimental results devoted to this new approach, have been accumulated over a short time. This review article focuses specifically on the concept and current case studies of the application of “enzyme evolution” to PHA biosynthesis.

Outline of enzyme evolution‐aided PHA production system.

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Outline of enzyme evolution‐aided PHA production system.