bluuebunny/crossref_metadata_2025 huggingface dataset:
Changes in protein kinase C (PKC) activity, isozyme translocation, and GAP‐43 phosphorylation in the rat hippocampal formation after a single‐trial contextual fear conditioning paradigm
Authors: Young Elizabeth , Cesena Teresa , Meiri Karina F. , Perrone‐Bizzozero Nora I.
Published: October 2002

The hippocampus plays an important role in spatial learning and memory. However, the biochemical alterations that subserve this function remain to be fully elucidated. In this study, rats were subjected to a single‐trial contextual fear conditioning (CFC) paradigm; the activation of different protein kinase C (PKC) subtypes and the levels and phosphorylation of the plasticity‐associated protein GAP‐43 were assayed in the hippocampus at varying times after training. We observed a rapid activation of hippocampal PKC (15 min through 24 h), with differential translocation of the PKC isotypes studied. At early times after CFC (15–90 min), PKCα and PKCγ translocated to the membrane, while PKCβII and PKCϵ moved more transiently (15 to 30 min) to the cytosol. These PKC isotypes returned to the membrane at later time points after CFC. Correlating with these changes in PKC translocation and activity, there was an early decrease in GAP‐43 phosphorylation followed by a more sustained increase from 1.5‐72 h. GAP‐43 protein levels were also increased after 3 h, and these levels remained elevated for at least 72 h. These changes in PKC and GAP‐43 were specific to the CFC trained animals and no changes were seen in animals exposed to the same stimuli in a non‐associative fashion. Comparison of translocation of different PKC isotypes with the changes in GAP‐43 phosphorylation suggested that PKCβII and PKCϵ may mediate both the early changes in the phosphorylation of this protein and the increases in GAP‐43 expression at later times after CFC. Hippocampus 2002;12:457–464. © 2002 Wiley‐Liss, Inc.
Object and spatial relational memory in adult rhesus monkeys is impaired by neonatal lesions of the hippocampal formation but not the amygdaloid complex
Authors: Alvarado Maria C. , Wright Anthony A. , Bachevalier Jocelyne
Published: October 2002

Adult rhesus monkeys with neonatal aspiration lesions of the hippocampal formation or the amygdaloid complex (including their respective subjacent cortices) and their age‐matched controls were tested on the transverse patterning problem (A+ vs. B−, B+ vs. C− and C+ vs. A−) and a spatial version of the delayed nonmatching‐to‐sample (DNMS) task with delays of 10 s to 30 s, 60 s, 120 s, and 600 s. Monkeys with neonatal damage to the amygdaloid complex learned both tasks and did not differ from controls at any delay of the spatial DNMS task. Monkeys with neonatal hippocampal damage, however, were unable to learn transverse patterning, though they easily transferred to a linear series (A+ vs. B−, B+ vs. C−, and C+ vs. X−). Three of the four were also unable to reach criterion on the spatial DNMS task within the limits of testing, and the performance of all four monkeys deteriorated with increasing choice delays. The data suggest a role of the primate hippocampal region in both object and spatial relational learning. Hippocampus 2002;12:421–433. © 2002 Wiley‐Liss, Inc.
Numerical simulation of thermal runaway phenomena in silicon semiconductor devices
Authors: Shioda Kazanori , Oobu Toshiharu , Kijima Kenji
Published: October 2002

A mathematical model for heat production due to thermal excitation of conductive electrons and positive holes in a semiconductor pn junction is derived and discussed. The model is applied to simulate the thermal runaway phenomena in power electronics semiconductor devices. Our discussion focuses especially on the modeling of unexpected huge currents due to an excessive temperature increase. Calculated dynamics of temperature distributions of a silicon wafer while cooling performance decreases proved it is possible for a silicon wafer to be heated over its melting point in a few milliseconds. Our results indicate that if a local hot spot arises in a wafer, the thermal intrinsic excitation carries an increased diffusion current of minor carriers and a recombination current in the depletion layer of a pn junction. Also it appears to be important that cooling performance should be uniform on the wafer to avoid the growth of hot spots and thermal‐runaway itself. © 2002 Wiley Periodicals, Inc. Heat Trans Asian Res, 31(6): 438–455, 2002; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/htj.10044
The acute nootropic effects of Ginkgo biloba in healthy older human subjects: a preliminary investigation
Authors: Nathan Pradeep J. , Ricketts Emily , Wesnes Keith , Mrazek Ludek , Greville Warick , Stough Con
Published: October 2002

Ginkgo biloba has been shown to have chronic memory enhancing effects in healthy subjects and patients with dementia. There is limited research on the acute nootropic effects of Ginkgo biloba in humans. The current study aimed to examine the acute effects of Ginkgo biloba (120 mg) on memory functioning in healthy older volunteers using the cognitive drug research (CDR) battery of memory tests and the Rey auditory verbal learning task (AVLT). The study was a double‐blind placebo‐controlled design, with each participant tested under both placebo and Ginkgo biloba treatment conditions. Testing was conducted pre‐ and 90 min post‐drug administration for each treatment condition. Treatment conditions were separated by a 7 day wash out period. No acute effects of Ginkgo biloba were found for any of the memory tests examined. The findings suggest that 120 mg of Ginkgo biloba has no acute nootropic effects in healthy older humans. Copyright © 2002 John Wiley & Sons, Ltd.
Diabetes is not a risk factor for tardive dyskinesia: a retrospective observational study
Authors: Raja Michele , Azzoni Antonella
Published: October 2002

The aim of this observational study was to compare the prevalence of tardive dyskinesia (TD) in diabetic and non diabetic patients. We compared 34 diabetic patients with 34 non diabetic controls, matched by sex, age and time of admission, who had received a complete neuropsychiatric evaluation and who had been exposed to antidopaminergic treatment for at least 3 months. Among them, 8 of 24 diabetic (33.3%) and 6 of 15 (40.0%) non diabetic patients presented TD. The prevalence of TD in the diabetic group is numerically lower than in the controls but the difference is not statistically significant (χ2 = 0.006; fd = 1; p = 0.937). These results suggest that diabetes is unlikely to play a major role in the pathogenesis of TD. Copyright © 2002 John Wiley & Sons, Ltd.
Tardive dyskinesia and impaired glucose tolerance
Authors: Chong Siow‐Ann , Mythily  , Lum Alvin , Yiong Huak Chan , Kane John
Published: October 2002

The authors examined the role of impaired glucose metabolism in the pathophysiology of tardive dyskinesia in schizophrenic patients with and without persistent TD. Glucose tolerance and insulin levels were determined in 86 patients with persistent tardive dyskinesia and in 108 patients without tardive dyskinesia. Dyskinesias were assessed by the abnormal involuntary movement scale (AIMS) and extrapyramidal symptoms by the Simpson—Angus rating scale (SARS). Fasting blood glucose levels were significantly lower while the first and second hour glucose levels did not reveal any differences in patients with tardive dyskinesia compared with those without tardive dyskinesia. Insulin levels did not differ in these two groups. Our cross‐sectional epidemiological study does not suggest hyperglycemia to be a risk factor for tardive dyskinesia. However, prospective long‐term studies with multiple assessment points are needed to clarify the role of glucose metabolism in the development of tardive dyskinesia. Copyright © 2002 John Wiley & Sons, Ltd.
Acute cholecystokinin effects on event‐related potentials in healthy volunteers
Authors: Knott Verner J. , Mahoney Colleen , Gunnarsson Tove , Bradwejn Jacques , Shlik Jakov
Published: October 2002

This study investigated the effects of a continuous slow infusion of cholecystokinin tetrapeptide (CCK‐4), a neuropeptide with panicogenic properties, on brain event‐related potentials (ERPs) in healthy adults. Twenty‐four volunteers, 15 females and 9 males, were assigned to infusion with either placebo or CCK‐4 in a randomized, double‐blind, parallel group design. ERPs, elicited within a standard auditory odd‐ball paradigm requiring the counting of rare (20%) occurring ‘deviant’ tones interspersed among more frequent (80%) occurring ‘standard’ tones, were assessed once before infusion, and at 10 min and 40 min after the onset of infusion. Compared with the placebo, CCK‐4 delayed the latencies of N100 and P200 components elicited by ‘deviant’ stimuli. No significant treatment differences were observed with respect to N200, P300b, mood or adverse symptoms. These preliminary findings suggest that CCK‐4 may interfere with information processing relating to the selection of significant stimuli and as such, may be of relevance to mechanisms underlying panic disorder. Copyright © 2002 John Wiley & Sons, Ltd.
Modelling overland flow based on Saint‐Venant equations for a discretized hillslope system
Authors: Wang Guang‐Te , Chen Shulin , Boll Jan , Stockle C. O. , McCooL D. K.
Published: October 2002

Mathematical modelling of overland flow is a critical task in simulating transport of water, sediment and other pollutants from land surfaces to receiving waters. In this paper, an overland flow routing method is developed based on the Saint‐Venant equations using a discretized hillslope system for areas with high roughness and steep slope. Under these conditions, the momentum equation reduces to a unique relationship between the flow depth and discharge. A hillslope is treated as a system divided into several subplanes. A set of first‐order non‐linear differential equations for subsequent subplanes are solved analytically using Chezy's formula in lieu of the momentum equation. Comparison of the analytical solution of the first‐order non‐linear ordinary differential equations and a numerical solution using the Runge‐Kutta method shows a relative error of 0·3%. Using runoff data reported in the literature, comparison between the new approach and a numerical solution of the full Saint‐Venant equations showed a close agreement. Copyright © 2002 John Wiley & Sons, Ltd.
Satellite‐derived indices of stream discharge in Taylor Valley, Antarctica
Authors: Dana Gayle L. , Davis Robert E. , Fountain Andrew G. , Wharton Robert A.
Published: October 2002

Temperature indices from NOAA–AVHRR data were developed to predict diurnal and seasonal streamflow in Taylor Valley, Antarctica. Masks of the primary land‐cover types (snow, glacier ice, lake ice, and soils) were created from Landsat TM data using binary threshold methods. The masks were then scaled to AVHRR resolution using modal selection of pixels and applied to both diurnal and seasonal time series of AVHRR temperature data from the 1994–95 austral summer season. A temperature area index (TAI) was calculated for different land‐cover types, as well as for the entire watershed. The TAI was defined as the number of pixels at or above a specified temperature. Diurnal discharge was well characterized by a linear relationship of streamflow to the TAI using a temperature threshold of 259 K from all land class pixels for lower Taylor Valley. Neither index successfully predicted seasonal discharge, most likely due to the mismatch of time scales between the satellite and streamflow data. The TAI has great potential for predicting diurnal streamflow in both Taylor Valley and other remote areas within the McMurdo Dry Valleys of Antarctica. Copyright © 2002 John Wiley & Sons, Ltd.
Effects of transient climate change on basin hydrology. 2. Impacts on runoff variability in the Arno River, central Italy
Authors: Burlando Paolo , Rosso Renzo
Published: October 2002

Potential climate change is expected to produce substantial effects on the hydrological cycle. In a companion paper (Burlando P, Rosso R. Hydrological Processes this issue) the effects on precipitation have been discussed, with particular attention being given to significant changes in storm characteristics, distributional and extremal properties. As a consequence, major changes in runoff and rainfall‐driven processes could occur. The analysis of such changes is discussed in this paper, focusing on the Arno River basin, central Italy, which can be representative of climatic environments of southern Europe. The effects of transient scenarios are simulated by a model that explicitly describes the processes involved in the rainfall–runoff transformation. Changes in the most significant components of the water balance are discussed with respect to seasonality, distributional properties and extremes. Finally, mention is also given to some potential implications on water resources. Copyright © 2002 John Wiley & Sons, Ltd.
The case for a World Lake Vision
Authors: Ballatore Thomas J. , Muhandiki Victor S.
Published: October 2002

The creation of a World Lake Vision is essential to achieving the sustainable management of the world's lakes. We discuss the value of lakes, how those values have historically led to changes in watershed land‐use and population, and how changes have resulted in stresses that impair lake value. Current approaches to lake management at both the local and international level are examined and their shortcomings highlighted. We illustrate how a World Lake Vision, along with the individual lake visions it promotes, can redress the shortcomings of current lake management approaches. Details of the World Lake Vision and the process of its creation are put forth. We end with a critique of the World Water Vision presented at the second World Water Forum in March 2000 from the perspective of lakes and conclude that, in its current form, the World Water Vision provides an inadequate guide for sustainable lake management. Copyright © 2002 John Wiley & Sons, Ltd.
Kinematic wave solutions for pollutant transport by runoff over an impervious plane, with instantaneous or finite‐period mixing
Authors: Singh Vijay P.
Published: October 2002

Kinematic wave solutions are derived for transport of a conservative non‐point‐source pollutant during a rainfall‐runoff event over an impervious plane. Rainfall is assumed to be steady, uniform and finite in duration. Prior to the start of rainfall, the pollutant is distributed uniformly over the plane. When rainfall occurs, the pollutant is washed off in a particular manner and the mixing of pollutant in the runoff water occurs either instantaneously or in a finite period of time under the assumption that the pollutant is soluble and is mixed completely in the runoff water. Copyright © 2002 John Wiley & Sons, Ltd.
In vitro and in vivo activity of MT201, a fully human monoclonal antibody for pancarcinoma treatment
Authors: Naundorf Stefanie , Preithner Susanne , Mayer Petra , Lippold Sandra , Wolf Andreas , Hanakam Frank , Fichtner Iduna , Kufer Peter , Raum Tobias , Riethmüller Gert , Baeuerle Patrick A. , Dreier Torsten
Published: October 2002

In our study, a novel, fully human, recombinant monoclonal antibody of the IgG1 isotype, called MT201, was characterized for its binding properties, complement‐dependent (CDC) and antibody‐dependent cellular cytotoxicity (ADCC), as well as for its in vivo antitumor activity in a nude mouse model. MT201 was found to bind its target, the epithelial cell adhesion molecule (Ep‐CAM; also called 17‐1A antigen, KSA, EGP‐2, GA733‐2), with low affinity in a range similar to that of the clinically validated, murine monoclonal IgG2a antibody edrecolomab (Panorex®). MT201 exhibited Ep‐CAM‐specific CDC with a potency similar to that of edrecolomab. However, the efficacy of ADCC of MT201, as mediated by human immune effector cells, was by 2 orders of magnitude higher than that of edrecolomab. Addition of human serum reduced the ADCC of MT201 while it essentially abolished ADCC of edrecolomab within the concentration range tested. In a nude mouse xenograft model, growth of tumors derived from the human colon carcinoma line HT‐29 was significantly and comparably suppressed by MT201 and edrecolomab. The fully human nature and the improved ADCC of MT201 with human effector cells will make MT201 a promising candidate for the clinical development of a novel pan‐carcinoma antibody that is superior to edrecolomab. © 2002 Wiley‐Liss, Inc.
Evaluation of downstaging in the detection of cervical neoplasia in Kolkata, India
Authors: Basu Parthasarathi , Sankaranarayanan Rengaswamy , Mandal Ranajit , Roy Chinmoy , Das Pradip , Choudhury Devhani , Datta Krabi , Karamakar Sanjib , Tsu Vivien , Chakrabarti Rohini Nandan , Siddiqi Maqsood ,   Calcutta Cervical Neoplasia Early Detection Study (CEDS) Group
Published: October 2002

Unaided visual inspection or “downstaging” has been suggested as a potential alternative method for cervical cancer screening in developing countries. Our study was designed to evaluate the accuracy of downstaging to detect cervical neoplasia in a low‐resource setting. A total of 6,399 women aged 30–64 years were screened with downstaging by trained nonmedical health workers. Two thresholds were used to define positive downstaging: “low threshold” when any visible abnormality on the cervix was considered positive and “high threshold” when selected abnormalities such as bleeding on touch, bleeding erosion, hypertrophied oedematous cervix, congested stippled cervix and growth or ulcer constituted the positive test. All women underwent a colposcopy examination. Biopsies were directed when colposcopy revealed abnormal lesions. True disease status was defined as histologically proven moderate dysplasia and worse lesions. Since all the participants received a diagnostic (reference) investigation (biopsy and/or colposcopy), sensitivity, specificity and predictive values were estimated directly. Low‐ and high‐threshold downstaging were positive in 1,585 (24.8%) and 460 (7.2%) women, respectively. The sensitivities of low‐ and high‐threshold downstaging to detect high‐grade precursors and invasive cancers were 48.9% and 31.9%, respectively. The specificities were 75.8% and 93.3%, respectively. These results indicate that downstaging is not suitable as an independent primary screening modality for cervical neoplasia. © 2002 Wiley‐Liss, Inc.
MICA triggering signal for NK cell tumor lysis is counteracted by HLA‐G1‐mediated inhibitory signal
Authors: Menier Catherine , Riteau Béatrice , Carosella Edgardo D. , Rouas‐Freiss Nathalie
Published: October 2002

MICA, a highly glycosylated membrane‐anchored cell–surface MHC Class I‐related chain, has recently been reported to activate NK cell cytolytic responses in epithelial tumors. Tumor cells may escape from NK lysis by counteracting NK cytotoxicity activating signals with inhibitory ones. Among the molecules that mediate an NK inhibitory signal, HLA‐G1, a non‐classical MHC Class I antigen, is of particular interest. HLA‐G1 is ectopically expressed in various tumors, including melanoma and constitutes the major NK inhibitory ligand in the M8 melanoma cell line when coexpressed with HLA‐A, ‐B, ‐C and ‐E molecules. We have evaluated the balance between 2 powerful signals that affect NK cell tumor lysis, one inhibitory and the other one activating, respectively HLA‐G1 and MICA. For this purpose, we transfected the M8 melanoma cell line, which spontaneously expresses MICA, with HLA‐G1 cDNA, using it as a target for the NKL effector. We carried out cytotoxicity assays, using antibodies that disrupt interactions between the MICA and HLA‐G1 ligands and their respective NK effector counterparts, the NKG2D activating and ILT2 inhibitory receptors. Results showed that 1) MICA expressed in the M8 melanoma cell line triggered NK cell tumor lysis and 2) HLA‐G1 coexpression mediated the inhibition of NK cytotoxicity by mitigating the MICA activating signal. HLA‐G1 expression in a tumor cell line in which MICA is switched on would therefore appear to be a powerful way to turn off NK cells, supporting the emerging idea that the balance between positive and negative NK cytolysis signals critically influences tumor progression. © 2002 Wiley‐Liss, Inc.
Expression of an IGF‐I receptor dominant negative mutant induces apoptosis, inhibits tumorigenesis and enhances chemosensitivity in Ewing's sarcoma cells
Authors: Scotlandi Katia , Avnet Sofia , Benini Stefania , Manara Maria Cristina , Serra Massimo , Cerisano Vanessa , Perdichizzi Stefania , Lollini Pier‐Luigi , De Giovanni Carla , Landuzzi Lorena , Picci Piero
Published: October 2002

IGF‐IR plays an essential role in the establishment and maintenance of the transformed phenotype of ES cells and interference with the IGF‐IR pathways causes reversal of the malignant potential of this neoplasm. In this report, we stably transfected a dominant negative IGF‐IR expression plasmid in an ES cell line to determine the effectiveness of this strategy against the in vitro and in vivo growth of ES cells. DXR sensitivity of TC‐71 cells expressing dominant negative mutants of IGF‐IR was also examined. The mutated IGF‐IR that we used carries a mutation in the ATP‐binding domain of the intracellular β subunit, while the extracellular, ligand‐binding α subunit remains unchanged. Cells carrying the dominant mutant IGF‐IR had a marked decrease in proliferation, a significant increase in anoikis‐induced apoptosis and a severely reduced ability to form colonies in soft agar. In vivo, when cells carrying dominant negative IGF‐IR were injected into nude mice, the tumor formation and metastatic abilities of ES cells were reduced and survival increased. Furthermore, transfected clones showed significantly higher sensitivity to DXR, a major drug in the treatment of ES. These results indicate that the IGF/IGF‐IR stimulation of ES cells may be inhibited by expression of mutated IGF‐IR on their surfaces and that this strategy may be considered a possible alternative to impair this important target of ES cells, whose therapeutic potential was further confirmed. © 2002 Wiley‐Liss, Inc.
Increased expression of a COOH‐truncated nucleophosmin resulting from alternative splicing is associated with cellular resistance to ionizing radiation in HeLa cells
Authors: Dalenc Florence , Drouet Jerome , Ader Isabelle , Delmas Caroline , Rochaix Philippe , Favre Gilles , Cohen‐Jonathan Elizabeth , Toulas Christine
Published: October 2002

We previously demonstrated that transfecting HeLa cells with the 24 kDa basic fibroblast growth factor‐2 (FGF‐2) isoform dramatically increased cell survival after irradiation. To investigate genes implicated in this radioresistance acquisition, we compared mRNA expression between radioresistant 24 kDa FGF‐2‐expressing cells (HeLa 3A) and radiosensitive control HeLa PINA cells using the differential display technique. Of the 32 differentially expressed mRNAs, 1 presented a significant homology with a known gene. This 378 bp fragment presented 100% identity with exon 11 and 12 of human nucleophosmin (NPM) but differed by including a part of intron 9 in its 5ℙ end. The differential expression of this fragment was confirmed using an RNase protection assay. We then cloned the entire corresponding mRNA and showed that it contained all the exons of NPM plus intron 9, suggesting that it was a splicing product of the NPM gene. This variant encoded for a 35‐amino acid truncated NPM (NPM2). NPM2 expression was increased in HeLa 3A. To investigate NPM2's role in radioresistance acquisition, we transfected HeLa cells with NPM2 cDNA and analyzed survival after irradiation of the clones obtained. After transfection with NPM2, radiosensitive HeLa cells exhibited a dramatic increase in cell survival after irradiation. Taken together, our results demonstrate that expression of a COOH‐truncated NPM form resulting from the alternative splicing of NPM mRNA is able to increase cell survival after irradiation and suggests that it might be involved in cellular response to ionizing radiation. © 2002 Wiley‐Liss, Inc.
Mutational analysis of the class I β‐tubulin gene in human breast cancer
Authors: Hasegawa Seiichi , Miyoshi Yasuo , Egawa Chiyomi , Ishitobi Makoto , Tamaki Yasuhiro , Monden Morito , Noguchi Shinzaburo
Published: October 2002

Non‐small cell lung cancers have a high incidence of somatic mutations of the β‐tubulin (class I) gene, suggesting involvement in the acquisition of resistance to taxanes, which exert their effects through binding to β‐tubulin. Since taxanes are often used in the treatment of breast cancer, we carried out a mutational analysis of the class I β‐tubulin (GenBank accession AF070600) gene in breast cancer. We paid special attention to the primer design so as not to amplify the pseudogenes. We identified 1 somatic mutation, codon 306 [Arg (CGC) to Cys (TGC)], and 2 genetic polymorphisms, codon 217 [Leu (CTG) to Leu (CTA)] and (C to T) at 57 bases downstream from exon 4. Our results suggest that acquisition of resistance to taxanes is unlikely to be explained by somatic mutations of the class I β‐tubulin gene in most breast cancers. In addition, the overestimation of the incidence of somatic mutations of the class I β‐tubulin gene due to the pseudogenes is discussed. © 2002 Wiley‐Liss, Inc.
Water resource hazard management system: assessing sustainable practices at the farm and catchment scales
Authors: Ochola W. O. , Kerkides P. , Argyrokastritis I.
Published: October 2002

Water quality is a pivotal environmental indicator of sustainable land management and environmental ‘health’. Hazards to water resource use at the farm and catchment scales have far‐reaching physical, biological, environmental and socio‐economic impacts. These impacts are exacerbated by on‐site and off‐site agricultural and non‐agricultural activities. A prototype water hazard management decision support system that uses an integrated framework to identify, by origin, hazards and related best water management practices guidelines is proposed. The system recommends best management guidelines with respect to the inherent water resource use mitigations. The system has been calibrated by and applied to expert knowledge and experimental and survey data from Kiumbu Catchment in central Kenya. Suggestions are made for the inclusion of GIS capabilities for the production of water resource assessment maps and other spatial water quality indicators. Copyright © 2002 John Wiley & Sons, Ltd.
Approaches to the assessment of toxicity data with endpoints related to endocrine disruption
Authors: Harvey Philip W. , Johnson Ian
Published: October 2002

There has been a substantial proliferation in the number of studies reporting endocrine effects as an endpoint. The vast majority have focused on oestrogenicity in vitro but, with recent recommendations by the USEPA Endocrine Disrupter Screening and Testing Advisory Committee, tests are now being developed for (anti)‐androgenicity and effects on the thyroid, largely because of the potential for altering reproduction or development via these mechanisms. Despite being a vital organ and involved in reproduction and development, there is currently no provision for assessing adrenocortical function. Similarly, the entire process of steroidogenesis poses multiple molecular targets for toxic disruption that are not included in current test strategies and at present there is no clear position on the significance of the data being generated. This review provides a framework for approaching endocrine data: that all the glands, tissues, receptors, transporter proteins and enzymes that comprise the endocrine system are targets for toxicity. They should be considered in much the same way as other target organs, with appropriate provision for the special cases of carcinogenesis and teratogenesis, and a pragmatic weight of evidence approach should be adopted considering all available data and recognizing its limitations. In this approach, structure–activity relationships and in vitro and targeted in vivo screens provide useful data but repeat‐dose regulatory studies with defined endpoints provide the most powerful tools for hazard assessment. Pragmatic consideration should be given to exposure issues (which may highlight the practical irrelevance, for example, of very low potency oestrogens) and subsequently whether endocrine disruption is the critical or most sensitive endpoint for a compound. Finally, endocrine disruption may be considered a mechanism and, as with other toxic endpoints, knowledge of effect and no‐observable‐effect levels and reversibility is as important as identifying the target tissue or any inherent hormone‐like property. Copyright © 2002 John Wiley & Sons, Ltd.
Analysis of the energetic state of heart cells after adenovirus‐mediated expression of hALC‐1
Authors: Zacharzowsky Udo B. , Wolff Gerhard , Kott Monika , Haase Hannelore , Bartsch Holger , Nuessler Andreas K. , Baltas Leonidas G. , Karawajew Leonid , Morano Ingo
Published: October 2002

Expression of the human atrial myosin light chain 1 (hALC‐1) in the cardiac ventricle in vivo as well as in primary cultivated adult cardiomyocytes caused a pronounced positive inotropic effect. Therefore, it is one of the most promising candidate gene to treat congestive heart failure (CHF). In this work, we investigated, whether hALC‐1 expression also modifies the energetic state of cardiomyocytes. Primary cultivated neonatal rat hearts cells (NRHC) were infected with adenoviral vectors (Ad vectors) containing a hALC‐1 cDNA (AdCMV.hALC‐1) or a control Ad vector. Infection efficiency of NRHC reached 100% at 50 multiplicity of infection (MOI). Interestingly and in contrast to primary cultures of liver cells, there were no cytotoxic side effects or induction of apoptosis up to MOI 50 in Ad vector infected NRHC. NRHC expressed large amounts of hALC‐1 upon infection with AdCMV.hALC‐1 which could easily been detected by protein staining and Western blot analysis. Analysis of intracellular hALC‐1 localization by double‐labeling immunofluorescence of AdCMV.hALC‐1 infected cardiomyocytes revealed the typical myofibrillar striation pattern, as well as co‐localization of hALC‐1 with myosin heavy chains. There was no difference in the oxygen consumption between controls and AdCMV.hALC‐1 infected NRHC. These data suggest that first: adenoviral vectors could be used as a safe and effective tool for gene transfer to cardiomyocytes, and second: that a positive inotropic effect of hALC‐1 is not associated with enhanced oxygen consumption. © 2002 Wiley‐Liss, Inc.
Differential effects of iron deficiency on the expression of CD80 and CD86 co‐stimulatory receptors in mitogen‐treated and untreated murine spleen cells
Authors: Kuvibidila Solo R. , Porretta Connie
Published: October 2002

The interaction of CD28 and its ligands (CD80, CD86) on antigen presenting cells and that of TCR/CD3‐MHC are required for T lymphocyte activation. To determine whether impaired lymphocyte proliferation associated with iron deficiency is due to reduced expression of these ligands, spleen cells obtained from eight to nine C57BL/6 mice/group of iron deficient (ID), iron replete (R), control (C), pair‐fed (PF), and high iron (HI) mice were labeled with anti‐CD80‐fluorescein isothiocyante (FITC) and anti‐CD86‐FITC. Diets differed only in iron concentration: 5, 50, and 125 mg/kg for the ID, C, and HI, respectively. Mean levels of hemoglobin and liver iron stores of ID and R mice were less than 50% those of C mice (P < 0.005). In non‐activated and concanavalin A‐treated cultures, significant differences were observed among groups in the percentage of CD80 + cells: ID>R > C = PF = HI (P < 0.05). The same trend was observed for CD86 + cells (P > 0.05). Fluorescence intensity (FI) of either marker did not significantly change by iron status. In vitro iron chelation by deferoxamine (20, 200 μg/ml) for 1, 2, and 24 h increased FI of both markers on unactivated B and T cells (P < 0.05). However, it had no effect on FI of either marker of mitogen‐treated cells presumably because the maximum levels are achieved by the mitogen. Lymphocyte proliferative responses to mitogens positively and significantly correlated with CD80 and CD86 FI (r = 0.41−0.59) but negatively correlated with the percentages of CD80 + cells (r = −0.48) (P < 0.05). Data suggest that impaired lymphocyte proliferation associated with iron deficiency is not due to reduced CD80 and CD86 expression. J. Cell. Biochem. 86: 571–582, 2002. © 2002 Wiley‐Liss, Inc.
Differential involvement of protein kinase C in basal versus acetylcholine‐regulated prolactin secretion in rat anterior pituitary cells during aging
Authors: Pu Hsiao‐Fung , Liu Tsuei‐Chu
Published: October 2002

Although it is well known that plasma concentration of prolactin (PRL) increases during aging in rats, how the anterior pituitary (AP) aging per se affects PRL secretion remains obscure. The objectives of this study were to determine if changes in the pituitary PRL responsiveness to acetylcholine (ACh; a paracrine factor in the AP), as compared with that to other PRL stimulators or inhibitors, contribute to the known age‐related increase in PRL secretion, and if protein kinase C (PKC) is involved. We also determined if replenishment with aging‐declined hormones such as estrogen/thyroid hormone influences the aging‐caused effects on pituitary PRL responses. AP cells were prepared from old (23–24‐month‐old) as well as young (2–3‐month‐old) ovariectomized rats. Cells were pretreated for 5 days with diluent or 17β‐estradiol (E2; 0.6 nM) in combination with or without triiodothyronine (T3; 10 nM). Then, cells were incubated for 20 min with thyrotropin‐releasing hormone (TRH; 100 nM), angiotensin II (AII; 0.2–20 nM), vasoactive intestinal peptide (VIP; 10−9–10−5 M), dopamine (DA; 10−9–10−5 M), or ACh (10−7–10−3 M). Cells were also challenged with ACh, TRH, or phorbol 12‐myristate 13‐acetate (PMA; 10−6 M) following PKC depletion by prolonged PMA (10−6 M for 24 h) pretreatment. We found that estrogen priming of AP cells could reverse the aging‐caused effects on pituitary PRL responses to AII and DA. In hormone‐replenished cells aging enhanced the stimulation of PRL secretion by TRH and PMA, but not by AII and VIP. Aging also reduced the responsiveness of cells to ACh and DA in suppressing basal PRL secretion, and attenuated ACh inhibition of TRH‐induced PRL secretion. Furthermore, ACh suppressed TRH‐induced PRL secretion mainly via the PMA‐sensitive PKC in the old AP cells, but via additional mechanisms in young AP cells. On the contrary, basal PRL secretion was PKC (PMA‐sensitive)‐independent in the old AP cells, but dependent in the young AP cells. Taken together, these results suggest differential roles of PMA‐sensitive PKC in regulating basal and ACh‐regulated PRL responses in old versus young AP cells. The persistent aging‐induced differences in AP cell responsiveness to ACh, DA, TRH, and PMA following hormone (E2/T3) replenishment suggest an intrinsic pituitary change that may contribute, in part, to the elevated in vivo PRL secretion observed in aged rats. J. Cell. Biochem. 86: 268–276, 2002. © 2002 Wiley‐Liss, Inc.
Effects of cell swelling on intracellular calcium and membrane currents in bovine articular chondrocytes
Authors: Yellowley Clare E. , Hancox Jules C. , Donahue Henry J.
Published: October 2002

Chondrocytes experience a dynamic extracellular osmotic environment during normal joint loading when fluid is forced from the matrix, increasing the local proteoglycan concentration and therefore the ionic strength and osmolarity. To exist in such a challenging environment, chondrocytes must possess mechanisms by which cell volume can be regulated. In this study, we investigated the ability of bovine articular chondrocytes (BAC) to regulate cell volume during a hypo‐osmotic challenge. We also examined the effect of hypo‐osmotic stress on early signaling events including [Ca2+]i and membrane currents. Changes in cell volume were measured by monitoring the fluorescence of calcein‐loaded cells. [Ca2+]i was quantified using fura‐2, and membrane currents were recorded using patch clamp. BAC exhibited regulated volume decrease (RVD) when exposed to hypo‐osmotic saline which was inhibited by Gd3+. Swelling stimulated [Ca2+]i transients in BAC which were dependent on swelling magnitude. Gd3+, zero [Ca2+]o, and thapsigargin all attenuated the [Ca2+]i response, suggesting roles for Ca2+ influx through stretch activated channels, and Ca2+ release from intracellular stores. Inward and outward membrane currents significantly increased during cell swelling and were inhibited by Gd3+. These results indicate that RVD in BAC may involve [Ca2+]i and ion channel activation, both of which play pivotal roles in RVD in other cell types. These signaling pathways are also similar to those activated in chondrocytes subjected to other biophysical signals. It is possible, then, that these signaling events may also be involved in a mechanism by which mechanical loads are transduced into appropriate cellular responses by chondrocytes. J. Cell. Biochem. 86: 290–301, 2002. © 2002 Wiley‐Liss, Inc.
Identification of PSF as a protein kinase Cα‐binding protein in the cell nucleus
Authors: Rosenberger Uwe , Lehmann Ingo , Weise Christoph , Franke Peter , Hucho Ferdinand , Buchner Klaus
Published: October 2002

Protein kinase C (PKC) isoforms are present in the cell nucleus in diverse cell lines and tissues. Since little is known about proteins interacting with PKC inside the cell nucleus, we used Neuro‐2a neuroblastoma cells, in which PKCα is present in the nucleus, to screen for nuclear binding partners for PKC. Applying overlay assays, we detected several nuclear proteins which bind to PKCα. Specificity of binding was shown by its dependence on PKC activation by phorbol ester, calcium, and phosphatidylserine. The PKC‐binding proteins were partially purified and analyzed by microsequencing and mass spectrometry. Four proteins could be identified: PTB‐associated splicing factor (PSF), p68 RNA helicase, and the heterogeneous nuclear ribonucleoprotein (hnRNP) proteins A3 and L. In the case of PSF, binding to PKC could also be demonstrated in a GST‐pull‐down assay using GST‐PKCα, expressed in insect cells. Phosphorylation experiments revealed that PSF is a weak in vitro substrate for PKCα. J. Cell. Biochem. 86: 394–402, 2002. © 2002 Wiley‐Liss, Inc.
ATP and UTP at low concentrations strongly inhibit bone formation by osteoblasts: A novel role for the P2Y2 receptor in bone remodeling
Authors: Hoebertz Astrid , Mahendran Siva , Burnstock Geoffrey , Arnett Timothy R.
Published: October 2002

There is increasing evidence that extracellular nucleotides act on bone cells via multiple P2 receptors. The naturally‐occurring ligand ATP is a potent agonist at all receptor subtypes, whereas ADP and UTP only act at specific receptor subtypes. We have reported that the formation and resorptive activity of rodent osteoclasts are stimulated powerfully by both extracellular ATP and its first degradation product, ADP, the latter acting at nanomolar concentrations, probably via the P2Y1 receptor subtype. In the present study, we investigated the actions of ATP, ADP, adenosine, and UTP on osteoblastic function. In 16–21 day cultures of primary rat calvarial osteoblasts, ADP and the selective P2Y1 agonist 2‐methylthioADP were without effect on bone nodule formation at concentrations between 1 and 125 μM, as was adenosine. However, UTP, a P2Y2 and P2Y4 receptor agonist, known to be without effect on osteoclast function, strongly inhibited bone nodule formation at concentrations ≥ 1 μM. ATP was inhibitory at ≥ 10 μM. Rat osteoblasts express P2Y2, but not P2Y4 receptor mRNA, as determined by in situ hybridization. Thus, the low‐dose effects of extracellular nucleotides on bone formation and bone resorption appear to be mediated via different P2Y receptor subtypes: ADP, signalling through the P2Y1 receptor on both osteoclasts and osteoblasts, is a powerful stimulator of osteoclast formation and activity, whereas UTP, signalling via the P2Y2 receptor on osteoblasts, blocks bone formation by osteoblasts. ATP, the ‘universal’ agonist, can simultaneously stimulate resorption and inhibit bone formation. These findings suggest that extracellular nucleotides could function locally as important negative modulators of bone metabolism, perhaps contributing to bone loss in a number of pathological states. J. Cell. Biochem. 86: 413–419, 2002. © 2002 Wiley‐Liss, Inc.
Improved semiempirical heats of formation through the use of bond and group equivalents
Authors: Repasky Matthew P. , Chandrasekhar Jayaraman , Jorgensen William L.
Published: October 2002

Deficiencies in energetics obtained using the common semiempirical methods, AM1, PM3, and MNDO, may partly be traced to the use of pseudoatomic equivalents for conversion of molecular energies to heats of formation at 298 K. We present an alternative scheme based on the use of bond and group equivalents. Values for the 61 bond and group equivalents necessary for treatment of molecules containing the common organic elements, hydrogen, carbon, nitrogen, and oxygen have been derived. For a set of 583 neutral, closed‐shell molecules mean absolute errors in AM1, PM3, and MNDO heats of formation are reduced from 6.6, 4.2, and 8.2 kcal/mol to 2.3, 2.2, and 3.0 kcal/mol, respectively. Several systematic problems are overcome in the present scheme including relative stabilities of branched hydrocarbons, energetics of conjugated systems, heats of formation of long chain hydrocarbons, and enthalpies of molecules containing multiple heteroatoms. Although the approach is restricted to molecules with well‐defined functional groups, the equivalents are easy to incorporate and are chemically relevant. This revised procedure allows semiempirical methods to be used for far more reliable evaluations of heats of reactions. Estimates are made of the errors inherent in these semiempirical formalisms, arising from integral approximations and the neglect of explicit treatment of electron correlation effects, while excluding those from inadequate parameterization. © 2002 Wiley Periodicals, Inc. J Comput Chem 23: 498–510, 2002; DOI 10.1002/jcc.2002;10023
Monte Carlo sampling algorithm for searching a scale‐transformed energy space of polypeptides
Authors: Nakamura Hideaki
Published: October 2002

A Monte Carlo sampling algorithm for searching a scale‐transformed conformational energy space of polypeptides is presented. This algorithm is based on the assumption that energy barriers can be overcome by a uniform sampling of the logarithmically transformed energy space. This algorithm is tested with Met‐enkephalin. For comparison, the entropy sampling Monte Carlo (ESMC) simulation is performed. First, the global minimum is easily found by the optimization of a scale‐transformed energy space. With a new Monte Carlo sampling, energy barriers of 3000 kcal/mol are frequently overcome, and low‐energy conformations are sampled more efficiently than with ESMC simulations. Several thermodynamic quantities are calculated with good accuracy. © 2002 Wiley Periodicals, Inc. J Comput Chem 23: 511–516, 2002; DOI 10.1002/jcc.10034
New method for parallel computation of Hessian matrix of conformational energy function in internal coordinates
Authors: Nakamura Shugo , Kyono Daisuke , Ikeguchi Mitsunori , Shimizu Kentaro
Published: October 2002

A new algorithm for parallel calculation of the second derivatives (Hessian) of the conformational energy function of biomolecules in internal coordinates is proposed. The basic scheme of this algorithm is the division of the entire calculation of the Hessian matrix (called “task”) into subtasks and the optimization of the assignment of processors to each subtask by considering both the load balancing and reduction of the communication cost. A genetic algorithm is used for this optimization considering the dependencies between subtasks. We applied this method to a glutaminyl transfer RNA (Gln‐tRNA) molecule for which the scalability of our previously developed parallel algorithm was significantly decreased when the large number of processors was used. The speedup for the calculation was 32.6 times with 60 processors, which is considerably better than the speedup for our previously reported parallel algorithm. The elapsed time for the calculation of subtasks, data sending, and data receiving was analyzed, and the effect of the optimization using the genetic algorithm is discussed. © 2002 Wiley Periodicals, Inc. J Comput Chem 23: 463–469, 2002; DOI 10.1002/jcc.10039
Quantum chemical geometry optimizations in proteins using crystallographic raw data
Authors: Ryde Ulf , Olsen Lars , Nilsson Kristina
Published: October 2002

A method is developed for the combination of quantum chemical geometry optimizations and crystallographic structure refinement. The method is implemented by integrating the quantum chemical software Turbomole with the crystallographic software Crystallography and NMR System (CNS), using three small procedures transferring information between the two programs. The program (COMQUM‐X)is used to study the binding of the inhibitor N‐methylmesoporphyrin to ferrochelatase, and we show that the method behaves properly and leads to an improvement of the structure of the inhibitor. It allows us to directly quantify in energy terms how much the protein distort the structure of the bound inhibitor compared to the optimum vacuum structure (4–6 kJ/mol). The approach improves the standard combined quantum chemical and molecular mechanics (QC/MM) approach by guaranteeing that the final structure is in accordance with experimental data (the reflections) and avoiding the risk of propagating errors in the crystal coordinates. The program can also be seen as an improvement of standard crystallographic refinement, providing an accurate empirical potential function for any group of interest. The results can be directly interpreted in standard crystallographic terms (e.g., R factors or electron density maps). The method can be used to interpret crystal structures (e.g., the protonation status of metal‐bound water molecules) and even to locally improve them. © 2002 Wiley Periodicals, Inc. J Comput Chem 23: 1058–1070, 2002
A comparison of predicted and experimental vibrational spectra in some small fluorocarbons
Authors: Jiang He , Appadoo Dominic , Robertson Evan , McNaughton Don
Published: October 2002

The vibrational assignments of difluoromethane, cis and trans 1,2‐difluoroethylene, trifluoroethylene, 2‐fluoropropene, 1,1‐difluoroethylene, and tetrafluoroethylene have been reassessed in the light of recent theoretical work. Inconsistent experimental wave number values and assignments have been clarified, and some changes to the experimental assignments are proposed. The assignments compare favorably with recent scaled density functional theory calculations using the hybrid three‐parameter B3‐PW91 density functional. © 2002 Wiley Periodicals, Inc. J Comput Chem 23: 1220–1225, 2002