bluuebunny/crossref_metadata_2025 huggingface dataset:
Development of rats' maternally directed orienting behaviors from birth to day 2
Authors: Polan H. J. , Milano D. , Eljuga L. , Hofer M. A.
Published: November 2004

We previously observed 2‐ to 3‐day‐old (P2–3) rats perform maternally directed orienting behaviors (MDOBs)—locomotion, torso flexion, rotation to the supine orientation, and snout probing, accompanied by audible vocalizations (barking)—under surfaces having properties of the dam's ventrum, which result in close contact between the pup's ventrum and the surface. We now report on the development of MDOBs from birth through P2. Wistar rats (24 litters) were tested at one of four times: 1 hr after birth before the first nursing bout (P0pre), on the day of birth after the first nursing bout (P0post), on postnatal Day 1 (P1), or on postnatal Day 2 (P2). Pups were placed prone on a warmed platform inside a corral where, for 4 min, they encountered one of two motherlike surfaces at head height: (a) a soft cotton roof or (b) a patch of the pup's own anesthetized dam's ventrum. Control pups received no overhead stimulus. The behaviors we had identified as MDOBs and others, including limb behaviors, are easily recognized at P0pre, and, in aggregate, are more frequent then than at any later age. The differential responsiveness of most of the behaviors to the dam's ventrum compared to the cotton roof or open condition is relatively low at P0pre, but rises rapidly through P2. Behavioral structure develops quickly from a rudimentary principal component with two behaviors at P0pre to two principal components incorporating seven behavior classes by P2. MDOBs are discussed in relation to late‐term fetal behaviors, early postnatal righting responses, and the oral nipple grasp, and as a model of the development of filial attachment in an altricial mammal. © 2002 Wiley Periodicals, Inc. Dev Psychobiol 40: 81–103, 2002. DOI 10.1002/dev.10015
A full belly and colostrum: Two major determinants of filial love
Authors: Val‐Laillet David , Simon Maud , Nowak Raymond
Published: November 2004

The establishment of filial bonding in sheep relies on the success of the first suckling bouts. Previous work has shown that colostrum ingestion facilitates the development of a preference for the mother. We conducted a series of experiments to investigate the influence of quantitative and qualitative factors linked to fluid ingestion. The first experiment demonstrated that the procedure was not detrimental to the mother–young interaction and did not cause any harm to the lamb. In a second experiment, lambs could interact with their dam, but were deprived of suckling during the first 12 hr after birth (The udder was covered.) and fed via a nasogastric tube in the presence of their mother on seven occasions. When tested in a two‐choice test at 12 hr of age, lambs that received either colostrum or saline (total amount = 10% birth weight) spent more time near their own dam than near an alien ewe. When smaller volumes of fluids were used (5% birth weight), lambs that received colostrum showed a preference for their mother while those ingesting saline did not. The differences persisted at 24 hr even though all the lambs had access to the maternal udder from 12 hr onward, and these differences were not related to weight variations or body temperature. In the choice test, the motor skills and vocal activity of the lambs were not affected by the treatment nor were the behaviors of the ewes. The third experiment showed that colostrum, and saline to a lesser extent, reduces the number of vocalizations emitted by lambs compared to sham‐intubated lambs, but did not otherwise influence mother–young interactions. This suggests that postingestive factors may facilitate the development of mother preference through a satiated or comforted state. This is the first evidence that depending on the amount of liquids ingested, a preferential relationship with the mother can be established via nutritional and nonnutritional signals originating from the gastrointestinal region, pointing out the major role played by the gut–brain axis in the development of filial bonding. © 2004 Wiley Periodicals, Inc. Dev Psychobiol 45: 163‐173, 2004.
Restricted innervation of uterus and placenta during pregnancy: Evidence for a role of the repelling signal Semaphorin 3A
Authors: Marzioni Daniela , Tamagnone Luca , Capparuccia Lorena , Marchini Cristina , Amici Augusto , Todros Tullia , Bischof Paul , Neidhart Sybille , Grenningloh Gabriele , Castellucci Mario
Published: November 2004

Because data from the literature suggest a lack of innervation of the placenta, we have investigated placenta, umbilical cord, and uterus to identify the molecules that play a role in regulating innervation in these organs. Neuropilin‐1 and Plexin‐A1 are cell surface proteins that form a receptor complex for Semaphorin 3A (Sema 3A), a secreted molecule mediating repelling signals for axonal growth cones. We have analyzed the expression of Neuropilin‐1, Plexin‐A1, and Semaphorin 3A in the above‐mentioned tissues on the hypothesis that these molecules could regulate innervation in these organs during gestation. We found that nervous fibers are only present in the proximal part of the umbilical cord, close to the newborn, and in nongestational uterine tissues. In contrast, nervous fibers are not present in the distal segment of the umbilical cord, in the placenta and in the uterine tissues during gestation. We also found that Sema 3A receptors, Neuropilin‐1 and Plexin‐A1, are expressed by the nervous fibers of the proximal part of the umbilical cord, whereas Sema 3A is secreted in the umbilical cord, in the placenta, and in gestational uterine tissues. We report that a factor secreted in the umbilical cord induces the collapse of neurite growth cones in vitro and provide evidence that this factor is Sema 3A. In summary, our results suggest that the chemorepulsive signals mediated by Sema 3A play an important role in preventing nerve fibers growth in the umbilical cord and in gestational uterine tissues. The inhibition of nerve growth into the myometrium as well as into the placenta could be considered fundamental processes to preserve the fetus from external stressful events. Developmental Dynamics 231:839–848, 2004. © 2004 Wiley‐Liss, Inc.
Nodal/activin signaling establishes oral–aboral polarity in the early sea urchin embryo
Authors: Flowers Vera Lynn , Courteau Girard R. , Poustka Albert J. , Weng Wei , Venuti Judith M.
Published: November 2004

Components of the Wnt signaling pathway are involved in patterning the sea urchin primary or animal–vegetal (AV) axis, but the molecular cues that pattern the secondary embryonic axis, the aboral/oral (AO) axis, are not known. In an analysis of signaling molecules that influence patterning along the sea urchin embryonic axes, we found that members of the activin subfamily of transforming growth factor‐β (TGF‐β) signaling molecules influence the establishment of AO polarities in the early embryo. Injection of activin mRNAs into fertilized eggs or treatment with exogenously applied recombinant activin altered the allocation of ectodermal fates and ventralized the embryo. The phenotypes observed resemble the ventralized phenotype previously reported for NiCl2, a known disrupter of AO patterning. Sensitivity to exogenous activin occurs between fertilization and the late blastula stage, which is also the time of highest NiCl2 sensitivity. These results argue that specification of fates along the embryonic AO axis involves TGF‐β signaling. To further examine TGF‐β signaling in these embryos, we cloned an endogenous TGF‐β from sea urchin embryos that is a member of the activin subfamily, SpNodal, and show through gain of function analysis that it recapitulates results obtained with exogenous activins and NiCl2. The expression pattern of SpNodal is consistent with a role for nodal signaling in the establishment of fates along the AO axis. Loss of function experiments using SpNodal antisense morpholinos also support a role for SpNodal in the establishment of the AO axis. Developmental Dynamics 231:727–740, 2004. © 2004 Wiley‐Liss, Inc.
PDGFR‐α signaling is critical for tooth cusp and palate morphogenesis
Authors: Xu Xun , Bringas Pablo , Soriano Philippe , Chai Yang
Published: November 2004

Platelet‐derived growth factor receptor alpha (PDGFR‐α) and PDGF ligands are key regulators for embryonic development. Although Pdgfrα is spatially expressed in the cranial neural crest (CNC)‐derived odontogenic mesenchyme, mice deficient for Pdgfrα are embryonic lethal, making it impossible to investigate the functional significance of PDGF signaling in regulating the fate of CNC cells during tooth morphogenesis. Taking advantage of the kidney capsule assay, we investigated the biological function of PDGF signaling in regulating tooth morphogenesis. Pdgfrα and Pdgfa are specifically and consistently expressed in the CNC‐derived odontogenic mesenchyme and the dental epithelium, respectively, throughout all stages of tooth development, suggesting a paracrine function of PDGF signaling in regulating tooth morphogenesis. Highly concentrated expression patterns of Pdgfrα and Pdgfa are associated with the developing dental cusp, suggesting possible functional importance of PDGF signaling in regulating cusp formation. Loss of the Pdgfrα gene does not affect proper odontoblasts proliferation and differentiation in the CNC‐derived odontogenic mesenchyme but perturbs the formation of extracellular matrix and the organization of odontoblast cells at the forming cusp area, resulting in dental cusp growth defect. Pdgfrα−/− mice have complete cleft palate. We show that the cleft palate in Pdgfrα mutant mice results from an extracellular matrix defect within the CNC‐derived palatal mesenchyme. The midline epithelium of the mutant palatal shelf remains functionally competent to mediate palatal fusion once the palatal shelves are placed in close contact in vitro. Collectively, our data suggests that PDGFRα and PDGFA are critical regulators for the continued epithelial–mesenchymal interaction during tooth and palate morphogenesis. Disruption of PDGFRα signaling disturbs the growth of dental cusp and interferes with the critical extension of palatal shelf during craniofacial development. Developmental Dynamics 232:75–84, 2005. © 2004 Wiley‐Liss, Inc.
Recursive computation‐based stereo matching and its implementation in VLSI
Authors: Miura Kiyoshi , Hariyama Masanori , Kameyama Michitaka
Published: November 2004

An important issue in the design of stereo vision, which is a necessity for robotic perception in three dimensions, is rapid determination of corresponding points in two shifted images. In this paper, we propose a method for identifying equivalent points on such images based on the computation of sums of absolute differences (SAD). Because the use of this method to identify such equivalent points can result in a rapidly increasing number of SAD computation cycles, reducing the number of these computations becomes an important issue. In this paper we propose a recursive algorithm for SAD computations that takes into account the existence of intermediate results that are common to several SAD operations. Our algorithm not only reduces the amount of computation, but also is suitable for VLSI implementation due to its regularity and ease of parallelization. Using this algorithm, we have reduced the amount of computation to 1.2% of the amount required when recursive computation is not used. In addition, we propose an allocation architecture based on scheduling that minimizes the memory capacity required for the intermediate results by minimizing the interconnections between computers. Our analysis reveals that performance levels 5000 times higher than the levels for special‐use processors are achievable. © 2004 Wiley Periodicals, Inc. Electron Comm Jpn Pt 2, 87(12): 19–27, 2004; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/ecjb.10191
The Role of Secondary Interactions in Group 11 Metal Complexes Containing the Ferrocene Ligand FcCH2NHpyMe in Supramolecular Structures
Authors: Barranco Eva M. , Crespo Olga , Gimeno M. Concepción , Jones Peter G. , Laguna Antonio
Published: November 2004

The ferrocene ligand 6‐[(ferrocenylmethyl)amino]‐2‐picoline, FcCH2NHpyMe [Fc = (η5‐C5H5)Fe(η5‐C5H4)], has been prepared by treatment of FcCH2NMe2 with 6‐amino‐2‐picoline, NH2pyMe. The coordination chemistry with group 11 metals has been studied and several complexes with coordination of the metal atom to the pyridine nitrogen atom, such as [M(PPh3)(FcCH2NHpyMe)]OTf, [Au(C6F5)(FcCH2NHpyMe)] and [AuR3(FcCH2NHpyMe)] (M = Au, Ag; R3 = (C6F5)3, (C6F5)2Cl; OTf = trifluoromethanesulfonate), have been obtained. The complex [Cu(FcCH2NHpyMe)2]PF6 has also been synthesised with a probable coordination of the copper centre to the pyridine and amine nitrogen atoms. Deprotonation of the NH group allows the synthesis of dinuclear complexes of the type [M2(PPh3)2(FcCH2NpyMe)]X or [Au2(C6F5)(PPh3)(FcCH2NpyMe)] with a mixed pyridine−amide ligand. Some of the complexes have been characterised by X‐ray diffraction and show the presence of intramolecular aurophilic interactions in the dinuclear complex and intermolecular interactions leading to chains in the mononuclear complex. Other types of secondary bonds are present in these structures, such as hydrogen bonding and weak η2 interactions between the gold centre and the cyclopentadienyl ring. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2004)
Biocatalysis for the Production of Cosmetic Ingredients
Authors: Veit T.
Published: November 2004

Biocatalysis has been proven to be a valuable and powerful tool for organic chemists, especially in the production of fine and pharmaceutical chemicals. When considering biocatalysis, terms like kinetic racemic resolution or chiral syntheses immediately come to mind. Application of enzymatic processes for the manufacture of bulk chemicals, e.g., in terms of care products, is not very widespread. Nevertheless, Degussa's Care Specialties business unit has set up a biocatalytic production process for the manufacture of special esters like cetyl ricinoleate, so‐called emollient esters, used as skin caring oils in cremes and lotions. Despite the installation of this process, biocatalysis may only be regarded as one catalysis tool among others, mainly because there are only a few off‐the‐shelf biocatalysts ready for use. The alternative, the development of proprietary biocatalysts within the chemical industry, is a long and laborious route which will definitely be followed in the future, but which for the moment remains a long term goal.
High‐Throughput‐Screening Systems for Hydrolases
Authors: Bornscheuer U.T.
Published: November 2004

In the past few years, several high‐throughput screening (HTS) formats have been developed, driven by the increasing number of biocatalysts available and the development of directed evolution methods as a novel tool in the design of enzymes. This article focusses on HTS‐methods to determine the activity and enantioselectivity of lipases and esterases. In addition, assays for amidase and racemase activity are included.
Screening for Novel Industrial Biocatalysts
Authors: Lorenz P. , Eck J.
Published: November 2004

Biocatalysis, the use of microbial cells or isolated enzymes in the production of fine chemicals, is steadily moving towards becoming accepted as an indispensable tool in the inventory of modern synthetic chemistry [1]. It is estimated that in 10 % of the cases biocatalysis will provide an overall superior synthetic strategy over traditional organic chemistry [2]. This remarkable development in a field coined “white biotechnology” is due to the growing recognition in the industry of the capabilities and performance of enzymes as exemplified in a growing number of implemented processes [3, 4], examples running at a scale of >1000 tons product/year. Breakthroughs in the key biotechnological areas of a) genetic resource access (explicitly the explorability of non‐cultivated microorganisms), b) enzyme screening and discovery and c) in vitro evolution of proteins to find and optimize enzymes to become near‐ideally suited biocatalysts have been instrumental in pushing industrial biocatalysis to where it stands today [5, 6]. With these technological options it seems that future use of biocatalysis is limited only by the availability of the biocatalyst [3], the screening for which is subject of this review.
Screening, Characterization and Application of Cyanide‐resistant Nitrile Hydratases
Authors: Gerasimova T. , Novikov A. , Osswald S. , Yanenko A.
Published: November 2004

Two new bacterial strains, Pseudomonas marginales MA32 and Pseudomonas putida MA113, containing nitrile hydratases resistant to cyanide were isolated from soil samples by an enrichment procedure. In contrast to known nitrile hydratases, which rapidly lose activity at low to moderate cyanide concentrations, the enzymes described in this paper tolerate up to 50 mM cyanide. They show a broad substrate spectrum including not only small substrates like acrylonitrile but also nitriles with longer side chains and even nitriles with quarternary alpha‐carbon atoms. Both characteristics are essential for the transformation of ketone cyanohydrins, which are much more instable and therefore releasing much higher amounts of prussic acid than cyanohydrins formed from aldehydes. P. marginales MA32 was used as a whole cell biocatalyst for the hydration of acetone cyanohydrin to α‐Hydroxyisobutyramide, which is a precursor of methacrylamide, an important pre‐polymer. After optimization of the process conditions a maximum amide concentration of more than 1.6 M could be reached within 5 hours with 5 g/L biocatalyst referred to cell dry weight.
Modulation by flavonoids of DNA damage induced by estrogen‐like compounds
Authors: Cemeli Eduardo , Schmid Thomas E. , Anderson Diana
Published: November 2004

Reactive oxygen species (ROS) are produced by a wide variety of exogenous chemicals and metabolic processes and cause a broad spectrum of damage to biological systems. As a consequence, ROS react with DNA, among many other biological targets, disrupting its structure and functionality. Estrogen‐like compounds mediate DNA damage by ROS generation, implying that their effects can be modulated by antioxidants such as catalase, superoxide dismutase, and vitamin C. We examined DNA damage in human lymphocytes and sperm after treatment with four estrogen‐like compounds (β‐estradiol, diethylstilbestrol, daidzein, and genistein) and its modulation by flavonoids (quercetin and kaempferol) using the Comet assay. The results indicated that quercetin and kaempferol reduced the DNA damage produced in sperm and lymphocytes by the four estrogenic compounds. The flavonoids also reduced the DNA damage induced by hydrogen peroxide, which was used as a positive control. Our results demonstrate that the antioxidant properties of flavonoids can protect the integrity of human sperm and lymphocyte DNA from ROS induced by estrogenic compounds. Environ. Mol. Mutagen., 2004. © 2004 Wiley‐Liss, Inc.
Non‐linear behavior of mass concrete in three‐dimensional problems using a smeared crack approach
Authors: Mirzabozorg H. , Ghaemian M.
Published: November 2004

A smeared crack approach has been proposed to model the static and dynamic behavior of mass concrete in three‐dimensional space. The proposed model simulates the tensile fracture on the mass concrete and contains pre‐softening behavior, softening initiation, fracture energy conservation and strain rate effects under dynamic loads. The validity of the proposed model has been checked using the available experimental results under static and dynamic loads. The direct and indirect displacement control algorithms have been employed under incremental increasing static loads. It was found that the proposed model gives excellent results and crack profiles when compared with the available data under static loads. The Koyna Dam in India has been used to verify the dynamic behavior of the proposed model. It was found that the resulting crack profiles were in good agreement with the available experimental results. Finally, the Morrow Point Dam was analyzed, including the dam–reservoir interaction effects, to consider its non‐linear seismic behavior. It was found that the resulting crack profiles were in good agreement with the contour of maximum principal stresses and no numerical instability occurred during the analysis. Copyright © 2004 John Wiley & Sons, Ltd.
Does the public have a role in evaluation? Surveys and democratic discourse
Authors: Henry Gary T.
Published: November 2004

Surveys can be a powerful tool for engaging the public in evaluation.
Understanding differences in people's answers to telephone and mail surveys
Authors: Dillman Don A. , Sangster Roberta L. , Tarnai John , Rockwood Todd H.
Published: November 2004

Because people sometimes answer similar questions differently in telephone and mail surveys, the increasing tendency to use both modes in survey designs makes an understanding of these patterns a pressing concern.
The design of templates as tools for formative evaluation
Authors: Loucks‐Horsley Susan
Published: November 2004

Templates are tools for program managers who wish to design, monitor, evaluate, and improve their programs in ways that reflect best practice. This chapter describes the purposes and evolution of templates, their anatomy and development process, and issues in their design.
Program managers' perspectives: Using the results of templates to manage and improve programs
Authors: Hays Irene D.
Published: November 2004

This chapter presents program managers' views on developing and using templates as a tool for evaluating U.S. Department of Energy‐sponsored education programs.
Roles for program templates in evaluation: a broader look
Authors: Ann Scheirer Mary
Published: November 2004

This chapter relates the nature and purposes of templates to several streams of current developments in evaluation practice and evaluation theory.
Integrating qualitative methods in a predominantly quantitative evaluation: A case study and some reflections
Authors: Mark Melvin M. , Feller Irwin , Button Scott B.
Published: November 2004

A review of qualitative methods used in a predominantly quantitative evaluation indicated a variety of roles for such a mixing of methods, including framing initial questions and method choices, revising evaluation questions during the inquiry, assessing the validity of measures, assessing adaptations in program implementation, and gauging the proper degree of uncertainty and generalizability of one's conclusions. These functions are considered in light of the stances on mixing paradigms that Greene and Caracelli describe in Chapter One.
Using performance measures for budgeting: A new beat, or is it the same old tune?
Authors: Joyce Philip G.
Published: November 2004

The challenges that face governments in developing performance‐based budgeting systems are described in this chapter. Performance‐based budgeting is placed in the context of past and current performance‐based management reforms.
Comments on the Guiding Principles
Authors: Chelimsky Eleanor
Published: November 2004

As useful as they are, the American Evaluation Association Guiding Principles fail to appreciate the precarious position of the evaluator who speaks truth to power, and their near total focus on elaborating further responsibilities for the evaluator may increase the evaluator's vulnerability to partisan attack.
Doing good and getting it right
Authors: Rossi Peter H.
Published: November 2004

Judging the American Evaluation Association principles to be weak statements, difficult to apply in concrete circumstances, and potentially misleading through ambiguity, the author argues for more concern with doing good research rather than getting everyone's approval.
Identifying and respecting differences among participants in evaluation studies
Authors: Mertens Donna M.
Published: November 2004

Drawing on literature from the field of ethics in research and on multicultural, feminist ethical principles, the author explores methodological implications for evaluation practice.
Coupling of Navier–Stokes equations with protein molecular dynamics and its application to hemodynamics
Authors: Liu Yaling , Zhang Lucy , Wang Xiaodong , Liu Wing Kam
Published: November 2004

The red blood cell (RBC) aggregation plays an important role in many physiological phenomena, in particular the atherosclerosis and thrombotic processes. In this research, we introduce a new modelling technique that couples Navier–Stokes equations with protein molecular dynamics to investigate the behaviours of RBC aggregates and their effects on the blood rheology. In essence, the Lagrangian solid mesh, which represents the immersed deformable cells, is set to move on top of a background Eulerian mesh. The effects of cell–cell interaction (adhesive/repulsive) and hydrodynamic forces on RBC aggregates are studied by introducing equivalent protein molecular potentials into the immersed finite element method. The aggregation of red blood cells in quiescent fluids is simulated. The de‐aggregation of a RBC cluster at different shear rates is also investigated to provide an explanation of the shear‐rate‐dependence of the blood viscoelastic properties. Finally, the influences of cell–cell interaction, RBC rigidity, and vessel geometry are addressed in a series of test cases with deformable cells (normal and sickle RBCs) passing through micro‐ and capillary vessels. Copyright © 2004 John Wiley & Sons, Ltd.
Different expression of P14ARF defines two groups of breast carcinomas in terms of TP73 expression and TP53 mutational status
Authors: Domínguez Gemma , Silva Javier , Silva Jose M. , García Jose M. , Larrondo Francisco J. , Vargas Javier , Sanfrutos Luis , Provencio Mariano , España Pilar , Bonilla Félix
Published: November 2004

In 95 breast carcinomas, we investigated P14ARF and TP73 mRNA expression and their relationship to TP53 mutations, determined by an immunohistochemical method, studying several clinicopathologic features of the tumors. P14ARF and TP73 mRNA levels were determined by semiquantitative reverse transcription polymerase chain reaction (RT‐PCR), using β‐actin as a control. P14ARF was overexpressed in 19% of the cases and underexpressed in 24%. TP73 was overexpressed in 22% of the tumors, and normal levels were found in the remaining 78%. The analysis of TP53 showed positive immunostaining in 38% of cases. The association of P14ARF and TP73 overexpression was statistically significant, as was the association between positive TP53 staining and TP73 overexpression. P14ARF was related to TP53 only in those cases in which there was low expression of P14ARF. Concomitant overexpression of P14ARF and TP73 was statistically related to positive TP53 immunostaining. The analysis of concomitant P14ARF and TP73 overexpression and clinicopathologic parameters of the tumors showed a statistically significant difference with respect to peritumoral vessel invasion (P = 0.01), lymph node metastasis (P = 0.03), negative ERBB2 expression (P = 0.005), and more advanced pathologic stages (P = 0.03). These results suggest that overexpression of P14ARF and TP73 could be implicated in breast carcinoma tumorigenesis and, ultimately, in the phenotypic features of these lesions. © 2001 Wiley‐Liss, Inc.
Identification of recurrent chromosomal rearrangements and the unique relationship between low‐level amplification and translocation in gliablastoma
Authors: Kubota Hisashi , Nishizaki Takafumi , Harada Katsumi , Harada Kei , Oga Atsunori , Ito Haruhide , Suzuki Michiyasu , Sasaki Kohsuke
Published: November 2004

To elucidate the structural abnormalities and the relationship between chromosome structural disorders and DNA copy number aberrations in tumor cells, we applied the techniques of spectral karyotyping (SKY), comparative genomic hybridization (CGH), and fluorescence in situ hybridization (FISH), using yeast artificial chromosome (YAC) probes for nine human glioblastoma cell lines. One striking finding was that independently derived cell lines had the same recurrent marker chromosomes. Seven recurrent chromosomes were detected by these cytogenetic methods. In particular, cell lines U251, SNB‐19, and U373‐MG showed very similar karyotypes. It is also interesting that regions of DNA amplification were found translocated and/or inserted at a high rate (91.7%). In all, there were 12 amplified loci in five of the nine cell lines. These amplified chromosomal bands were scattered on the chromosomes, including the normal chromosome, with one exception (7q32‐qter in U373‐MG). FISH with YAC clones mapping to these chromosomal regions as DNA probes often showed DNA probe signals not only at original chromosomal sites but also in translocated or inserted segments. This form of DNA amplification was characterized by low‐level increases (four‐ to 10‐fold) and by translocation or insertion of the relevant chromosomal locus. These studies shed light on typical derivative chromosomes and the relationship between DNA amplification and chromosomal translocation in glioblastoma. © 2001 Wiley‐Liss, Inc.
Suppression of tumorigenicity in the human prostate cancer cell line M12 via microcell‐mediated restoration of chromosome 19
Authors: Astbury Caroline , Jackson‐Cook Colleen K. , Culp Stephen H. , Paisley Thomas E. , Ware Joy L.
Published: November 2004

Previously we immortalized human, nontransformed prostate epithelial cells with SV40 large T‐antigen (SV40TAg) and derived increasingly aggressive sublines from the immortalized line. The progression of the tumorigenic sublines to metastatic capacity was accompanied by the formation of an unbalanced translocation between chromosomes 16 and 19, resulting in loss of 19p and proximal 19q. To test whether the tumorigenic and/or metastatic phenotype was causally related to this genetic alteration, we restored a neo‐tagged human chromosome 19 to M12 cells by microcell‐mediated transfer and assessed their growth. In vitro, the resultant hybrids grew more slowly in monolayer culture and showed a significant reduction in anchorage‐independent growth as compared to M12neo controls. In vivo, all mice (13/13) injected subcutaneously (SC) with control M12neo cells developed tumors after 9–15 days. In contrast, 9/15 mice injected SC with microcell‐transferred chromosome 19 hybrid cells failed to form tumors, with 6/15 producing very small tumors after 120 days. Analysis of three of these six tumors showed consistent, new chromosomal changes. Furthermore, in one of the tumors, loss of a chromosome 19 was noted in 40% of the cells. After intraprostatic injections of the hybrid cells, only 2/7 mice developed microscopic tumors, with no metastases. These data suggest the presence of a gene or genes on chromosome 19 that function to suppress growth. © 2001 Wiley‐Liss, Inc.
Liver regeneration in fulminant hepatitis as evaluated by serum transforming growth factor α levels
Authors: Tomiya T , Fujiwara K
Published: November 2004

Transforming growth factor α (TGF α) is supposed to act as a mitogen for hepatocytes in an autocrine manner in vitro and in vivo. Retarded liver regeneration is a possible reason for poor prognosis of fulminant hepatitis (FH). We analyzed serum TGF α levels in patients with FH and patients with acute nonfulminant hepatitis (AH). Also, the relation of those levels to serum hepatocyte growth factor (HGF) levels and their changes after glucagon-insulin (G-I) therapy were studied. Maximal serum TGF α levels achieved in each case after admission until recovery from disease or death were correlated positively with maximal serum alanine transaminase (ALT) and total bilirubin levels in patients with AH, but negatively with maximal total bilirubin levels in patients with FH. Maximal serum TGF α levels in patients with FH were significantly higher in survivors than in nonsurvivors. Maximal serum HGF levels were positively correlated with maximal serum TGF α levels in patients with AH, but not in patients with FH. Multiple regression analysis indicated that G-I therapy was related to the increment of serum TGF α levels in patients with FH. These results suggest that serum TGF α levels are increased in accordance with liver regeneration after necrosis in patients with AH, but such liver regeneration may be retarded, depending on the extent of liver damage in patients with FH. G-I therapy seems to stimulate liver regeneration after liver damage. The possible contribution of TGF α and HGF to liver regeneration merits consideration for recovery from AH.
Cirrhosis and muscle cramps: Evidence of a causal relationship
Authors: Angeli P , Albino G , Carraro P , Pria M D , Merkel C , Caregaro L , De Bei E , Bortoluzzi A , Plebani M , Gatta A
Published: November 2004

The aim of the study was to define the features, prevalence, and pathophysiology of therapy for muscle cramps in cirrhotic patients. The first protocol study included 294 cirrhotic patients and 194 age- and sex-matched controls. Controls were defined as inpatients or outpatients without any clinical and laboratory evidence of liver disease. Features and prevalence of muscle cramps were defined on the basis of a standard questionnaire. As far as the pathophysiological associations of muscle cramps were concerned, the following parameters were evaluated: mean arterial pressure (MAP), nutritional status, liver function tests, plasma volume (PV), plasma renin activity (PRA), and electrolyte, mineral, and acid-base status. The prevalence of cramps was higher in cirrhotic patients than in controls, and it was related to the duration of recognized cirrhosis and to the severity of liver function impairment. At a multiple regression analysis, the presence of ascites, low values of MAP, and high values of PRA were the independent predictive factors for the occurrence of cramps in cirrhosis. In the second protocol study, the effects of a sustained expansion of the effective circulating volume induced by intravenous infusion of human albumin were compared with those of a placebo in 12 cirrhotic patients with more than three cramp crises a week. Compared with the placebo, albumin reduced the cramp frequency (P < .01). In conclusion, an increased prevalence of true muscle cramps occurs in patients with cirrhosis. Our data indicate that the pathophysiological link between cirrhosis and cramps may be represented by the reduction of the effective circulating volume. They also indicate that weekly infusion of human albumin may be an effective treatment for cramps in cirrhosis.
Selective portal vein embolization with absolute ethanol induces hepatic hypertrophy and makes more extensive hepatectomy possible
Authors: Ogasawara K , Uchino J , Une Y , Fujioka Y
Published: November 2004

Portal vein embolization has been used recently to decrease the amount of the liver to be resected and to enhance the function of the remaining hypertrophied lobes. We have observed a strong contact destructivity of absolute ethanol and used it for portal vein embolization. The present study was performed to produce hepatic hypertrophy and to show histopathologic changes that follow ethanol embolization of rat liver. Hepatic proliferation and histopathology were studied in rats receiving low and high doses of absolute ethanol via portal vein and rats undergoing 70% hepatectomy alone. The liver weight of the unresected and unembolized lobes increased rapidly after embolization and hepatectomy. Although the increase was more rapid in the high-dose group than the low-dose group in early days, the final results were not different from each other and were almost equal to those after hepatectomy. Complete obstruction of portal venous branches and massive necrosis were the main histopathologic observations after portal vein embolization with all doses of ethanol. Because the mortality rate in the low-dose group was lower than in the high-dose group and extensive necrosis of the liver parenchyma and subsequent regeneration was sufficient, using minimum dose of ethanol was much safer. Based on the biochemical and hematologic parameters, portal vein embolization with low-dose ethanol did not impair liver function more than hepatectomy alone during the initial 14 days. Portal vein embolization with absolute ethanol makes more extensive hepatectomy possible by reducing the volume necessary to resect and preserves the function of the remaining liver.
Adenine arabinoside monophosphate coupled to lactosaminated human albumin administered for 4 weeks in patients with chronic type B hepatitis decreased viremia without producing significant side effects
Authors: Cerenzia M T , Fiume L , De Bernardi Venon W , Lavezzo B , Brunetto M R , Ponzetto A , Di Stefano G , Busi C , Mattioli A , Gervasi G B , Bonino F , Verme G
Published: November 2004

A conjugate of adenine arabinoside monophosphate (ara-AMP) with the liver-targeting molecule lactosaminated human serum albumin (L-HSA) was administered by intravenous infusion for 28 days to eight patients with chronic type B hepatitis. The daily dose varied among the patients, ranging from 34 mg/kg to 53 mg/kg (equal to 1.5 and 2.3 mg/kg ara-AMP, respectively). Pharmacokinetic analysis indicated that, at every dose tested, the conjugate was disposed of without accumulation. Viral DNA serum levels fell markedly during treatment; values rose again when treatment was ceased. The L-HSA-ara-AMP conjugate did not cause either the neurotoxic side effects of free ara-AMP or other adverse clinical reactions. It produced a significant increase both in serum alkaline phosphatase activity and platelet number, and a small but significant decrease in erythrocyte number. These laboratory parameters returned to normal levels within 2 months after treatment. The conjugate induced the production of small amounts of antibodies (˜ 20 pmol of conjugate bound by 1 mL of serum) in one patient only. In conclusion, the present results indicate that the L-HSA-ara-AMP conjugate can exert the antiviral activity of ara-AMP in chronic type B hepatitis patients without producing the neurotoxic side effects which hamper a 4-week period of treatment with the free drug.
Alcoholism in hereditary hemochromatosis revisited: Prevalence and clinical consequences among homozygous siblings
Authors: Adams P C , Agnew S
Published: November 2004

The relationship between alcoholism and hereditary hemochromatosis remains controversial. Previous studies have included patients with alcoholic siderosis rather than hereditary hemochromatosis. In this retrospective study, the clinical features, iron status, alcohol history, liver histology, and long-term survival were reviewed in 105 homozygotes for hemochromatosis using rigid diagnostic criteria including an HLA identical sibling with iron overload. Heavy alcohol consumption (>80 g ethanol/day) was found in 15% of hemochromatosis patients. Histological features of alcoholic liver disease (Mallory’s hyaline bodies, pericentral fibrosis, polymorphonuclear infiltrate, and fatty infiltration) were uncommon in hemochromatosis. Hemochromatosis patients with heavy alcohol consumption had a higher prevalence of cirrhosis than hemochromatosis patients without heavy alcohol consumption. Hepatic iron concentration and hepatic iron index did not significantly differ between these two hemochromatosis groups. Long-term survival was significantly reduced in patients with heavy alcohol consumption (mean follow-up, 9.22 years). This suggests that chronic alcohol consumption has an additive hepatotoxic effect despite the paucity of histological features of alcoholic liver disease.
Nitric oxide is not involved in hepatocyte killing by neutrophils activated by N -formyl-methionyl-leucyl-phenylalanine or phorbol myristate acetate in vitro
Authors: Wagner J G , Ganey P E , Roth R A
Published: November 2004

Polymorphonuclear leukocytes (PMNS) have been implicated as cellular mediators of hepatic injury in models of inflammation in vivo. In vitro, hepatocyte killing by activated PMNs is mediated in part by proteases, but the role of nitric oxide is unknown. NO is produced by PMNs and hepatocytes and can act either to damage or protect in various models of toxicity. Therefore, we tested the hypothesis that NO is important in PMN-mediated hepatocyte killing in vitro. Freshly isolated hepatocytes from rat liver and PMNs elicited from rat peritoneum were cultured together or alone for 16 hours. Both cell types spontaneously released NO, estimated as its stable breakdown product, nitrite. Accumulation of nitrite in medium from hepatocyte cultures was augmented threefold by incubation with L-arginine and was completely inhibited by treatment with the nitric oxide synthase (NOS) inhibitor NG-methyl-L-arginine (NMA). Nitrite release in PMN cultures was unaffected by L-arginine addition and only partially inhibited by NMA. In PMN: hepatocyte cocultures (10:1), accumulation of nitrite was additive relative to cells cultured separately. Incubation with NMA blocked nitrite production completely in cocultures, whereas L-arginine caused a two-fold increase in nitrite. Addition of PMN stimulants, N-formyl-methionyl-leucyl-phenylalanine (FMLP), or phorbol myristate acetate (PMA), caused increased release of alanine aminotransferase (ALT) activity into medium from hepatocytes cultured with PMNs but not from hepatocytes cultured alone; this indicated that injury to hepatocytes was due to activated PMNS. However, neither FMLP nor PMA significantly altered nitrite release from cocultures. Despite the alterations in NO production induced by addition of NMA or L-arginine, neither agent altered the release of ALT from hepatocytes in coculture with activated PMNs. Thus, PMNs and hepatocytes provided NO in vitro, but neither suppression nor elevation of NO production affected PMN-mediated hepatocyte killing. Accordingly, NO is not involved in the mechanisms by which FMLP-or PMA-stimulated PMNs mediate hepatocyte injury in vitro.
Transforming growth factor β1-induced cell death in preneoplastic foci of rat liver and sensitization by the antiestrogen tamoxifen
Authors: Mullauer L , Grasl-Kraupp B , Bursch W , Schulte-Hermann R
Published: November 2004

Previous studies have shown 5- to 10-fold higher rates of apoptosis in prestages of liver cancer (putative preneoplastic cell foci [PPF]) than in unaltered liver; fasting or withdrawal of tumor promoters enhanced apoptosis even further. We studied whether transforming growth factor β1 (TGF-β1), an inducer of apoptosis in normal liver, might be involved in induction of apoptosis in PPF. PPF were produced in 7-week- old female Sprague-Dawley rats with a single oral dose of the genotoxic carcinogen 7,12-dimethylbenz(a)anthracene (DMBA). At 24 weeks of age, TGF-β1 was injected into animals (40 micro g/kg intravenously) either once and they were killed 4 hours later (single-dose experiment) or eight times at 24-hour intervals and they were killed 24 hours after the last administration (multiple-dose experiment). Further subgroups received daily subcutaneous injections of tamoxifen (TAM) (8 mg/kg) for 4 consecutive weeks before TGF-β1 treatment. In normal liver, the apoptosis incidence was low in solvent- and TAM-only-treated animals, in the single- as well as the multiple-dose experiment. TGF-β1, increased the apoptosis incidence severalfold, and the combined administration of TGF-β1 with TAM caused a further strong increase. The already-elevated basal apoptotic incidence in PPF was further increased by TGF-β1, and particularly by TGF-β1 plus TAM treatments, which resulted in a reduction of foci number and size. In summary, these results show that TGF-β1 can induce apoptosis in PPF. This apoptosis-inducing activity is strongly enhanced by the additional treatment with the antiestrogen TAM, which by itself does not have any cell death-inducing effect in the liver or PPF. The elevated apoptotic activity of PPF in response to TGF-β1 can lead to a selective reduction of the liver load with preneoplastic cells.
Impact of essential fatty acid deficiency on hepatic sterol metabolism in rats
Authors: Levy E , Garofalo C , Rouleau T , Gavino V , Bendayan M
Published: November 2004

The major aim of the current investigation was to define whether essential fatty acid (EFA) deficiency modifies the intrahepatic metabolism and biliary output of sterols in rats. EFA-deficient diet caused an impoverishment in linoleic, arachidonic, and docosahexaenoic acids, and a marked enrichment in the eicosatrienoic acid of the plasma, liver, and hepatic microsomes. During a short term of biliary drainage, a significant decline of the pool size of biliary sterols was noted in EFA-deficient rats compared with control rats. To assess the biosynthesis of biliary components, the common bile duct was cannulated and the pool size depleted (24 hours). Subsequently, a 6-hour bile collection disclosed a significant decrease (nmoles/min/g liver) in bile acids (4.8 +/- 0.3 vs. 8.4 +/- 0.7, P < .005), cholesterol (0.26 +/- 0.01 vs. 0.34 +/- 0.02, P < .05), and phospholipids (1.49 +/- 0.11 vs. 2.82 +/- 0.32, P < .005) in EFA-deficient rats compared with controls (n = 6/group). When cholesterogenesis was measured by the incorporation of [14C]acetate and 3H20 into cholesterol, using liver slices, it was also found to be significantly (P < .001) reduced in EFA-deficient rats. The activity of hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase, the rate-limiting enzyme in cholesterol biosynthesis, was consistently lower (80 percent, P < .001) in EFA-deficient rats. In parallel experiments, the direct measurement of microsomal acyl-Co A: cholesterol acyl-transferase (ACAT) showed a decrease averaging 52 percent (P < .001). This is in striking contrast to the elevated activity (157 percent, P < .005) of cholesterol 7 alpha-hydroxylase, the rate-limiting enzyme in bile acid biosynthesis. Current experiments also suggest that the enzyme regulation involving phosphorylation and dephosphorylation is modified by EFA deficiency. Among the structural alterations observed in the morphology of hepatocytes in EFA-deficient rats, the lumen of bile canaliculi was reduced in size. These results underline the importance of EFA in the major mechanisms involved in the maintenance of hepatocyte sterol balance.
Inhibition of Hepatitis B Virus Replication by Targeted Pretreatment of Complexed Antisense Dna In Vitro
Authors: Nakazono K , Ito Y , Wu C H , Wu G Y
Published: November 2004

We have shown that antisense oligonucleotides can be targeted to hepatitis B virus (HBV)–infected cells, resulting in specific inhibition of viral protein synthesis and replication

in vitro

. The targeting system was based on the internalization of DNA complexes by highly selective receptors for galactose–terminal glycoproteins, asialoglycoproteins, on the surface of hepatocytes. Our objective in this study was to determine whether antisense DNA could be targeted to hepatocytes to prevent subsequent infection by HBV. A 21–mer phosphorothioate–linked oligo DNA complementary to the HBV polyadenylation signal and 5′–upstream sequences was complexed to a targetable DNA carrier consisting of asialoglycoprotein coupled to polylysine. Pretreatment of Huh7, asialoglycoprotein receptor (+) cells, with antisense complexes before lipofection with an HBV–plasmid at a level of 6.5 × 106 copies of plasmid per cell inhibited the amount of newly synthesized, core–associated viral DNA in Huh7 cells to undetectable levels, less than 0.1 pg, as assessed by quantitative polymerase chain reaction (PCR). Hepatitis B viral RNA transcripts were decreased by 60% compared with controls as detected by RNase protection assays, and HBV surface antigen (HBsAg) accumulation was inhibited by 97%. The inhibition lasted for 6 days and was dose dependent. Controls consisting of antisense alone and a random oligo complex showed no significant effect on any of the parameters under identical conditions. We conclude that preexposure of cells to targeted complexed antisense DNA can substantially block viral gene expression and viral replication after transfection of HBV DNA.