bluuebunny/crossref_metadata_2025 huggingface dataset:
Density functional investigation on electron‐transfer catalysis of cycloreversion of cyclobutane: Radical anion mechanism
Authors: Qu Zheng‐Wang , Zhu Hui , Zhang Xing‐Kang , Zhang Qi‐Yuan
Published: January 2003

The mechanism of cycloreversion of cyclobutane radical anion (c‐C4H) has been investigated at the UB3LYP/6‐31++G(d,p) level, and compared with those of neutral c‐C4H8 and c‐C4H radical cation. Although both c‐C4H and C2H4 are shown to be Rydberg states unstable with respect to electron ejection, the activation barrier for the “rotating” cycloreversion of c‐C4H (37.3 kcal/mol) is lower by about 25.2 kcal/mol than that of c‐C4H8, and even the intervention of tetramethylene radical anion intermediate may reduce the activation barrier for the cycloreversion of c‐C4H8 by about 8.4 kcal/mol, mainly due to stronger electron‐deficiency of intermediate biradical species than close‐shell cyclobutanes. For the cycloreversion for c‐C4H, side isomerization reaction may be efficiently prevented by the low kinetic stability of tetramethylene radical anion intermediate towards dissociation, just different from the radical cation case. Our theoretical results have suggested the possibility of electron‐attachment catalysis of the cycloreversion of some electron‐deficient substituted cyclobutanes. © 2003 Wiley Periodicals, Inc. J Comput Chem 24: 340–344, 2003
Anab initiostudy of intermolecular interactions of nitromethane dimer and nitromethane trimer
Authors: Li Jinshan , Zhao Feng , Jing Fuqian
Published: January 2003

Different geometries of nitromethane dimer and nitromethane trimer have been fully optimized employing the density functional theory B3LYP method and the 6‐31++G** basis set. Three‐body interaction energy has been obtained with theab initiosupermolecular approach at the levels of MP2/6‐31++G**//B3LYP/6‐31++G** and MP2/aug‐cc‐pVDZ//B3LYP/6‐31++G**. The internal rotation of methyl group induced by intermolecular interaction has been observed theoretically. For the optimized structures of nitromethane dimer, the strength of CH…ON H‐bond ranges from −9.0 to −12.4 kJ mol−1at the MP2/aug‐cc‐pVDZ//B3LYP/6‐31++G** level, and the B3LYP method underestimates the interaction strength compared with the MP2 method, while MP2/6‐31++G**//B3LYP/6‐31++G** calculated ΔECis within 2.5 kJ mol−1of the corresponding value at the MP4(SDTQ)/6‐31G**//B3LYP/6‐31++G** level. The analytic atom–atom intermolecular potential has been successfully regressed by using the MP2/6‐31++G**//B3LYP/6‐31++G** calculated interaction energies of nitromethane dimer. For the optimized structures of nitromethane trimer the three‐body interaction energies occupy small percentage of corresponding total binding energies, but become important for the compressed nitromethane explosive. In addition, it has been discovered that the three‐body interaction energy in the cyclic nitromethane trimer is more and more negative as intermolecular distances decrease from 2.2 to 1.7 Å. © 2003 Wiley Periodicals, Inc. J Comput Chem 24: 345–352, 2003
Ab initio study on the structures of fluorinated formates and hydrogen abstraction reaction with OH radical
Authors: Chandra Asit K. , Uchimaru Tadafumi , Sugie Masaaki , Sekiya Akira
Published: January 2003

The conformational potential energy surfaces for mono‐ and difluoromethyl formate have been determined by using a modified G2(MP2) level of calculations. The structures and vibrational frequencies for the conformers of mono‐ and difluoromethyl formate have been reported. The hydrogen abstraction reaction channels between these two formates and OH radicals have been studied at the same level of theory. Using the standard transition state theory and taking into account the effect of tunneling across the reaction barrier, we have estimated the rate constant for hydrogen abstraction by OH radical. The effect of successive fluorine substitution for methyl hydrogen on the conformational stability and on the hydrogen abstraction rate has been analyzed. © 2003 Wiley Periodicals, Inc. J Comput Chem 24: 396–407, 2003
Extended method for adiabatic mode reordering
Authors: Tautermann Christofer S. , Voegele Andreas F. , Loerting Thomas , Kaps Peter , Liedl Klaus R.
Published: January 2003

The task of vibrational mode reordering is very important for reaction valley studies and for the determination of small curvature tunneling effects. An extended algorithm for adiabatic mode reordering is presented. It is based on the method introduced by Konkoli et al. [J Comput Chem 1997, 18, 1282], which is shown to suffer from numerical problems in the region of frequency‐crossings and avoided crossings. One improvement is the use of cubic splines for the interpolation of the projected matrix of force constants, which allows larger step sizes between the discrete points along the reaction path, where vibrational analysis is performed. The main improvement is the use of perturbation theory to resolve crossings and avoided crossings. Within this theoretical framework it becomes clear why the method of the maximal overlap between the normal modes cannot work properly, as eigenvectors associated with nearby eigenvalues tend to become “wobbly”. Thus a perturbative procedure is designed that is used for all cases where two harmonic frequencies approach each other and the overlap of the associated normal modes is of no practical use. Advantages of the new procedure are the use of larger step sizes along the minimum energy path and the much more reliable resolution of mode‐crossings and avoided crossings independent of the systems symmetry. In addition to that it is shown that one should be very cautious in all computational situations when working with eigenvectors associated with nearby eigenvalues. © 2003 Wiley Periodicals, Inc. J Comput Chem 24: 386–395, 2003
A practical and efficient method to calculate AIM localization and delocalization indices at post‐HF levels of theory
Authors: Wang Yi‐Gui , Werstiuk Nick Henry
Published: January 2003

A practical and efficient method is proposed for calculating localization and delocalization indices at post‐Hartree‐Fock levels, and the method is tested at the CISD/6‐311G++(2d,2p) level for a large set of molecules. Our method, which utilizes wave functions written in the natural molecular orbital format and obtained with GAUSSIAN 94 or GAUSSIAN 98, convincingly extends the concepts established at the HF level. © 2003 Wiley Periodicals, Inc. J Comput Chem 24: 379–385, 2003
Boundary element solution of the linear Poisson–Boltzmann equation and a multipole method for the rapid calculation of forces on macromolecules in solution
Authors: Bordner A. J. , Huber G. A.
Published: January 2003

The Poisson–Boltzmann equation is widely used to describe the electrostatic potential of molecules in an ionic solution that is treated as a continuous dielectric medium. The linearized form of this equation, applicable to many biologic macromolecules, may be solved using the boundary element method. A single‐layer formulation of the boundary element method, which yields simpler integral equations than the direct formulations previously discussed in the literature, is given. It is shown that the electrostatic force and torque on a molecule may be calculated using its boundary element representation and also the polarization charge for two rigid molecules may be rapidly calculated using a noniterative scheme. An algorithm based on a fast adaptive multipole method is introduced to further increase the speed of the calculation. This method is particularly suited for Brownian dynamics or molecular dynamics simulations of large molecules, in which the electrostatic forces must be calculated for many different relative positions and orientations of the molecules. It has been implemented as a set of programs in C++, which are used to study the accuracy and speed of this method for two actin monomers. © 2003 Wiley Periodicals, Inc. J Comput Chem 24: 353–367, 2003
Coordination number of zinc ions in the phosphotriesterase active site by molecular dynamics and quantum mechanics
Authors: Koca Jaroslav , Zhan Chang‐Guo , Rittenhouse Robert C. , Ornstein Rick L.
Published: January 2003

We have run several molecular dynamics (MD) simulations on zinc‐containing phosphotriesterase (PTE) with two bound substrates, sarin and paraoxon, and with the substrate analog diethyl 4‐methylbenzylphosphonate. A standard nonbonded model was employed to treat the zinc ions with the commonly used charge of +2. In all the trajectories, we observed a tightly bound water (TBW) molecule in the active site that was coordinated to the less buried zinc ion. The phosphoryl oxygen of the substrate/inhibitor was found to be coordinated to the same zinc ion so that, considering all ligands, the less buried zinc was hexa‐coordinated. The hexa‐coordination of this zinc ion was not seen in the deposited X‐ray pdb files for PTE. Several additional MD simulations were then performed using different charges (+1, +1.5) on the zinc ions, along with ab initio and density functional theory (DFT) calculations, to evaluate the following possibilities: the crystal diffraction data were not correctly interpreted; the hexa‐coordinated zinc ion in PTE is only present in solution and not in the crystal; and the hexa‐coordinated zinc ion in PTE is an artifact of the force field used. A charge of +1.5 leads to a coordination number (CN) of 5 on both zinc ions, which is consistent with the results from ab initio and DFT calculations and with the latest high resolution X‐ray crystal structure. The commonly used charge of +2 produces a CN of 6 on the less buried zinc. The CN on the more buried zinc ion is 5 when the substrate/inhibitor is present in the simulation, and increases to 6 when the substrate/inhibitor is removed prior to the simulation. The results of both of the MD and quantum mechanical calculations lead to the conclusion that the zinc ions in the PTE active site are both penta‐coordinated, and that the MD simulations performed with the charge of +2 overestimate the CN of the zinc ions in the PTE active site. The overall protein structures in the simulations remain unaffected by the change in zinc charge from +2 to +1.5. The results also suggest that the charge +1.5 is the most appropriate for the molecular dynamics simulations on zinc‐containing PTE when a nonbonded model is used and no global thermodynamic conclusion is sought. We also show that the standard nonbonded model is not able to properly treat the CN and energy at the same time. A preliminary, promising charge‐transfer model is discussed with the use of the zinc charge of +1.5. © 2003 Wiley Periodicals, Inc. J Comput Chem 24: 368–378, 2003
INI1 expression induces cell cycle arrest and markers of senescence in malignant rhabdoid tumor cells
Authors: Reincke Britta S. , Rosson Gary B. , Oswald Betty W. , Wright Cynthia F.
Published: January 2003

The INI1 gene, which encodes a functionally uncharacterized protein component of the hSWI/SNF chromatin remodeling complex, is often mutated or deleted in malignant rhabdoid tumor (MRT). Two isoforms of INI1, that differ by the variable inclusion of nine amino acids, potentially are produced by differential RNA splicing. To determine the effect of the two INI1 isoforms on cell growth, INI1‐devoid (MRT) and INI1‐expressing cell lines were transfected separately with mammalian expression vectors or transduced with adenoviruses. Transfection of the short form of INI1 into either INI1‐deficient or expressing cell lines resulted in complete suppression of cell growth in colony formation assays. The longer splice variant induced moderate to severe growth suppression of MRT cells, but had a far milder effect on non‐MRT cells. Transduction of MRT cells with adenoviruses expressing either isoform of INI1 led to a dramatic change in morphology, growth suppression, and cell cycle arrest. Furthermore, senescence‐associated proteins were up‐regulated after transduction, while levels of proteins implicated in cell cycle progression were down‐regulated. Adenoviral delivery of INI1 into a non‐MRT cell line, however, had no demonstrable effect on any of these parameters. These results support the genetic evidence that INI1 is a tumor suppressor gene gone awry in MRT cells, and also suggest that delivery of the INI1 gene to MRT cells by adenoviruses may lead to a more effective treatment of this highly aggressive malignancy. © 2003 Wiley‐Liss, Inc.
Expression of Smad4 in the FaDu cell line partially restores TGF‐beta growth inhibition but is not sufficient to regulate fibronectin expression or suppress tumorigenicity
Authors: Hummer B. Timothy , Bartlett Christopher , Henry Erin , Weissman Bernard E.
Published: January 2003

Mutations of the Smad4 gene, a member of a group of TGF‐β signal transduction components, occur in several types of cancer suggesting that its inactivation significantly affects TGF‐β responsiveness in these tumors. To further investigate the role of Smad4 with respect to TGF‐β signaling and carcinogenesis, we re‐expressed the Smad4 gene in the Smad4‐deficient cancer cell line FaDu by microcell‐mediated chromosome transfer (MMCT) and retroviral infection to closely approximate physiological protein levels. The Smad4‐expressing FaDu clones were then evaluated for TGF‐β responsiveness to assess the role of Smad4 in TGF‐β‐induced growth inhibition and target gene regulation. We found that the re‐expression of the Smad4 gene by either method partially restored TGF‐β responsiveness in FaDu cells with respect to both growth inhibition and expression of p21WAF1/CIP1 and p15INK4B. However, only the microcell hybrids showed growth retardation in organotypic raft culture and an enhanced ability to upregulate fibronectin. In contrast, the re‐expression of Smad4 by either method failed to suppress tumorigenicity. These results suggest that in addition to a homozygous deletion of Smad4, FaDu cells contain additional defects within the TGF‐β signaling pathway, thereby limiting the extent of TGF‐β responsiveness upon Smad4 re‐expression and perhaps accounting for the inability to induce p15INK4B to a high level. They also demonstrate the advantages of providing a physiological extracellular environment, when assessing TGFβ responsiveness. © 2003 Wiley‐Liss, Inc.
Dissociation of focal adhesion kinase and paxillin tyrosine phosphorylation induced by bombesin and lysophosphatidic acid from epidermal growth factor receptor transactivation in Swiss 3T3 cells
Authors: Salazar Eduardo Perez , Hunger‐Glaser Isabel , Rozengurt Enrique
Published: January 2003

Tyrosine phosphorylation of the nonreceptor tyrosine kinase p125 focal adhesion kinase (FAK) and the adapter protein paxillin is rapidly increased by multiple agonists, including bombesin (BOM) and lysophosphatidic acid (LPA), through heptahelical G protein‐coupled receptors (GPCRs). The pathways involved remain incompletely understood. The experiments presented here were designed to test the role of epidermal growth factor receptor (EGFR) transactivation in the rapid increase of tyrosine phosphorylation of FAK and paxillin induced by GPCR agonists. Our results show that treatment with the selective EGFR tyrosine kinase inhibitor AG 1478, at concentrations that completely blocked the increase in tyrosine phosphorylation of these proteins induced by EGF, did not affect the stimulation of tyrosine phosphorylation of either FAK or paxillin induced by multiple GPCR agonists including LPA, BOM, vasopressin, bradykinin, and endothelin. Similar results were obtained when Swiss 3T3 cells were treated with another highly specific inhibitor of the EGF receptor kinase activity, PD‐158780. Collectively, our results clearly dissociate EGFR transactivation from the tyrosine phosphorylation of FAK and paxillin induced by multiple GPCR agonists. © 2002 Wiley‐Liss, Inc.
α‐Lipoic acid induces p27Kip‐dependent cell cycle arrest in non‐transformed cell lines and apoptosis in tumor cell lines
Authors: Mark Karyn van de , Chen James S. , Steliou Kosta , Perrine Susan P. , Faller Douglas V.
Published: January 2003

α‐Lipoic acid is a naturally‐occurring co‐factor found in a number of multi‐enzyme complexes regulating metabolism. We report here that α‐lipoic acid induces hyperacetylation of histones in vivo and has differential effects on the growth and viability of normal versus transformed cell lines. The human tumor cell lines FaDu and Jurkat, as well as a Ki‐v‐Ras‐transformed Balb/c‐3T3 murine mesenchymal cell line, all initiated apoptosis following exposure to α‐lipoic acid. In contrast, treatment of non‐transformed cell lines with α‐lipoic acid resulted only in reversible cell cycle arrest in G0/G1. Treatment with butyrate, another short‐chain fatty acid, induced a G0/G1 arrest in both transformed and non‐transformed cell lines. α‐Lipoic acid caused a post‐translational elevation in the levels of the cyclin‐dependent kinase inhibitor p27Kip1. Studies using p27Kip1‐deficient MEF cells demonstrated that p27Kip1 was required for the α‐lipoic acid‐mediated cell cycle arrest. The mechanism of apoptosis was independent of Fas‐mediated signaling, as α‐lipoic acid‐treated Jurkat cell mutants deficient in Fas or FADD retained sensitivity to apoptosis. The differential selectivity of the pro‐apoptotic effects of α‐lipoic acid for transformed cells supports its potential use in the treatment of neoplastic disorders. © 2003 Wiley‐Liss, Inc.
Role of CTGF/HCS24/ecogenin in skeletal growth control
Authors: Takigawa Masaharu , Nakanishi Tohru , Kubota Satoshi , Nishida Takashi
Published: January 2003

Connective tissue growth factor/hypertrophic chondrocyte‐specific gene product 24 (CTGF/Hcs24) is a multifunctional growth factor for chondrocytes, osteoblasts, and vascular endothelial cells. CTGF/Hcs24 promotes the proliferation and maturation of growth cartilage cells and articular cartilage cells in culture and hypertrophy of growth cartilage cells in culture. The factor also stimulates the proliferation and differentiation of cultured osteoblastic cells. Moreover, CTGF/Hcs24 promotes the adhesion, proliferation, and migration of vascular endothelial cells, as well as induces tube formation by the cells and strong angiogenesis in vivo. Because angiogenesis is critical for the replacement of cartilage with bone at the final stage of endochondral ossification and because gene expression of CTGF/Hcs24 predominates in hypertrophic chondrocytes in the physiological state, a major physiological role for this factor should be the promotion of the entire process of endochondral ossification, with the factor acting on the above three types of cells as a paracrine factor. Thus, CTGF/Hcs24 should be called “ecogenin: endochondral ossification genetic factor.” In addition to hypertrophic chondrocytes, osteoblasts activated by various stimuli including wounding also express a significantly high level of CTGF/Hcs24. These findings in conjunction with in vitro findings about osteoblasts mentioned above suggest the involvement of CTGF/Hcs24 in intramembranous ossification and bone modeling/remodeling. Because angiogenesis is also critical for intramembranous ossification and bone remodeling, CTGF/Hcs24 expressed in endothelial cells activated by various stimuli including wounding may also play important roles in direct bone formation. In conclusion, although the most important physiological role of CTGF/Hcs24 is ecogenin action, the factors also play important roles in skeletal growth and modeling/remodeling via its direct action on osteoblasts under both physiological and pathological conditions. © 2003 Wiley‐Liss, Inc.
HES and HERP families: Multiple effectors of the notch signaling pathway
Authors: Iso Tatsuya , Kedes Larry , Hamamori Yasuo
Published: January 2003

Notch signaling dictates cell fate and critically influences cell proliferation, differentiation, and apoptosis in metazoans. Multiple factors at each step—ligands, receptors, signal transducers and effectors—play critical roles in executing the pleiotropic effects of Notch signaling. Ligand‐binding results in proteolytic cleavage of Notch receptors to release the signal‐transducing Notch intracellular domain (NICD). NICD migrates into the nucleus and associates with the nuclear proteins of the RBP‐Jκ family (also known as CSL or CBF1/Su(H)/Lag‐1). RBP‐Jκ, when complexed with NICD, acts as a transcriptional activator, and the RBP‐Jκ‐NICD complex activates expression of primary target genes of Notch signaling such as the HES and enhancer of split [E(spl)] families. HES/E(spl) is a basic helix‐loop‐helix (bHLH) type of transcriptional repressor, and suppresses expression of downstream target genes such as tissue‐specific transcriptional activators. Thus, HES/E(spl) directly affects cell fate decisions as a primary Notch effector. HES/E(spl) had been the only known effector of Notch signaling until a recent discovery of a related but distinct bHLH protein family, termed HERP (HES‐related repressor protein, also called Hey/Hesr/HRT/CHF/gridlock). In this review, we summarize the recent data supporting the idea of HERP being a new Notch effector, and provide an overview of the similarities and differences between HES and HERP in their biochemical properties as well as their tissue distribution. One key observation derived from identification of HERP is that HES and HERP form a heterodimer and cooperate for transcriptional repression. The identification of the HERP family as a Notch effector that cooperates with HES/E(spl) family has opened a new avenue to our understanding of the Notch signaling pathway. © 2003 Wiley‐Liss, Inc.
Chemical sympathectomy‐induced changes in TH‐, VIP‐, and CGRP‐immunoreactive fibers in the rat mandible periosteum: Influence on bone resorption
Authors: Cherruau M. , Morvan F.O. , Schirar A. , Saffar J.L.
Published: January 2003

The expression of neurotransmitter receptors by bone cells supports the concept that the nervous system is a regulator of bone metabolism. The discrimination of the respective roles of the sensory and sympathetic nervous systems requires evidence of topographic relationships between the corresponding fibers and the cells involved in bone turnover, in vivo. In this study, the influence of the sympathetic system on bone resorption was assessed by using a synchronized model of cortical resorption along the mandible. The sympathetic system was destroyed by daily injections of guanethidine (40 mg/kg) for 25 days; a resorption wave was induced on day 21. The distribution of periosteal tyrosine‐hydroxylase (TH)‐, vasoactive intestinal polypeptide (VIP)‐, and calcitonin gene‐related peptide (CGRP)‐immunoreactive (IR) fibers was studied by compartmentalizing the periosteum. Most fibers were located in the distal, non‐osteogenic compartment. TH‐IR fibers were located perivascularly, VIP‐IR fibers were gathered at the boundary with the osteogenic compartment, and CGRP‐IR fibers were scattered. Sympathectomy decreased the number of TH‐ and VIP‐IR fibers and increased the number of CGRP‐IR fibers, without changing their topography. After the injection of Fast blue, a retrograde fluorescent marker, over the periosteum, fluorescent neuronal cell bodies were found in the superior cervical ganglion (SCG). Many neurons were TH‐IR and very few were VIP‐IR. Sympathectomy decreased the numbers of fluorescent and TH‐IR cell bodies. It also decreased the number of preosteoclasts and osteoclasts, which had a drastic effect on the cortical bone surface, as assessed by scanning electron microscopy. These data indicate that VIP‐IR fibers have a strategic position close to the most peripheral and less differentiated, osteogenic cells, pointing to a functional relationship. As poorly differentiated osteogenic cells support preosteoclast differentiation, VIP‐IR fibers may be involved in this process, as suggested by the smaller number of preosteoclasts in sympathectomized rats. Although VIP is predominantly a parasympathetic mediator, it seemed to be conveyed by sympathetic fibers, as shown by the marked effect of guanethidine treatment. Nevertheless, these fibers did not originate from the SCG, contrary to TH‐IR fibers. © 2003 Wiley‐Liss, Inc.
Role of PP2A in the regulation of p38 MAPK activation in bovine aortic endothelial cells exposed to cyclic strain
Authors: Lee Taeseung , Kim Sang Joon , Sumpio Bauer E.
Published: January 2003

We have previously reported that cyclic strain results in rapid phosphorylation of p38 mitogen activated protein kinase (MAPKs). The aim of this study was to examine the role of protein phosphatase type 2A (PP2A) in regulating p38 MAPK activation in bovine aortic endothelial cells exposed to cyclic strain. In this study, we demonstrate that the catalytic subunit of PP2A is tyrosine phosphorylated by cyclic strain, resulting in inhibition of phosphatase activity. Okadaic acid, an inhibitor of PP2A at lower concentrations increased phosphorylation of p‐38. Phospho‐p38 MAPK physically associated with the catalytic subunit, PP2Ac. Phospho‐p38 MAPK was dephosphorylated by purified PP2Ac in cell lysates, but if pretreated with okadaic acid, phospho‐p38 MAPK was maintained. Taken together, our result suggests that PP2A plays a regulatory role in p38 MAPK activation in endothelial cells exposed to cyclic strain. J. Cell. Physiol. 194: 349–355, 2003. © 2003 Wiley‐Liss, Inc.
Pathophysiological implications of stroma pattern formation in uveal melanoma
Authors: Clarijs Ruud , Ruiter Dirk J. , de Waal Robert M.W.
Published: January 2003

Clinical outcome of cancer patients is mainly determined by the rate of metastasis and, also by primary tumor growth. Formation of extracellular matrix and interactions of neoplastic and non‐neoplastic (host) cells in solid tumors have been shown to be essential for these processes. One result of such interactions is the outgrowth of new blood vessels from existing ones, angiogenesis, to provide the tumor tissue with oxygen and nutrients. It is assumed that the neovascular bed also facilitates the escape of metastatic cells from the primary lesions. In addition, recent reports suggested the existence of blood‐conducting channels lined by melanoma cells (so‐called “vascular channels”) accompanied by depositions of extracellular matrix patterns in cutaneous and uveal melanoma. Since the presence of these matrix structures has been negatively associated with prognosis, we hypothesize that they play a role in melanoma outgrowth or metastasis. In this review, we will discuss the morphological and functional properties of the extracellular matrix patterns in that may underlie these clinical phenomena. © 2003 Wiley‐Liss, Inc.
Cancer/testis‐associated genes: Identification, expression profile, and putative function
Authors: Zendman Albert J.W. , Ruiter Dirk J. , Van Muijen Goos N.P.
Published: January 2003

Cancer/testis‐associated genes (CTAs) are a subgroup of tumor antigens with a restricted expression in testis and malignancies. During the last decade, many of these immunotherapy candidate genes have been discovered using various approaches. Most of these genes are localized on the X‐chromosome, often as multigene families. Methylation status seems to be the main, but not the only regulator of their specific expression pattern. In testis, CTAs are exclusively present in cells of the germ cell lineage, though there is a lot of variation in the moment of expression during different stages of sperm development. Likewise, there is also a lot of heterogeneity in the expression of CTAs in melanoma samples. Clues regarding functionality of CTAs for many of these proteins point to a role in cell cycle regulation or transcriptional control. Better insights in the function of these genes may shed light on the link between spermatogenesis and tumor growth and could be of use in anti‐tumor therapies. This review outlines the CTA family and focuses on their expression and putative function during male germ cell development and melanocytic tumor progression. © 2003 Wiley‐Liss, Inc.
HPV16 E6 and E7 oncoproteins regulate Notch‐1 expression and cooperate to induce transformation
Authors: Weijzen Sanne , Zlobin Andrei , Braid Mike , Miele Lucio , Kast W. Martin
Published: January 2003

Notch receptor signaling has been implicated in cellular transformation. Notch‐1 receptor expression is increased during the progression from cervical intraepithelial lesions (CIN) to invasive cervical carcinoma. Moreover, the main cellular localization of Notch‐1 protein changes from cytoplasmic to nuclear with the transition from CIN III to microinvasive carcinoma. Since the E6 and E7 proteins encoded by human papilloma virus (HPV) are a causative agent of cervical carcinoma, this study determined whether E6 and E7 protein expression causes the observed upregulation in Notch‐1 expression. Mouse and human primary cell lines were transfected with HPV16 E6 and E7 and Notch‐1 expression and activity were analyzed. We show that Notch‐1 expression and activity are upregulated by E6 and E7 independently. This was due to both transcriptional and post‐transcriptional mechanisms. A protein involved in Notch processing, Presenilin‐1 (PS‐1), was also upregulated by E6 and E7. In the presence of E6 and E7, Notch‐1 protein expression is localized in the cytoplasm. Downregulation of Notch‐1 expression in a human cervical carcinoma cell line expressing E6/E7 caused striking inhibition of proliferation in vitro and tumorigenicity in vivo. These data suggest that E6‐ and E7‐mediated upregulation of Notch signaling may contribute to disruption of regular cell growth in cervical cancer. © 2003 Wiley‐Liss, Inc.
Cl− channel blockers inhibit transition of quiescent (G0) fibroblasts into the cell cycle
Authors: Zheng Ya‐Juan , Furukawa Tetsushi , Tajimi Kimitaka , Inagaki Nobuya
Published: January 2003

Modulation of ion permeability during the cell cycle is one of the key events in cell cycle progression. We have compared the effects of K+ and Cl− channel blockers on the cell cycle in synchronous and asynchronous NIH3T3 cells. The Cl− channel blocker 5‐N‐2‐(3‐phenylpropylamino) benzoic acid (NPPB; 0.2 mM) inhibited entry into S phase in synchronous cells but not in asynchronous cells, while the K+ channel blocker 4‐aminopyridine (4‐AP) showed similar inhibitory effects in both conditions. In NIH3T3 cells synchronized by serum deprivation/replenishment, G0‐to‐G1 transition occurred within 8 h after serum addition, and the G1/S checkpoint at 10–14 h. NPPB applied only at 0–8 or 8–14 h after serum addition inhibited entry into S phase. Cl− permeability measured as 125I efflux increased at 4 and 10 h after serum addition. Ki‐67‐negative cells, which represent quiescent G0 phase cells, progressively decreased in number until 8 h after serum addition. The Cl− channel blockers (NPPB and 4,4′‐diisothiocyanatostilbene‐2,2′‐disulfonic acid [DIDS]) but not the K+ channel blocker (4‐AP) significantly decreased the rate of reduction in number of Ki‐67‐negative cells. These data indicate that an increase in Cl− permeability plays an important role in reentry of quiescent cells into the proliferating phase, in addition to the known effects on passage through the G1/S checkpoint. © 2003 Wiley‐Liss, Inc.
Subcellular localization of Photofrin® determines the death phenotype of human epidermoid carcinoma A431 cells triggered by photodynamic therapy: When plasma membranes are the main targets
Authors: Hsieh Ya‐Ju , Wu Chih‐Ching , Chang Cheng‐Jen , Yu Jau‐Song
Published: January 2003

Photodynamic therapy (PDT) is a kind of photochemo‐therapeutic treatment that exerts its effect mainly through the induction of cell death. Distinct types of cell death may be elicited by different PDT regimes. In this study, the mechanisms involved in the death of human epidermoid carcinoma A431 cells triggered by PDT with Photofrin® (a clinically approved photosensitizer) were characterized. Photofrin distributes dynamically in A431 cells; the plasma membranes and Golgi complex are the main target sites of Photofrin after a brief (3 h) and prolonged (24 h) incubation, respectively. Cells with differentially localized Photofrin displayed distinct death phenotypes in response to PDT. The effects of PDT on cells with plasma membrane‐localized Photofrin were further studied in details. Cells stopped proliferating post PDT at Photofrin dose >7 μg/ml, and at higher dose (28 μg/ml) plasma membrane disruption and cell swelling were observed immediately after PDT. Dramatic alterations of several important signaling events were detected in A431 cells post Photofrin®‐PDT, including (i) immediate formation of reactive oxygen species (ROS), (ii) rapid activation of c‐Jun N‐terminal kinase, (iii) delayed activation of caspase‐3 and cleavage of polyADP‐ribose polymerase and p21‐activated kinase 2, and (iv) loss of mitochondrial membrane potential. Intriguingly, the characteristics of typical apoptosis such as phosphatidylserine externalization and DNA fragmentation were not detected in the cell death process caused by this PDT regime. In conclusion, our results show that when plasma membranes are the main targets, Photofrin‐PDT can lead to instant ROS formation and subsequent activation of downstream signaling events similar to those elicited by many apoptotic stimuli, but the damage of plasma membranes renders the death phenotype more necrosis like. © 2003 Wiley‐Liss, Inc.
Structural and functional aspects of Bufo americanus spermatozoa: Effects of inactivation and reactivation
Authors: Kouba Andrew J. , Vance Carrie K. , Frommeyer Mark A. , Roth Terri L.
Published: January 2003

Very little is known about the effects of manipulating toad sperm activity in vitro, and such information is important in the development of a genetic resource bank for bufonid species. The specific objectives of this study were to: 1) identify the optimal inactivation and reactivation solutions for toad spermatozoa colleted in urine; 2) establish the length of time toad spermatozoa can be exposed to an inactivation buffer and still resume motility upon reactivation; 3) evaluate the consequence of inactivation on specific sperm characteristics; and 4) characterize the sperm mitochondria vesicle (MV) and its relationship to motility. Reactivated sperm motility was similar after inactivation in either Simplified Amphibian Ringers (SAR) solution or DeBoer's (DB) solution. Diluting the buffer by 80% with water provided the best method for reactivating sperm. Dilutions with NaCl solutions (10–50 mM) produced inferior results. SAR‐inactivated spermatozoa could remain suspended up to 4 hr and still regain 25% of initial motility upon reactivation in water. Compared to the controls, sperm motility was greater (P<0.01) over time for samples treated with SAR, although forward progression was significantly lower. Furthermore, SAR treatment resulted in sperm samples with a greater number of viable, morphologically normal, and intact MVs over time. Electron microscopy and fluorescent staining confirmed that the toad sperm's MV contains a large number of active mitochondria with very few other cytoplasmic structures. Nearly all spermatozoa exhibiting motility had an intact MV, and dissociation of this structure was clearly related to motility loss. In conclusion, toad spermatozoa can be effectively inactivated and reactivated by varying the osmolality of the external solutions and, although sperm forward progression is reduced, all other characteristics are well maintained. Moreover, the increased number of spermatozoa with intact MV after inactivation suggests the process may help preserve this important structure. J. Exp. Zool. 295A:172–182, 2003. © 2003 Wiley‐Liss, Inc.
Damage to the gills, skin and other tissues by lysenin and the coelomic fluid of the earthworm Eisenia foetida in two teleosts, Tanichthys albonubes and Oreochromis mossambicus
Authors: Ohta Naoshi , Aizu Masako , Kaneko Toyoji , Sato Tetsuji , Kobayashi Hideshi
Published: January 2003

Lysenin is a 33‐kDa protein found in the coelomic fluid (CF) of the earthworm Eisenia foetida. Purified lysenin binds specifically to sphingomyelin (SM). In the present studies, we found that the white cloud mountain minnow Tanichthys albonubes and the Mozambique tilapia Oreochromis mossambicus died in solutions of lysenin (at concentrations above 2.5 µg/ml) and CF (0.6%, v/v) within 2 h. The gills of both species of fish were damaged similarly by lysenin and by CF. Most gill lamellae became irregularly bent or curled, with swelling of the epithelial cells of the lamellae. Red blood cells in the lamellar vascular sinuses, in the central venous sinuses, and in the blood vessels of the entire body became swollen and lysed, choking the sinuses. Epithelial cells in the skin were also damaged. When fish of both species were treated with lysenin or CF that had been incubated with SM‐liposomes, they did not die. Their behavior remained normal and there was no damage to any cells or tissues. These findings suggest that SM might be involved in the lethal effects of lysenin and CF. It is likely that purified lysenin and lysenin in CF bound to SM in the cell membranes of the tissues mentioned above, damaging the cells. The presence of SM in the gills and skin was confirmed, supporting this hypothesis. The damage to gills and hemolysis might have resulted in lethal respiratory problems. Damage to the skin might disturb the exchange of ions through the skin, hastening death. Damage by lysenin and CF to epithelial cells of the cornea and the wall of the oral cavity was also recognized, but there was no such damage to the intestine. J. Exp. Zool. 295A:117–126, 2003. © 2003, Wiley‐Liss, Inc.
Storage of red deer epididymides for four days at 5°C: Effects on sperm motility, viability, and morphological integrity
Authors: Soler Ana J. , Pérez‐guzmán Maria D. , Garde José J.
Published: January 2003

The purpose of this study was to assess the sperm motility, the plasma membrane integrity and the morphology of red deer spermatozoa when maintained within epididymides stored for 4 days at 5°C, and to evaluate whether such stored spermatozoa are able to withstand a refrigeration process. Thirty pairs of testes, with attached epididymides, were collected from 30 hunter‐killed mature stags (Cervus elaphus hispanicus), and spermatozoa from each one of the pairs were immediately collected in Triladyl™ medium, evaluated and refrigerated (Control Group). The remaining testes and epididymides were gradually cooled to 5°C and stored for 1, 2, 3, and 4 days (Experimental Groups), after which spermatozoa were processed as described previously for the control group. The effects on spermatozoa that had been stored within epididymides for various times were determined by assaying sperm motility index (SMI), plasma membrane integrity and sperm morphology (SM). In the same way, SMI and SM were assessed after spermatozoa refrigeration at 5°C for 3 hours in different groups (SMI‐R, SM‐R). There was no significant decrease in plasma membrane integrity of spermatozoa recovered from epididymides stored at 5°C for 4 days. Similarly, the percentage of morphologically normal spermatozoa remained unaffected during the first 3 days of storage. In contrast, during storage sperm motility evaluation revealed significantly (P<0.05) lower SMI values for samples from epididymides stored 2, 3, and 4 days (47.7±3.6, 45.5±4.4, 44.1±5.2) than that of the control group (57.6±1.6). Similar results were obtained after refrigeration of spermatozoa in Triladyl at 5°C. These data suggest that it might be possible to recover functional spermatozoa from red deer epididymides stored at 5°C during several days when epididymal spermatozoa cannot be collected and cryopreserved immediately. J. Exp. Zool. 295A:188–199, 2003. © 2003, Wiley‐Liss, Inc.
Comparisons of calcium regulation in fish larvae
Authors: Chen Yi‐yen , Lu Fu‐i , Hwang Pung‐pung
Published: January 2003

The purpose of the present study was to compare the ability of larvae of different species, goldfish (Carassius auratus), zebrafish (Danio rerio), and ayu (Plecoglossus altivelis), to regulate their calcium balance. Whole‐body Ca2+ content and Ca2+ influx in the larvae of the three species, which were incubated in low‐ (0.02 mM), mid‐ (0.2 mM), and high‐ (2.0 mM) Ca2+ artificial fresh water from embryonic stages, were compared. The Ca2+ uptake kinetics were determined in zebrafish and goldfish incubated in high‐ or low‐Ca2+ artificial fresh water. Ca2+ content of both zebrafish and ayu acclimated to low‐Ca2+ media were significantly lower than those acclimated to mid‐ or high‐Ca2+ media. However, Ca2+ contents of goldfish in low‐, mid‐, and high‐Ca2+ groups showed no significant differences. In goldfish, Ca2+ influx in the low‐Ca2+ group was significantly higher than those of the mid‐ and high‐Ca2+ groups. In contrast, the Ca2+ influx rate in the low‐Ca2+ group was significantly lower than those in the mid‐ and high‐Ca2+ groups in zebrafish and ayu. Compared to the high‐Ca2+ group, the low‐Ca2+ group of goldfish showed a 13% increase in the maximal velocity (Jmax) and an 84% decrease in the Michaelis constant (Km) for Ca2+ influx. Smaller changes, i.e., an 8% increase in Jmax and a 67% decrease in Km, were found in zebrafish larvae. Goldfish possess a more effective Ca2+ regulatory capacity than do zebrafish and ayu. Differences in the strategies for Ca2+ balance may be associated with different development patterns and environments in which these fish naturally occur. J. Exp. Zool. 295A:127–135, 2003. © 2003 Wiley‐Liss, Inc.
Molting in the Djungarian hamster (Phodopus sungorus Pallas): Seasonal or continuous process?
Authors: Kuhlmann Michael T. , Clemen Günter , Schlatt Stefan
Published: January 2003

After transfer into a short daylight regimen, the brownish summer pelage of the Djungarian hamster (Phodopus sungorus) changes into the whitish winter phenotype. Although changes in serum prolactin levels are identified as the initiating hormonal signal, morphological data about molting in that species are sparse. The aim of this study was to characterize in detail the summer and winter pelage of the Djungarian hamster and to analyze the alterations in the skin and pelage induced by photoperiodic changes. The main difference between summer and winter hair types is the pattern of pigmentation. In contrast to other mammalian species showing seasonal changes, the winter coat of the Djungarian hamster is not characterized by an increase in hair density. Molting patches were observed at all times, even in the winter coat, showing that the light regimen does not control the process of molting itself but the pattern of pigmentation and eventually the loss of hair during the single molting wave. J. Exp. Zool. 295A:160–171, 2003. © 2003 Wiley‐Liss, Inc.
Distribution and characteristics of D‐amino acid and D‐aspartate oxidases in fish tissues
Authors: Sarower Mohammed Golam , Matsui Takashi , Abe Hiroki
Published: January 2003

The distributions of D‐amino acid oxidase (D‐AAO, EC 1.4.3.3) and D‐aspartate oxidase (D‐AspO, EC 1.4.3.1) activities were examined on several tissues of various fish species. Both enzyme activities were commonly high in kidney and liver and low in intestine with some exceptions. After oral administration of D‐alanine at 5 μmol /g body weight−1day−1 to carp for 30 days, D‐AAO activity increased by about 8‐, 3‐, and 1.5‐fold in intestine, hepatopancreas, and kidney, respectively, whereas no increase was found in brain. In contrast, oral administration of D‐glutamate or D‐aspartate did not show any increase of D‐AspO activity in any tissues. D‐AAO and D‐AspO of common carp kidney and hepatopancreas were subcellularly localized in peroxisomes, as clarified in mammals. D‐proline was the best substrate for D‐AAO in rainbow trout kidney, common carp kidney, and hepatopancreas, followed by D‐alanine and D‐phenylalanine. N‐methyl‐D‐aspartate was the best substrate for D‐AspO in rainbow trout kidney and common carp hepatopancreas. The optimal pH for D‐AAO in rainbow trout kidney was broad, from 7.4 to 8.2, and that for D‐AspO was around 10. D‐AAO was inhibited by benzoate known as D‐AAO inhibitor and D‐AspO was strongly inhibited by meso‐tartarate as D‐AspO inhibitor. From these results, at least D‐AAO in fish is considered to work as a metabolizing agent of exogenous and endogenous free D‐alanine that is abundant in aquatic invertebrates such as crustaceans and bivalve mollusks, which are potential food sources of these fishes. J. Exp. Zool. 295A:151–159, 2003. © 2003 Wiley‐Liss, Inc.
Lipids as energy source during salinity acclimation in the euryhaline crab Chasmagnathus granulata dana, 1851 (crustacea‐grapsidae)
Authors: Luvizotto‐santos Ricardo , Lee James tony , Branco Zelionara pereira , Bianchini Adalto , Nery Luiz eduardo maia
Published: January 2003

Lipids seem to be the major energy store in crustaceans. Moreover, they are extremely important in maintaining structural and physiological integrity of cellular and sub cellular membranes. During salinity adaptation, energy‐demanding mechanisms for hemolymph osmotic and ionic regulation are activated. Thus, the main goal of this work was to verify the possible involvement of lipids as an energy source in the osmotic adaptation process. The estuarine crab Chasmagnathus granulata was captured and acclimated to salt water at 20‰ salinity and 20 ± 2°C, for 30 days. After acclimation, crabs were divided into groups of ten and transferred to fresh water (0‰), salt water at 40‰ salinity, or maintained in salt water at 20‰ salinity (control group), without feeding. Before and seven days after the salinity change, wet weight and lipid concentration in gills, muscle, hepatopancreas, and hemolymph were determined according to the colorimetric assay of sulphophosphovanilin. Results show that hepatopancreas lipids were not mobilized during osmotic stress regulation. Gill and muscle lipids were significantly lower in crabs subjected to hypo‐osmotic stress than those subjected to the hyper‐osmotic stress or maintained at the control salinity. Our results point to the occurrence of lipid mobilization and involvement of these compounds in the osmotic acclimation process in C. granulata, but with differences between tissues and the osmotic shock (hypo or hyper) considered. J. Exp. Zool. 295A:200‐205, 2003. © 2003 Wiley‐Liss, Inc.
Prenatal sex ratios influence sexual dimorphism in a reptile
Authors: Uller Tobias , Olsson Mats
Published: January 2003

The prenatal environment influences offspring traits in a variety of ways and in a wide range of taxa. For example, maternal allocation of steroids to the eggs influences offspring traits in birds, and in some mammals the intrauterine position influences morphological, behavioural, and physiological traits due to sex‐related steroid transfer between sibling fetuses. We show that similar phenomena occur in the common lizard (Lacerta vivipara), a viviparous reptile. Females developing in male‐biased clutches had a more masculine allometry (relatively larger heads) at parturition than females developing in female‐biased clutches. Males were correspondingly feminized in female‐biased clutches. The effects could either be due to diffusion of steroids produced by the offspring or by a general tendency for females to allocate steroids according to the sex ratio of her clutch. Subsequent to parturition, the sexes differed in their growth trajectories depending on sex ratio environment. In males, the difference in allometry between sex ratio environments remained over time, whereas in females the corresponding effect disappeared. J. Exp. Zool. 295A:183–187, 2003. © 2003 Wiley‐Liss, Inc.