bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of endothelial cell gap formation and barrier dysfunction: Role of myosin light chain phosphorylation
Authors: Garcia Joe G. N. , Davis Harold W. , Patterson Carolyn E.
Published: February 2005

Endothelial cell (EC) contraction results in intercellular gap formation and loss of the selective vascular barrier to circulating macromolecules. We tested the hypothesis that phosphorylation of regulatory myosin light chains (MLC) by Ca2+/calmodulin‐dependent myosin light chain kinase (MLCK) is critical to EC barrier dysfunction elicited by thrombin. Thrombin stimulated a rapid (<15 sec) increase in [Ca2+]i which preceded maximal MLC phosphorylation (60 sec) with a 6 to 8‐fold increase above constitutive levels of phosphorylated MLC. Dramatic cellular shape changes indicative of contraction and gap formation were observed at 5 min with maximal increases in albumin permeability occurring by 10 min. Neither the Ca2+ ionophore, A23187, nor phorbol myristate acetate (PMA), a direct activator of protein kinase C (PKC), alone or in combination, produced MLC phosphorylation. The combination was synergistic, however, in stimulating EC contraction/gap formation and barrier dysfunction (3 to 4‐fold increase). Down‐regulation or inhibition of PKC activity attenuated thrombin‐induced MLC phosphorylation (∼40% inhibition) and both thrombin‐ and PMA‐induced albumin clearance (∼50% inhibition). Agents which augmented [cAMP]i partially blocked thrombin‐induced MLC phosphorylation (∼50%) and completely inhibited both thrombin‐ and PMA‐induced EC permeability (100% inhibition). Furthermore, cAMP produced significant reduction in the basal levels of constitutive MLC phosphorylation. Finally, MLCK inhibition (with either ML‐7 or KT 5926) or Ca2+/calmodulin antagonism (with either trifluoperazine or W‐7) attenuated thrombin‐induced MLC phosphorylation and barrier dysfunction. These results suggest a model wherein EC contractile events, gap formation and barrier dysfunction occur via MLCK‐dependent and independent mechanisms and are significantly modulated by both PKC and cAMP‐dependent protein kinase A activities. © 1995 Wiley‐Liss, Inc.
Differential regulation of integrin‐mediated proplatelet formation and megakaryocyte spreading
Authors: Leven Robert M.
Published: February 2005

Guinea pig bone marrow megakaryocytes were cultured on a type I rat tail collagen gel which stimulated proplatelet formation. Proplatelet formation was inhibited by monoclonal antibody LM609 to the alphav beta3 integrin (VnR), but not by monoclonal antibodies to the alpha5, alpha6, beta1, or IIb beta3(GPIIb–IIIa) integrin proteins. Megakaryocytes cultured on a plastic surface and stimulated with thrombin undergo a spreading and an adhesion reaction. This reaction is blocked in a dose‐dependent manner by the tetrapeptide RGDS and by the monoclonal antibody PG2 to the GPIIb–IIIa integrin, but not by the monoclonal antibody LM609 to the VnR. Immunoprecipitation and affinity chromatography experiments demonstrate that guinea pig megakaryocytes have distinct GPIIb–IIIa and VnR integrins with similar electrophoretic mobility. Spreading was significantly inhibited in a dose‐dependent fashion by drugs which elevate cellular cyclic AMP, including forskolin, dibutyryl cAMP, and isobutylmethylxanthine. In contrast to spreading, megakaryocyte proplatelet formation was stimulated by these agents in a dose‐dependent manner. Megakaryocyte spreading was stimulated by the protein kinase C (PKC) activator phorbol myristate acetate (PMA) and inhibited by the PKC inhibitors Calphostin C and K5720 in a dose‐dependent manner. PKC inhibitors did not inhibit megakaryocyte proplatelet formation. These results demonstrate that the closely related VnR and GPIIb‐IIIa integrins regulate different aspects of megakaryocyte morphological change and appear to be associated with different second messenger systems. © 1995 Wiley‐Liss, Inc.
Negative regulation of a special, double AP‐1 consensus element in the vimentin promoter: Interference by the retinoic acid receptor
Authors: Van De Klundert Francy A. J. M. , Jansen Hans J. , Bloemendal Hans
Published: February 2005

The growth‐regulated vimentin gene contains a functional double AP‐1 binding site formed by two nearly perfect inverted repeats. We present evidence for down‐regulation of vimentin expression by the retinoic acid receptor (RAR) in two mesodermally derived cell types. By mutation analysis we prove that the double consensus element is responsible for this negative regulation. From in vitro protein‐DNA interaction studies we conclude that AP‐1 binding is inhibited at RAR amounts required for occupation of the cognate RAR binding site in nuclear extracts from 3T3 cells and differentiated embryonal carcinoma cells. Furthermore, we show that, unlike in other cases, trans‐activation of the vimentin AP‐1 enhancer element can occur in undifferentiated embryonal carcinoma cells, despite the low amount of Jun and Fos proteins present in these cells. Here, however, down‐regulation by retinoic acid cannot be detected. © 1995 Wiley‐Liss, Inc.
Perturbation of the platelet‐derived growth factor receptor signaling by dibutyryl‐camp in human astrocytoma cells
Authors: Tsai Chon‐Haw , Hung Li‐Man , Chen Jan‐Kan
Published: February 2005

It has been shown that cAMP may perturb the polypeptide growth factor‐induced nuclear events. However, the possible interactions of the cAMP‐protein kinase A (cAMP‐PKA) and receptor tyrosine kinase pathways in the cytosol have not been fully elucidated. In this study, we use human astrocytoma cells as a model to investigate this issue. The results show that platelet‐derived growth factor (PDGF)‐induced receptor autophosphorylation in human astrocytoma cells is suppressed by dibutyryl‐cAMP pretreatment and such suppression is not due to changes in the ligand‐receptor binding properties. Further studies show that PDGF‐induced tyrosine phosphorylation of phospholipase C_γ1 (PLC_γ1) and phosphatidylinositol 3‐kinase (PI 3‐kinase) are also suppressed in dibutyryl‐cAMP‐pretreated cells. The suppression of PLC_γ1 tyrosine phosphorylation was accompanied by a decreased production of water soluble inositol phosphates. In contrast, similar treatment with normal human astrocytes potentiates the tyrosine phosphorylation of PLC_γ1 and PI 3‐kinase. The results indicate that cAMP can either negatively or positively modulate the PDGF receptor tyrosine kinase activity depending on the cell types examined. © 1995 Wiley‐Liss, Inc.
Thermotolerance and nuclear protein aggregation: Protection against initial damage or better recovery?
Authors: Stege G. J. J. , Brunsting J. F. , Kampinga H. H. , Konings A. W. T.
Published: February 2005

Heat‐induced nuclear protein aggregation and subsequent disaggregation were measured in nonpreheated and preheated (thermotolerant) HeLa S3 cells. The effect of thermotolerance on the formation of and recovery from heat‐induced nuclear protein aggregates was related to the cellular levels of hsp27, hsp60, hsp70, hsc70, and hsp90. Cells heated at different time points after the thermotolerance trigger showed various levels of protection against heat‐induced nuclear protein aggregation. This protection, however, did not parallel the development and decay of thermotolerance on cell survival. The protection was maximal when the thermotolerance level already had started to decay. The level of protection against nuclear protein aggregation did however parallel the cellular level of hsp70 indicating that hsp70 may be involved in this process. At all stages during the development and decay, thermotolerant cells showed a more rapid recovery (disaggregation) from the heat‐induced nuclear protein aggregates than non‐thermotolerant cells. The rates of disaggregation during development and decay of thermotolerance paralleled the cellular levels of hsp27 suggesting that hsp27 is somehow involved in this recovery process from heat‐induced nuclear protein aggregates. The total cellular levels of none of the individual hsp's completely correlate with development and decay of thermotolerance, indicating that overexpression of any of these hsp's alone does not determine the level of thermotolerance. Clonogenic cell survival paralleled the rates of disaggregation, leading to the notion that recovery processes are the most important determinant for the thermotolerant state of HeLa S3 cells. The best corelation with clonogenic survival was found when both initial aggregation and subsequent disaggregation were taken into account, suggesting that the combined action of various hsp's in these two processes have to be included in thermotolerance development and decay. © 1995 Wiley‐Liss, Inc.
Identification of a chemotactic epitope in human transforming growth factor‐β1 spanning amino acid residues 368–374
Authors: Postlethwaite Arnold E. , Seyer Jerome M.
Published: February 2005

TGF‐b̃1 plays a critical role in inflammatory and repair processes due in part to its ability to provide a potent chemotactic stimulus for inflammatory cells such as neutrophils and monocytes and for fibroblasts which initiate the fibrogenic response. In the present study, we have used synthetic oligopeptides representing the amino acid sequence of the 12.1 kDa monomer of human TGF‐b̃1 in an effort to identify a chemotactic epitope on the molecule. A seven residue peptide containing residues 368‐374, Val Tyr Tyr Val Gly Arg Lys, was demonstrated to be capable of inducing chemotactic migration of human peripheral blood neutrophils, monocytes, monocyte leukemia cell line THP‐1, and infant foreskin fibroblasts. Furthermore, larger peptides from the carboxy‐terminal portion of TGF‐b̃1 that contained residues 368–374 also induced migration of these cell types. None of the peptides representing the complete amino acid of TGF‐b̃1 monomer were able to compete with [125I]hrTGF‐b̃1 for binding to TGF‐b̃ cell surface receptors or fibroblasts or THP‐1 cells. Implications of these observations are discussed. © 1995 Wiley‐Liss, Inc.
Mast cells are potent regulators of endothelial cell adhesion molecule ICAM‐1 and VCAM‐1 expression
Authors: Meng Hong , Tonnesen Marcia G. , Marchese Mary J. , Clark Richard A. F. , Bahou Wadie F. , Gruber Barry L.
Published: February 2005

To investigate the possible role of mast cells (MC) in regulating leukocyte adhesion to vascular endothelial cells (EC), microvascular and macrovascular EC were exposed to activated MC or MC conditioned medium (MCCM). Expression of intercellular and vascular adhesion molecules (ICAM‐1 and VCAM‐1) on EC was monitored. Incubation of human dermal microvascular endothelial cells (HDMEC) and human umbilical vein endothelial cells (HUVEC) with activated MC or MCCM markedly increased ICAM‐1 and VCAM‐1 surface expression, noted as éarly as 4 hr. Maximal levels were observed at 16 hr followed by a general decline over 48 hr. A dose‐dependent response was noted using incremental dilutions of MCCM or by varying the number of MC in coculture with EC. At a ratio as low as 1:1,000 of MC:EC, increased ICAM‐1 was observed. The ICAM‐1 upregulation by MCCM was >90% neutralized by antibody to tumor necrosis factor alpha (TNF‐α), suggesting that MC release of this cytokine contributes significantly to inducing EC adhesiveness. VCAM‐1 expression enhanced by MCCM was partly neutralized (70%) by antibody to TNF‐α; thus other substances released by MC may contribute to VCAM‐1 expression. Northern blot analysis demonstrated MCCM upregulated ICAM‐1 and VCAM‐1 mRNA in both HDMEC and HUVEC. To evaluate the function of MCCM‐enhanced EC adhesion molecules, T cells isolated from normal human donors were used in a cell adhesion assay. T‐cell binding to EC was increased significantly after exposure of EC to MCCM, and inhibited by antibodies to ICAM‐1 or VCAM‐1. Intradermal injection of allergen in human atopic volunteers known to develop late‐phase allergic reactions led to marked expression of both ICAM‐1 and VCAM‐1 at 6 hr, as demonstrated by immunohistochemistry. These studies indicate that MC play a critical role in regulating the expression of EC adhesion molecules, ICAM‐1 and VCAM‐1, and thus augment inflammatory responses by upregulating leukocyte binding. © 1995 Wiley‐Liss Inc.
Inhibition of the arachidonic acid pathway prevents induction of IL‐8 mRNA by phorbol ester and changes the release of IL‐8 from HL 60 cells: Differential inhibition of induced expression of IL‐8, TNF‐α, IL‐1α, and IL‐1β
Authors: Meier R. W. , Niklaus G. , Dewald B. , Fey M. F. , Tobler A.
Published: February 2005

The promyelocytic HL 60 cell line can be used as an in vitro model system to study hematopoetic cell differentiation and inflammatory events. We studied the signal transduction pathway of induced interleukin (IL)‐8 expression and compared it with those of tumor necrosis factor alpha (TNF‐α), IL‐1α, and IL‐1β. The differentiation of HL 60 cells to macrophage‐like cells by PMA resulted in a rapid and marked induction of these inflammatory cytokines. The up‐regulation occurred in the absence of ongoing protein synthesis, but cycloneximide‐sensitive gene products modulated their induction kinetics. Staurosporine, a potent inhibitor of protein kinases, strongly inhibited their gene expression. Phosphorylation may not act directly on latent transcription factors, since bromophenacyl bromide, an inhibitor for the release of arachidonic acid from phorbol‐12 myristate 13‐acetate (PMA)‐stimulated HL 60 cells, markedly depressed the induced mRNAs for IL‐8, TNF‐α, and IL‐1α and ‐β. Similarly, 5,8,11,14 eicosatetraynoic acid (ETYA), another inhibitor of the arachidonic acid pathway, blocked the induction of transcripts for TNF‐α, and both IL‐1 genes in phorbol ester‐stimulated HL 60 cells. In contrast, ETYA increased the induced IL‐8 RNA levels and stimulated the release for IL‐8. Also, ketoconazole, an inhibitor of 5‐lipoxygenase and indomethacin, an inhibitor of cyclooxygenases did not block the induction of IL‐8 mRNA. However, the release of IL‐8 protein was regulated by indomethacin and ketoconazole. Our results indicate that arachidonic acid metabolites are mediators in the signal transduction pathway of IL‐8 expression and that the involved second messengers are different from those which are important for the induction of TNF‐α and IL‐1β expression. © 1995 Wiley‐Liss Inc.
Ca2+ influx, phosphoinositide hydrolysis, and histamine release induced by lysophosphatidylserine in mast cells
Authors: Lloret Sergio , Moreno Juan Jose
Published: February 2005

We have previously demonstrated that snake venom phospholipases A2 (PLA2s) and mammalian PLA2s induced inflammatory processes. This effect was correlated with the activity of the enzymes and the release of lipid mediators. We have now determined the role of lysophosphatidylserine (LysoPS) as an inflammatory lipid mediator. Thus, we have studied the possibility that intracellular calcium concentration, phosphoinositide hydrolysis, and the subsequent histamine release in mast cells is due to the action of lysophosphatidylserine. Lysophosphatidylserine‐stimulated release of histamine was significantly higher than release by other lysophospholipids. The contribution of increased phospholipase C activity and the intracellular Ca2+ influx were therefore examined. LysoPS increased mast cell calcium concentration, and this increment was associated with phospholipase C activation and release of inositol phosphates. The increase in intracellular calucium and histamine degranulation induced by LysoPS were inhibited by apomorphine. Pretreatment of mast cells with pertussis toxin decreased the secretagogic effect of LysoPS and compound 48/80 without modifying the effect of the ionophore A23187. These results suggest that pertussis toxinsensitive G‐protein might be involved in the mast cell degranulation produced by lysophosphatidylserine and allow the increase in phospholipase C activity, thus enhancing intracellular calcium concentration, which then induces exocytosis of histamine. © 1995 Wiley‐Liss Inc.
Effect of 1,25‐dihydroxyvitamin D3 on induction of scavenger receptor and differentiation of 12‐O‐tetradecanoylphorbol‐13‐acetate‐treated THP‐1 human monocyte like cells
Authors: Suematsu Yuji , Nishizawa Yoshiki , Shioi Atsushi , Hino Masayuki , Tahara Hideki , Inaba Masaaki , Morii Hirotoshi , Otani Shuzo
Published: February 2005

We investigated the effect of 1,25‐dihydroxyvitamin D3 (1,25(OH)2D3) on the expression of scavenger receptors in human monocytic cell line (THP‐1 cells) treated for 24 h with 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) which induces their differentiation into macrophages. The capacity to degrade 125I‐labeled acetyl low density lipoprotein (LDL) was developed in accordance with macrophage differentiation. The treatment with 10 nM 1,25(OH)2D3 for 72 h inhibited the degradation of acetyl LDL by THP‐1 macrophages in a dose‐dependent manner, suggesting that 1,25(OH)2D3 inhibits scavenging function in macrophages. In order to clarify the mechanism of its inhibitory effect on degradation of acetyl LDL, we performed the ligand binding assay using 125I‐labeled acetyl LDL. Scatchard analysis revealed that 1,25(OH)2D3 decreased the number of scavenger receptors without changing the affinity for acetyl LDL. We next examined the effect of 1,25(OH)2D3 on the expression of scavenger receptor mRNA. The mRNA of type I scavenger receptor was first detected in THP‐1 cells 4 days after the treatment with TPA, the mRNA level increased up to 6 days, and then decreased. The treatment with 1,25(OH)2D3 for 72 h dramatically decreased the mRNA levels after the acquisition of macrophage phenotypes as evidenced by nonspecific esterase staining. However, 1,25(OH)2D3 did not affect the activity of non‐specific esterase nor the induction of interleukin‐1β mRNA by lipopolysaccharide in THP‐1 macrophages. These findings suggest that 1,25(OH)2D3 exclusively decreases the expression of scavenger receptors in TPA‐induced THP‐1 macrophages without affecting the basic cellular functions as macrophages. © 1995 Wiley‐Liss Inc.
Differential gene expression in migrating renal epithelial cells after wounding
Authors: Pawar Shashi , Kartha Sreedharan , Toback F. Gary
Published: February 2005

An in vitro model of wound healing was used to study cell migration that is independent of proliferation during renal regeneration after acute tubular necrosis. Monolayer cultures of high‐density, quiescent renal epithelial cells of the BSC‐1 line were subjected to scrape wounding and then Northern blot analysis was employed to identify genes that mediate cell migration. After wounding the monolayer, there is maximal induction of the immediate‐early genes Egr‐1, c‐fos, NAK‐1, and gro at 1 hour, followed by peak induction of connective tissue growth factor (CTGF) and c‐myc at 4 hours. Message levels of urokinase‐type plasminogen activator (u‐PA) and its inhibitor (PAI‐1) and heat shock protein (HSP)‐70 are markedly raised 4–8 hours after wounding. Constitutive expression is repressed at 1 hour for transcripts that encode receptors for fibronectin (FN), epidermal growth factor, and hepatocyte growth factor (c‐met), and the secreted proteins FN and osteopontin. Expression of genes encoding transforming growth factor (TGF)‐β2, retinoic acid receptor α, int‐1, int‐2, and gap junction protein which can play a role in cell movement, appeared unchanged after wounding. Differential expression of genes was a function of cell location relative to the wound; NAK‐1, PAI‐1, and HSP‐70 were induced or stimulated only in cells at the wound edge, u‐PA was stimulated in cells away from the wound, and CTGF was induced in each of these populations suggesting that cell‐to‐cell communication may regulate gene expression after wounding. Adenosine diphosphate, a potent stimulator of cell migration which enhances expression of u‐PA and PAI‐1 in nonwounded cultures, additively stimulates these genes after wounding and may thereby potentiate wound healing. Thus scrape wounding of renal epithelial cells is followed by induction, stimulation, or repression of specific genes with distinct responses in different populations of cells. © 1995 Wiley‐Liss Inc.
Homologous desensitization of ATP‐stimulated mitogenesis: Mechanism involves desensitization of arachidonic acid release and cAMP elevation but not the activation of protein kinase A
Authors: Huang Ning‐Na , Wang Ding‐Ji , Heller Elizabeth , Heppel Leon A.
Published: February 2005

Prolonged incubation of quiescent 3T3, 3T6, and A431 cells with the P2Y purinoceptor agonists ATP, ADP, or AMPPNP reduced the mitogenic responses of target cells to a further challenge by these agonists, as measured by [3H]thymidine incorporation. The mitogenic desensitization was agonist‐specific, for no effect was seen on DNA synthesis stimulated by epidermal growth factor, insulin, bombesin, 12‐0‐tetradecanoyl‐phorbol‐12 acetate (TPA), or adenosine. The desensitization was completely reversible, since after a 24 hr incubation in the absence of ATP, the cells responded fully to the mitogenic action of ATP. The presence of a low level of cycloheximide blocked recovery, suggesting that down‐regulation of the P2Y receptor may have occurred during desensitization. In Swiss 3T3 cells, stimulation of DNA synthesis occurs predominantly by activation of arachidonic acid release, followed by its oxidation to prostaglandin E2 and stimulation of adenylyl cyclase. Interestingly, prolonged preincubation with ATP produced a similar degree of desensitization of DNA synthesis and of ATP‐dependent arachidonic acid release and cAMP accumulation. Furthermore, this was true for both wild type cells and mutants with a defective cAMP‐dependent protein kinase (PKA). We conclude that homologous desensitization is likely due to uncoupling of the P2Y purinoceptor from phospholipase A2, and this process does not require activation of protein kinase A. © 1995 Wiley‐Liss Inc.
Constructions of bipartite graphs from finite geometries
Authors: Mellinger Keith E. , Mubayi Dhruv
Published: February 2005

We construct an incidence structure using certain points and lines in finite projective spaces. The structural properties of the associated bipartite incidence graphs are analyzed. These n × n bipartite graphs provide constructions of C6‐free graphs with Ω(n4/3 edges, C10‐free graphs with Ω(n6/5) edges, and Θ(7,7,7)‐free graphs with Ω(n8/7) edges. Each of these bounds is sharp in order of magnitude. © 2005 Wiley Periodicals, Inc. J Graph Theory 49: 1–10, 2005
Characterizing the electrospray ionization mass spectral fragmentation pattern of indole derivatives synthesized from 2‐keto glycosides
Authors: Cao Shuxia , Liu Hongmin , Xu Wen , Liao Xincheng , Zhao Yufen
Published: February 2005

Four indole derivatives synthesized from 2‐keto glycosides were analyzed by electrospray ionization tandem mass spectrometry (ESI‐MSn) with low‐energy collision‐induced dissociation to establish a general structural elucidation of indole derivatives. Their fragmentation pathways are proposed on the basis of the MSn studies and deuterium‐labeled experiments. Indole derivatives undergo complicated gas‐phase rearrangements in addition to simple bond cleavages. A rearrangement, which involves a contraction of the six‐membered ring, was observed and a mechanism was proposed. The observations may have some potential applications in the interpretation of the mass spectra of indole derivatives. Copyright © 2005 John Wiley & Sons, Ltd.
Expression of the nm23 homologues nm23‐H4, nm23‐H6, and nm23‐H7 in human gastric and colon cancer
Authors: Seifert M , Welter C , Mehraein Y , Seitz G
Published: February 2005

Eight members of the nm23‐gene family have been described. The involvement of nm23‐H1 and nm23‐H2 in tumour progression and metastasis, as well as in gene regulation and apoptosis, has been shown in numerous studies. Whether nm23‐H4, ‐H6, and ‐H7 play a role in tumours is, however, largely unknown. This study describes data on the expression of these three nm23 homologues in human colon and gastric cancer by real‐time RT‐PCR and immunohistochemistry. Increased expression of these genes, most strikingly nm23‐H4 and ‐H7, was observed in the majority of tumours analysed. No correlation with tumour stage according to the TNM classification was found. In contrast, by immunohistochemical analysis, nm23‐H4 and ‐H6 overexpression correlated with the intestinal tumour type in gastric cancer tissues, whereas no increased immunoreactivity for the three nm23 proteins was noted in the diffuse type tumour specimens. These findings indicate that nm23‐H6, and particularly nm23‐H4 and ‐H7, may be involved in the development of colon and gastric carcinoma, the latter possibly in a type‐specific manner. A contribution to tumour progression or metastasis could not, however, be proven. Elucidation of the specific mechanisms by which the nm23 homologues nm23‐H4, ‐H6, and ‐H7 are involved in tumour development requires further studies. Copyright © 2005 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Analysis of GTPases carrying hydrophobic amino acid substitutions in lieu of the catalytic glutamine: Implications for GTP hydrolysis
Authors: Mishra Rajeev , Gara Sudheer Kumar , Mishra Shambhavi , Prakash Balaji
Published: February 2005

Ras superfamily GTP‐binding proteins regulate important signaling events in the cell. Ras, which often serves as a prototype, efficiently hydrolyzes GTP in conjunction with its regulator GAP. A conserved glutamine plays a vital role in GTP hydrolysis in most GTP‐binding proteins. Mutating this glutamine in Ras has oncogenic effects, since it disrupts GTP hydrolysis. The analysis presented here is of GTP‐binding proteins that are a paradox to oncogenic Ras, since they have the catalytic glutamine (Glncat) substituted by a hydrophobic amino acid, yet can hydrolyze GTP efficiently. We term these proteins HAS‐GTPases. Analysis of the amino acid sequences of HAS‐GTPases reveals prominent presence of insertions around the GTP‐binding pocket. Homology modeling studies suggest an interesting means to achieve catalysis despite the drastic hydrophobic substitution replacing the key Glncat of Ras‐like GTPases. The substituted hydrophobic residue adopts a “retracted conformation,” where it is positioned away from the GTP, as its role in catalysis would be unproductive. This conformation is further stabilized by interactions with hydrophobic residues in its vicinity. These interacting residues are strongly conserved and hydrophobic in all HAS‐GTPases, and correspond to residues Asp92 and Tyr96 of Ras. An experimental support for the “retracted conformation” of Switch II arises from the crystal structures of Ylqf and hGBP1. This conformation allows us to hypothesize that, unlike in classical GTPases, catalytic residues could be supplied by regions other than the Switch II (i.e., either the insertions or a neighboring domain). Proteins 2005. © 2005 Wiley‐Liss, Inc.
Cation–π interactions in protein–protein interfaces
Authors: Crowley Peter B. , Golovin Adel
Published: February 2005

Arginine is an abundant residue in protein–protein interfaces. The importance of this residue relates to the versatility of its side chain in intermolecular interactions. Different classes of protein–protein interfaces were surveyed for cation–π interactions. Approximately half of the protein complexes and one‐third of the homodimers analyzed were found to contain at least one intermolecular cation–π pair. Interactions between arginine and tyrosine were found to be the most abundant. The electrostatic interaction energy was calculated to be ∼3 kcal/mol, on average. A distance‐based search of guanidinium:aromatic interactions was also performed using the Macromolecular Structure Database (MSD). This search revealed that half of the guanidinium:aromatic pairs pack in a coplanar manner. Furthermore, it was found that the cationic group of the cation–π pair is frequently involved in intermolecular hydrogen bonds. In this manner the arginine side chain can participate in multiple interactions, providing a mechanism for inter‐protein specificity. Thus, the cation–π interaction is established as an important contributor to protein–protein interfaces. Proteins 2005. © 2005 Wiley‐Liss, Inc.
Solution structure of APETx1 from the sea anemone Anthopleura elegantissima: A new fold for an HERG toxin
Authors: Chagot Benjamin , Diochot Sylvie , Pimentel Cyril , Lazdunski Michel , Darbon Hervé
Published: February 2005

APETx1 is a 42‐amino acid toxin purified from the venom of the sea anemone Anthopleura elegantissima. This cysteine‐rich peptide possesses three disulfide bridges (C4–C37, C6–C30, and C20–C38). Its pharmacological target is the Ether‐a‐gogo potassium channel. We herein determine the solution structure of APETx1 by use of conventional two‐dimensional 1H‐NMR techniques followed by torsion angle dynamics and refinement protocols. The calculated structure of APETx1 belongs to the disulfide‐rich all‐beta structural family, in which a three‐stranded anti‐parallel β‐sheet is the only secondary structure. APETx1 is the first Ether‐a‐gogo effector discovered to fold in this way. We therefore compare the structure of APETx1 to those of the two other known effectors of the Ether‐a‐gogo potassium channel, CnErg1 and BeKm‐1, and analyze the topological disposition of key functional residues proposed by analysis of the electrostatic anisotropy. The interacting surface is made of a patch of aromatic residues (Y5, Y32, and F33) together with two basic residues (K8 and K18) at the periphery of the surface. We pinpoint the absence of the central lysine present in the functional surface of the two other Ether‐a‐gogo effectors. Proteins 2005. © 2005 Wiley‐Liss, Inc.
Flexibility of metal binding sites in proteins on a database scale
Authors: Babor Mariana , Greenblatt Harry M. , Edelman Marvin , Sobolev Vladimir
Published: February 2005

Protein metal binding sites in the pre‐bound (apo) state, and their rearrangements upon metal binding were not analyzed previously at a database scale. Such a study may provide valuable information for metal binding site prediction and design. A high resolution, nonredundant dataset of 210 metal binding sites was created, containing all available representatives of apo–holo pairs for the most populated metals in the PDB. More than 40% of the sites underwent rearrangements upon metal binding. In 30 cases rearrangements involved the backbone. The tendency for side‐chain rearrangement inversely correlates with the number of first‐shell residues. Analysis of side‐chain reorientations as a result of metal binding showed that in 95% of the rigid‐backbone binding sites at most one side chain moved. Thus, in general, part of the first coordination shell is already in place in the pre‐bound form. The frequencies of side‐chain reorientation directly correlated with metal ligand flexibility and solvent accessibility in the apo state. Proteins 2005. © 2005 Wiley‐Liss, Inc.
Metal–insulator transition in manganese‐doped InSb crystals
Authors: Obukhov S. A.
Published: February 2005

Transport properties of manganese‐doped InSb crystals with hole concentration close to the critical concentration Ncr of the metal–insulator transition (MIT) in p‐InSb(Mn) was found to be different from MIT in semiconductors doped with non‐magnetic impurities such as p‐Ge(Ga), p‐Si(P) and p‐InSb(Ge). On the insulator side of the transition (NMn ≤ Ncr = 2 × 1017 cm–3) in the range of hole concentration p = 1–2 × 1017 cm–3 an activation type of conductivity with energy gap Δ = 7 × 10–6–10–3 eV was observed. Thus, the usually observed variable range hopping conductivity in crystal semiconductors doped with non‐magnetic impurities at low and super‐low temperatures was not revealed in p‐InSb(Mn) at p ≤ Ncr. An external magnetic field decreased Δ and caused a giant decrease of specific resistivity (ρ0/ρB ∼ 104 at T ≤ 0.3 K and B ≥ 3 T). On cooling to temperatures below T = 1.5 K the Hall constant changed sign at B < 3 T. The change of Hall constant sign with decrease of temperature means that conductivity of p‐InSb(Mn) and crystals compensated with Te – p‐InSb(Mn, Te) – at low temperatures was caused by electrons. The proposed model associated an activation energy Δ at p ∼ Ncr with the energy gap between sub‐bands formed in the impurity band. (© 2005 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)
Binding of singlet oxygen with a stacked guanine dimer
Authors: Kushwaha P. S. , Mishra P. C.
Published: February 2005

Geometry optimization calculations were performed using the B3LYP/6‐31+G* method on the complexes of 1O2 and 3O2 molecules with a stacked dimer of planar guanine, varying the distance (D) between the planes of the guanine molecules. In this process, geometries of the guanine molecules were held fixed, D was fixed at different values, while the bond lengths of 1O2 and 3O2 as well as their orientations with respect to the guanine molecules were optimized for each value of D. The complexes in their most stable geometries were solvated in water using the integral equation formalism of the polarized continuum model of the self‐consistent reaction field theory. In gas phase, the most stable complex between 1O2 and the guanine dimer (2G.1O2) is formed when D is about 6 Å, while the most stable complex between 3O2 and the guanine dimer (2G.3O2) is formed when D is about 3.75 Å. In the minimum total energy geometry of 2G.1O2, 1O2 is located between the guanine molecules, above the imidazole ring of one of them. However, in the minimum total energy geometry of 2G.3O2, 3O2 is located outside the stack of guanine molecules, near the amino group of one of them. The solvation calculations showed that in aqueous media, 1O2 would bind with the stacked guanine dimer more strongly than in gas phase, while 3O2 would not bind with the same. The mode of binding of 1O2 with the stacked guanine dimer is such that it seems that 1O2 would replace one basepair in DNA, as happens in the intercalative mode of binding of drugs and other molecules, and it can lead to the formation of 8‐oxoguanine that has a mutagenic nature. © 2004 Wiley Periodicals, Inc. Int J Quantum Chem, 2005
Statistical thermodynamics of binary systems with variable valent states of one of the components
Authors: Zakharov A. Yu. , Zakharov M. A. , Udovsky A. L. , Oonk H. A. J.
Published: February 2005

This report contains a generalization of the usual lattice model of multicomponent systems. The generalization allows us to take into account the following factors: (i) the short‐range parts of interatomic repulsions; which are not identical for different pairs of atoms (therefore, it is impossible to take into account the repulsions by means of usual ideal lattice introduction); (ii) the long‐range interatomic potentials are taken into account by means of effective fields approximation; (iii) the existence of the vacancies as one of the components in the system; (iv) the existence of comparatively stable multi‐atomic complexes; and (v) some other factors due to specifics of constituents and their interactions, such as variable valence states and chemical reactions. © 2005 Wiley Periodicals, Inc. Int J Quantum Chem, 2005
Collectivity, shell openness indices, and complexity measures of multiconfigurational states: Computations within full CI scheme
Authors: Luzanov A. V. , Zhikol O. A.
Published: February 2005

It is shown that the multiconfigurational character of the N‐electron wave function can be treated by a new quantitative index, N, computed as a special matrix invariant of the full wave function. Along with N, a simpler index N is given due to the Kutzelnigg and Smith open‐shell theory. The indices are compared with an effective number of unpaired electrons introduced by Takatsuki, Fueno, and Yamaguchi. Full configuration interaction (FCI) computations on such quantities realized in the present work for atoms and dissociation reactions lead to the conclusion that all indices give a qualitatively similar picture. It is stressed that N as a more complicated quantity reflects a global complexity of the electronic state, while other indices represent the electron structure peculiarities only at the one‐electron level. © 2005 Wiley Periodicals, Inc. Int J Quantum Chem, 2005
Effects of thermal modulation on tunneling through a fluctuating barrier in presence of bichromatic electromagnetic field
Authors: Saha Aparna , Sarkar Pranab
Published: February 2005

The effects of thermal modulation on the tunneling dynamics of a particle moving in a bistable potential with a fluctuating barrier in the presence of bichromatic electromagnetic field are examined. This study brings to light various interesting features. While rate constant increases steadily with increasing fluctuation strength, a stochastic resonance‐like behavior is exhibited when tunneling rate is plotted as a function of barrier fluctuation frequency. The tunneling rate constant decreases with the increase in the differences of the strength of the two electric fields; on the other hand, it displays a maxima when plotted as a function of the ratio of the frequencies of the two fields. When the rate constant is plotted as a function of temperature, it increases and eventually settles down to a saturation value. We have also studied the effect of the phase difference between the two electric fields on the tunneling behavior in the presence of thermal modulation. © 2005 Wiley Periodicals, Inc. Int J Quantum Chem, 2005
Theoretical evaluation of the nanocarrier properties of two families of functionalized dendrimers
Authors: Evangelista‐Lara Aurelio , Guadarrama Patricia
Published: February 2005

Two families of dendrimers, denamide (oxy‐amide) and denurea (oxy‐urea), of second and third generation were designed as possible nanocarriers for high‐spectral parasiticide drugs of the family of macrocyclic lactones. Their geometric description was carried out by Connolly's algorithm, obtaining molecular volumes and sizes of cavities. According to these calculations, about 50% of the total dendrimeric molecular volume in both families corresponds to cavities that are able to shelter guest drugs, forming complexes in ratios higher than 1:1. The molecular shape suffers more notorious changes, going through higher dendrimeric generations; therefore, third‐generation dendrimers with two carbon atoms in their fractal patterns are more spherical than second‐generation dendrimers with six carbon atoms in their fractal patterns. The experimentally studied host–guest system formed by PAMAM and ibuprofen (analgesic drug) was taken as a reference in the present study. Molecular dynamics and mechanics calculations were able to reproduce some experimental observations. Nevertheless, the interaction energies of some of the 1:1 complexes were calculated at the density functional theory (DFT) level. Comparing the fractal patterns of the denamide and denurea families with polyamidoamine dendrimers (PAMAM), the observed order of efficiency, in terms of favorable interactions with guest molecules, is as follows: denamide > PAMAM > denurea. The differences in functionalization are responsible for such behavior. © 2005 Wiley Periodicals, Inc. Int J Quantum Chem, 2005
Potential of laser ablation and laser desorption mass spectrometry to characterize organic and inorganic environmental pollutants on dust particles
Authors: Carré Vincent , Aubriet Frédéric , Scheepers Paul T. , Krier Gabriel , Muller Jean‐François
Published: February 2005

Stainless steel factories are known to release particles into the atmosphere. Such particulate matter contains significant amounts of heavy metals or toxic inorganic compounds and organic pollutants such as, for example, Cr(VI) and polycyclic aromatic hydrocarbons (PAHs). The investigation of Cr(VI) and PAHs is often complicated by the associated matrix. Organic and inorganic pollutants present in stainless steel dust particles have been investigated with the same laser microprobe mass spectrometer according to two original methodologies. These analytical methods do not require time‐consuming pretreatment (extraction, solubilization) or preconcentration steps. More specifically, experiments are conducted with a Fourier transform ion cyclotron resonance mass spectrometer coupled to an ArF (193 nm) or a tripled frequency Nd‐YAG (355 nm) laser. Experiments at 355 nm allow the nature of the most frequently occurring Cr(III)/Cr(VI) compounds in dust particles to be identified. Examination of PAHs at 193 nm is assisted by the formation of π‐complexes with 7,7′,8,8′‐tetracyanoquinodimethane to prevent their evaporation in the mass spectrometer during analysis and to ensure an increase in sensitivity. Copyright © 2005 John Wiley & Sons, Ltd.
A new method to analyze matrix‐assisted laser desorption/ionization time‐of‐flight peptide profiling mass spectra
Authors: Dekker Lennard J. , Dalebout Johannes C. , Siccama Ivar , Jenster Guido , Sillevis Smitt Peter A. , Luider Theo M.
Published: February 2005

In protein and peptide mass spectrometry in which profiling of peaks is involved, their masses and intensities are important characteristics. Because of the relative low reproducibility of peak intensities associated with complex samples in matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOFMS), it is difficult to accurately assess the number of peaks and their intensities. In this study we evaluate these two characteristics for tryptic digests of cerebro‐spinal fluid. We observed that the reproducibility of peak intensities was relatively poor (CV = 42%) and that additional normalization or spiking did not lead to a large improvement (CV = 30%). Moreover, at least seven mass spectra per sample were required to obtain a reliable peak list. An improvement of the sensitivity (i.e., eventually more peaks are detected) is observed if more replicates per sample are measured. We conclude that the reproducibility and sensitivity of peptide profiling can be significantly improved by a combination of measuring at least seven spectra per sample with a dichotomous scoring of the intensities. This approach will aid the analysis of large numbers of mass spectra of patient samples in a reproducible way for the detection and validation of candidate biomarkers. Copyright © 2005 John Wiley & Sons, Ltd.
Generating survival times to simulate Cox proportional hazards models
Authors: Bender Ralf , Augustin Thomas , Blettner Maria
Published: February 2005

Simulation studies present an important statistical tool to investigate the performance, properties and adequacy of statistical models in pre‐specified situations. One of the most important statistical models in medical research is the proportional hazards model of Cox. In this paper, techniques to generate survival times for simulation studies regarding Cox proportional hazards models are presented. A general formula describing the relation between the hazard and the corresponding survival time of the Cox model is derived, which is useful in simulation studies. It is shown how the exponential, the Weibull and the Gompertz distribution can be applied to generate appropriate survival times for simulation studies. Additionally, the general relation between hazard and survival time can be used to develop own distributions for special situations and to handle flexibly parameterized proportional hazards models. The use of distributions other than the exponential distribution is indispensable to investigate the characteristics of the Cox proportional hazards model, especially in non‐standard situations, where the partial likelihood depends on the baseline hazard. A simulation study investigating the effect of measurement errors in the German Uranium Miners Cohort Study is considered to illustrate the proposed simulation techniques and to emphasize the importance of a careful modelling of the baseline hazard in Cox models. Copyright © 2005 John Wiley & Sons, Ltd.
Introducing an innovative semi‐captive environment for the Philippine tarsier (Tarsius syrichta)
Authors: Jachowski David S. , Pizzaras Carlito
Published: February 2005

In response to demands for research, captive breeding, and tourism, a semi‐captive environment was created for the Philippine tarsiers (Tarsius syrichta) in Corella, Bohol, Philippines. The 7600‐m2 enclosure was continuous with the surrounding habitat, and utilized a unique predator control fence design and a lighting system to attract nocturnal insects. During 2 years of observation, the locations of over 500 tarsier sleeping sites were recorded. Tarsiers were found to prefer dense, low‐level vegetation in secondary forests, with perching sites averaging 2 m above the ground. Up to 10 tarsiers were observed within the enclosure at one time, which is a high density compared to densities based on home‐range estimates for wild tarsiers in the vicinity of the study site. In addition, the tarsiers were observed to be more social than previously reported. Zoo Biol 24:101–109, 2005. © 2005 Wiley‐Liss, Inc.
Husbandry, overwinter care, and reproduction of captive striped skunks (Mephitis mephitis)
Authors: Larivière Serge , Hwang Yeen Ten , Gorsuch Wanda A. , Medill Sarah A.
Published: February 2005

The striped skunk (Mephitis mephitis) is the focus of research interest for a variety of reasons, including its roles as a rabies reservoir, urban pest, predator of bird eggs, and parasite host. Some research programmes may require husbandry and breeding of captive animals, and because skunks are inactive in the winter in northern environments, special methods are needed to maintain the animals over winter. We report on the establishment of a colony of striped skunks kept in captivity in the northern part of their range. Our goals were to develop simple and effective methods to keep skunks in captivity over winter, and successfully breed skunks in a manner that resembles conditions in the wild. In the spring of 2002, three wild‐caught, pregnant females gave birth in captivity (mean litter size=7). During the first year, 11 of 12 females kept in captivity over winter reproduced successfully in the spring of 2003, with a mean litter size of 5.5 (range=4–8, n=11). In the summer of 2003, 91 of 93 juveniles (97.9%) survived >60 days past weaning. Our high survival and reproduction rates testify to the success of this program, and provide insights into the husbandry of a species that is inactive during winter. Zoo Biol 24:83–91, 2005. © 2005 Wiley‐Liss, Inc.
A sensitive and rapid immunochemical method for quantitation of proteins
Authors: Mawal M. R. , Mawal Y. R. , Ranadive S. N. , Ranjekar P. K.
Published: February 2005

A sensitive and rapid ELISA for quantitation of seed globulins is described. This method employs conjugation of pigeon pea (Cajanus cajan) globulin antibodies and the enzyme peroxidase together with dextran. Using this conjugate, proteins as low as 0.1 ng were detected. Dextran conjugate has a ten-fold greater efficiency of quantitating pigeon pea globulins than the commercial goat anti-rabbit IgG conjugate, and is three-fold more efficient than pigeon pea globulin IgG peroxidase conjugate. The method can be conveniently adapted for quantitation of other proteins also.
The reaction of bisulfite with liposomal membranes
Authors: Finch L. , Southerland W. M.
Published: February 2005

Bisulfite has been shown to induce leakage of encapsulated substances from liposomal vesicles. The bisulfite induced leakage of either DNP-tyrosine, potassium ferricyanide, or [3H]glycine was observed to be greater with lipsomes composed of phospholipids containing unsaturated fatty acids. The leakage of encapsulated substances from liposomes was found to be concentration dependent when incubated for a constant time interval and time dependent when incubated at a constant bisulfite concentration. In addition, bisulfite caused the leakage of approximately 5 times more [3H]glycine from unilamellar liposomes than from multilamellar liposomes. These findings are consistent with the interaction of bisulfite with liposomal membranes via reaction with sites of unsaturation.