bluuebunny/crossref_metadata_2025 huggingface dataset:
VEGF expression by ganglion cells in central retina before formation of the foveal depression in monkey retina: Evidence of developmental hypoxia
Authors: Sandercoe Trent M. , Geller Scott F. , Hendrickson Anita E. , Stone Jonathan , Provis Jan M.
Published: May 2003

In macaque monkeys the foveal depression forms between fetal day (Fd) 105 and birth (Fd 172 of gestation). Before this, the incipient fovea is identified by a photoreceptor layer comprising cones almost exclusively, a multilayered ganglion cell layer (GCL), and a “domed” profile. Vessels are absent from the central retina until late in development, leading to the suggestion that the GCL in the incipient fovea may be transitorily hypoxic. Vascular endothelial growth factor (VEGF), expressed by both glial and neuronal cells and mediated by the hypoxia‐inducible transcription factor (HIF)‐1, is the principal factor involved in blood vessel growth in the retina. We examined VEGF expression in macaque retinas between Fd 85 and 4 months postnatal. Digoxygenin‐labeled riboprobes were generated from a partial‐length human cDNA polymerase chain reaction fragment, detected using fluorescence confocal microscopy, and quantified using Scion Image. High levels of VEGF mRNA were detected in astrocytes associated with developing vessels. We also detected strong expression of VEGF mRNA in the GCL at the incipient fovea prior to Fd 105, with peak labeling in the incipient fovea that declined with distance in nasal and temporal directions. By Fd 152 peak labeling was in two bands associated with development of the inner nuclear layer (INL) capillary plexus: in the inner INL where Müller and amacrine cell somas are located, and in the outer INL where horizontal cells are found. The findings suggest that at the incipient fovea the GCL is hypoxic, supporting the hypothesis that the adaptive significance of the fovea centralis is in ensuring adequate oxygen supply to neuronal elements initially located within the avascular region. J. Comp. Neurol. 462:42–54, 2003. © 2003 Wiley‐Liss, Inc.
Thalamocortical and intracortical connections of monkey cingulate motor areas
Authors: Hatanaka Nobuhiko , Tokuno Hironobu , Hamada Ikuma , Inase Masahiko , Ito Yumi , Imanishi Michiko , Hasegawa Naomi , Akazawa Toshikazu , Nambu Atsushi , Takada Masahiko
Published: May 2003

Although there has been an increasing interest in motor functions of the cingulate motor areas, data concerning their input organization are still limited. To address this issue, the patterns of thalamic and cortical inputs to the rostral (CMAr), dorsal (CMAd), and ventral (CMAv) cingulate motor areas were investigated in the macaque monkey. Tracer injections were made into identified forelimb representations of these areas, and the distributions of retrogradely labeled neurons were analyzed in the thalamus and the frontal cortex. The cells of origin of thalamocortical projections to the CMAr were located mainly in the parvicellular division of the ventroanterior nucleus and the oral division of the ventrolateral nucleus (VLo). On the other hand, the thalamocortical neurons to the CMAd/CMAv were distributed predominantly in the VLo and the oral division of the ventroposterolateral nucleus‐the caudal division of the ventrolateral nucleus. Additionally, many neurons in the intralaminar nuclear group were seen to project to the cingulate motor areas. Except for their well‐developed interconnections, the corticocortical projections to the CMAr and CMAd/CMAv were also distinctively preferential. Major inputs to the CMAr arose from the presupplementary motor area and the dorsal premotor cortex, whereas inputs to the CMAd/CMAv originated not only from these areas but also from the supplementary motor area and the primary motor cortex. The present results indicate that the CMAr and the caudal cingulate motor area (involving both the CMAd and the CMAv) are characterized by distinct patterns of thalamocortical and intracortical connections, reflecting their functional differences. J. Comp. Neurol. 462:121–138, 2003. © 2003 Wiley‐Liss, Inc.
Localisation of specific heparan sulfate proteoglycans during the proliferative phase of brain development
Authors: Ford‐Perriss Miriam , Turner Kirsty , Guimond Scott , Apedaile Anwyn , Haubeck Hans‐Dieter , Turnbull Jeremy , Murphy Mark
Published: May 2003

Early brain development is characterised by the proliferation of neural precursor cells. Several families of signalling molecules such as the fibroblast growth factors (FGFs) and Wnts are known to play important roles in this early phase of brain development. Accumulating evidence demonstrates that signalling of these molecules requires the presence of heparan sulfate chains attached to a proteoglycan core protein (HSPG). However, the specific identity of the HSPG components in the developing brain is unknown. To determine which HSPGs might be involved at this early phase, we analysed the expression of the major cell surface HSPG families in the developing brain at a time of most active proliferation. Syndecan‐1 and glypican‐4 were the most highly expressed in the developing brain during the time of peak proliferation and localise to ventricular regions of the brain, where the precursor cells are proliferating. Syndecan‐4, although less abundant, also localises to cells in the ventricular zone. We have also examined HSPG involvement in brain development using cultures of embryonic neural precursor cells. We find that FGF2 stimulation of proliferation is inhibited in the presence of sodium chlorate, an inhibitor of heparan sulfate synthesis, and is rescued by addition of exogenous heparan sulfate. These data support a requirement for heparan sulfate in FGF signalling for proliferation of brain precursor cells. The expression of these specific HSPGs within the proliferative zone of the brain suggests that they may be involved in regulation of early brain development, such as FGF‐stimulated proliferation. Developmental Dynamics 227:170–184, 2003. © 2003 Wiley‐Liss, Inc.
Analysis of regulatory elements of E‐cadherin with reporter gene constructs in transgenic mouse embryos
Authors: Stemmler Marc P. , Hecht Andreas , Kinzel Bernd , Kemler Rolf
Published: May 2003

Proper regulation of E‐cadherin–mediated cell adhesion is important during early embryonic development and in organogenesis. In mice, E‐cadherin is expressed from the fertilized egg onward and becomes down‐regulated during gastrulation in mesoderm and its derivatives, but its expression is maintained in all epithelia. E‐cadherin promoter analyses led to the identification of binding sites for two transcriptional repressors, Snail and SIP1, which are able to mediate down‐regulation in vitro, but little is known about the regulatory elements that govern E‐cadherin transcriptional activity in vivo. Here, we compared the developmentally regulated expression of a series of lacZ‐reporter transgenes fused to different sequences of the murine E‐cadherin gene between −6 kb, including the promoter, and +16 kb, covering one third of intron 2. Four different segments with distinct regulatory properties were identified. The promoter fragment from +0.1 to −1.5 kb remains inactive in most cases but occasionally induces ectopic expression in mesodermal tissues, although it contains binding sites for the repressors Snail and SIP1. This promoter fragment also lacks positive elements needed for the activation of transcription in ectoderm and endoderm. Sequences from −1.5 to −6 kb harbor regulatory elements for brain‐specific expression and, in addition, insulator or silencer elements, because they are consistently inactive in the mesoderm. Only if sequences from +0.1 to +11 kb are combined with the promoter fragments is E‐cadherin–specific transgene expression observed in endoderm and certain epithelia. Sequences between +11 and +16 kb contain cis‐active elements that generally enhance transcription. Our analyses show that E‐cadherin expression is governed by a complex interplay of multiple regulatory regions dispersed throughout large parts of the locus. Developmental Dynamics 227:238–245, 2003. © 2003 Wiley‐Liss, Inc.
Intracellular calcium plays an essential role in cardiac development
Authors: Porter George A. , Makuck Ryan F. , Rivkees Scott A.
Published: May 2003

Intracellular calcium signaling plays an essential role in cardiac physiology and modulates cardiac gene expression. However, the role that intracellular calcium signaling plays during cardiac development is not known. To address this issue, we examined the effects of altered intracellular calcium levels on cardiac morphogenesis. In acutely cultured mouse embryos, L‐type calcium channel blockade decreased resting intracellular calcium levels and inhibited calcium transients. Embryos cultured at embryonic day (E) 7.5–8.5 in the presence of the L‐type calcium channel blockers nifedipine and verapamil developed hearts that had a large left ventricle, lacked a right ventricle and had a long, thin outflow tract. If embryos were cultured at E7.5, calcium channel blockade also induced an abnormal, anterior cardiac loop. These alterations in development were not due to altered cardiac function, as heart rates at the end of the culture period were not affected by calcium channel blockade and blood flow was observed. Treatment with nifedipine altered the mRNA expression of the transcription factor Gata4, which was absent in the developing ventricles, and the sarcomeric protein Mylpc (myosin light chain 2V), which was decreased distal to the left ventricle and was absent at the site of the developing right ventricle. In contrast, the expression pattern of other cardiac transcription factor (Hand1, Hand2, Mef2c, Nkx2‐5) and cytoskeletal protein (Myhca, Tagln) mRNA did not change with calcium channel blockade. These data demonstrate that proper intracellular calcium signaling is essential for normal cardiac looping, gene expression, and organ development. Developmental Dynamics 227:280–290, 2003. © 2003 Wiley‐Liss, Inc.
A Compact and Lipophilic Enantiomer Showing a Bilayer Crystal Structure − Free Energy of Spontaneous Resolution of the Racemic Compound into the Crystalline Enantiomers
Authors: Roux María Victoria , Jiménez Pilar , Vacas Araceli , Cano Félix H. , Apreda‐Rojas María del Carmen , Ros Francisco
Published: May 2003

The melting points and the enthalpies and entropies of fusion of (R)‐1 and rac‐1, with the racemate both as the conglomerate and as the true racemic compound, were determined by differential scanning calorimetry: the melting points were 113.2 ± 0.3, 90.7 ± 0.4, and 80.5 ± 0.6 °C, respectively, the ΔHfus values 8.82 ± 0.03, 7.94 ± 0.27, and 5.98 ± 0.59 kcal·mol−1, respectively, and the ΔSfus values 22.8 ± 0.1, 21.8 ± 0.7, and 16.9 ± 1.7 cal·deg−1·mol−1, respectively. The specific heat of fusion (ΔCfus) of the pure enantiomer 1 calculated from these data is 39.1 cal·deg−1·mol−1. The ΔHfus, ΔSfus, and ΔCfus values of these enantiomers are consistently high relative to average values for organic compounds. The enthalpy, entropy, and free energy of formation of the racemic compound from the crystalline enantiomers are 1.56 kcal·mol−1, 3.8 cal·deg−1·mol−1, and 0.22 kcal·mol−1, as calculated from the above data. The positive sign of the free energy of formation bears out the observed spontaneous resolution of the racemic compound into the crystalline enantiomers. A statistical thermodynamic model of melting, weighing the different mechanical contributions to the crystalline and the liquid states, is described in conjunction with the interpretation of the data. It is concluded that both the interaction potential energy of the crystal of the pure enantiomer 1 and the vibrational mobility of the crystal structure are low, features pertinent to a low enthalpy and entropy of the crystal, respectively. X‐ray crystallography of (R)‐1 revealed a simple bilayer arrangement of the roughly ellipsoidal molecules: a characteristic bilayer consists of two monomolecular layers with interactions between the lipophilic groups in between, featuring a methyl−phenyl interaction, while the cyano groups project towards the outsides of the bilayer, which stacks by means of dipolar interactions to a constant distance. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2003)
ZnBr2/CuCN‐Promoted, Highly Regioselective SN2′ Reactions of Some Functionalised Organolithium Compounds with Allylic and Propargylic Halides
Authors: Yus Miguel , Gomis Joaquín
Published: May 2003

Treatment of lithium (2‐lithiomethylphenyl)methoxide (2a) and lithium 2‐(2‐lithiomethylphenyl)ethoxide (2b) [easily prepared by ring opening of phthalan (1a) and isochroman (1b), respectively, by the use of lithium and a catalytic amount of 4,4′‐di‐tert‐butylbiphenyl (DTBB)] with zinc bromide and copper cyanide, both in equimolecular amounts, in THF at room temperature, followed by treatment with different allylic chlorides or bromides 3 at −78 or 20 °C, almost exclusively provides (after hydrolysis with water) the corresponding alcohols 4′ resulting from an SN2′ displacement, the process being highly regioselective. The same procedure with intermediates 2a and 2b was applied to propargylic chlorides or bromides 5, exclusively yielding the corresponding allenylic alcohols 6′, resulting from an SN2′ reaction. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2003)
Astrocytes as stem cells: Nomenclature, phenotype, and translation
Authors: Steindler Dennis A. , Laywell Eric D.
Published: May 2003

Recently discovered multipotent astrocytic stem cells are discussed in light of current nomenclature for glial precursor and lineage‐associated cells in the developing, postnatal, and adult mammalian brain. Defining the phenotype of any immature cell in the nervous system is a challenge, and a position is stated that includes the need for categorizing cells within a continuum of differentiation potential. The possibility for dedifferentiating glial cells into clonogenic stem‐like cells offers numerous possibilities for translating knowledge and technology from this subfield of stem cell biology to regenerative medicine. Along with the need for developing a new lexicon for defining the cellular players that contribute to the generation of glia and neurons in the developing and mature central nervous system, the relationships also need to be established among potency, repopulation attempts, and tumorigenesis of cells meeting the criteria of glial stem cells. Finally, it is possible that understanding the normal differentiation, de‐ and transdifferentiation potential of glial stem‐like cells in the mature central nervous system will provide insights into the possible use of these cells, or biogenic factors associated with their growth and differentiation, in therapeutic approaches for a variety of neurological disorders. GLIA 43:62–69, 2003. © 2003 Wiley‐Liss, Inc.
Radial glia and somal translocation of radial neurons in the developing cerebral cortex
Authors: Nadarajah Bagirathy
Published: May 2003

A series of recent studies have demonstrated that radial glia are neural precursors in the developing cerebral cortex. These studies have further implied that these cells are the sole precursor constituents of the dorsal forebrain ventricular zone that generate the projection neurons of the cortex. In view of these new findings, this review discusses radial neurons, a progeny of cortical neurons that are generated by radial glia and adopt somal translocation as the mode of migration. GLIA 43:33–36, 2003. © 2003 Wiley‐Liss, Inc.
Radial glia diversity: A matter of cell fate
Authors: Kriegstein Arnold R. , Götz Magdalena
Published: May 2003

Early in development of the central nervous system, radial glial cells arise from the neuroepithelial cells lining the ventricles around the time that neurons begin to appear. The transition of neuroepithelial cells to radial glia is accompanied by a series of structural and functional changes, including the appearance of “glial” features, as well as the appearance of new signaling molecules and junctional proteins. However, not all radial glia are alike. Radial glial lineages appear to be heterogeneous both within and across different brain regions. Subtypes of neurogenic radial glia within the cortex, for example, may have restricted potential in terms of the cell types they are able to generate. Radial glia located in different brain regions also differ in their expression of growth factors, a diverse number of transcription factors, and the cell types they generate, suggesting that they are involved in regionalization of the developing nervous system in several aspects. These findings highlight the important but complex role of radial glia as participants in key steps of brain development. GLIA 43:37–43, 2003. © 2003 Wiley‐Liss, Inc.
Use of a biomimetic strategy to engineer bone
Authors: Holy C. E. , Fialkov J. A. , Davies J. E. , Shoichet M. S.
Published: May 2003

Engineering trabecular‐like, three‐dimensional bone tissue throughout biodegradable polymer scaffolds is a significant challenge. Using a novel processing technique, we have created a biodegradable scaffold with geometry similar to that of trabecular bone. When seeded with bone‐marrow cells, new bone tissue, the geometry of which reflected that of the scaffold, was evident throughout the scaffold volume and to a depth of 10 mm. Preseeded scaffolds implanted in non‐healing rabbit segmental bone defects allowed new functional bone formation and bony union to be achieved throughout the defects within 8 weeks. This marks the first report of successful three‐dimensional bone‐tissue engineering repair using autologous marrow cells without the use of supplementary growth factors. We attribute our success to the novel scaffold morphology. © 2003 Wiley Periodicals, Inc. J Biomed Mater Res 65A: 447–453, 2003
Adhesion of human leukocytes to biomaterials: An in vitro study using alkanethiolate monolayers with different chemically functionalized surfaces
Authors: Barbosa Judite N. , Barbosa Mário A. , Águas Artur P.
Published: May 2003

The adhesion of human leukocytes to self‐assembled monolayers of well‐defined surface chemistry was investigated in vitro. Polymorphonuclear (PMN) and mononuclear leukocytes were isolated from human blood by centrifugation techniques. The effect on adhesion of cell activation produced by pre‐incubation of leukocytes with phytohemagglutinin (PHA) and phorbol 12‐myristate 13‐acetate (PMA) was also studied. Gold substrates were modified by treatment with alkanethiols with three different terminal chemical groups: COOH, OH, and CH3. After incubation with the two subpopulations of leukocytes, the monolayers were washed, treated with fixative, stained with a Giemsa method, and observed by light microscopy to quantify the number of attached leukocytes. Comparative quantification of the density of leukocyte adhesion to the three types of self‐assembled monolayers was determined. The hydrophobic surface expressing CH3 was found to be the one that induced the highest adhesion density of leukocytes, both of PMN and mononuclear cells. In vitro activation of both mononuclear and PMN leukocytes further increased cell adhesion to the chemically defined monolayers that were used. This enhancement was higher for PHA‐activated than for PMA‐stimulated mononuclear cells, whereas PMA treatment of neutrophils resulted in a higher rate of adhesion of these cells than PHA stimulation. © 2003 Wiley Periodicals, Inc. J Biomed Mater Res 65A: 429–434, 2003
Engineering integrin‐specific surfaces with a triple‐helical collagen‐mimetic peptide
Authors: Reyes Catherine D. , García Andrés J.
Published: May 2003

Integrin‐mediated cell adhesion to extracellular matrix proteins anchors cells and triggers signals that direct cell function. The integrin α2β1 recognizes the glycine‐phenylalanine‐hydroxyproline‐glycine‐glutamate‐arginine (GFOGER) motif in residues 502–507 of the α1(I) chain of type I collagen. Integrin recognition is entirely dependent on the triple‐helical conformation of the ligand similar to that of native collagen. This study focuses on engineering α2β1‐specific bioadhesive surfaces by immobilizing a triple‐helical collagen‐mimetic peptide incorporating the GFOGER binding sequence onto model nonadhesive substrates. Circular dichroism spectroscopy verified that this peptide adopts a stable triple‐helical conformation in solution. Passively adsorbed GFOGER‐peptide exhibited dose‐dependent HT1080 cell adhesion and spreading comparable to that observed on type I collagen. Subsequent antibody blocking conditions verified the involvement of integrin α2β1 in these adhesion events. Focal adhesion formation was observed by immunofluorescent staining for α2β1 and vinculin on MC3T3‐E1 cells. Model functionalized surfaces then were engineered using three complementary peptide‐tethering schemes. These peptide‐functionalized substrates supported α2β1‐mediated cell adhesion and focal adhesion assembly. Our results suggest that this peptide is active in an immobilized conformation and may be applied as a surface modification agent to promote α2β1–specific cell adhesion. Engineering surfaces that specifically target certain integrin–ligand interactions and signaling cascades provides a biomolecular strategy for optimizing cellular responses in biomaterials and tissue engineering applications. © 2003 Wiley Periodicals, Inc. J Biomed Mater Res 65A: 511–523, 2003
Local infusion of FGF‐2 enhances bone ingrowth in rabbit chambers in the presence of polyethylene particles
Authors: Goodman Stuart B. , Song Yong , Yoo Juh Yung , Fox Nora , Trindade Michael C.D. , Kajiyama Glen , Ma Ting , Regula Donald , Brown Jim , Smith R. Lane
Published: May 2003

Osseointegration of porous‐coated implants during revision arthroplasty procedures is often impeded due to the presence of residual granuloma, particulate debris, and a sclerotic, dysvascular bone bed. We hypothesized that local infusion of recombinant fibroblast growth factor (FGF‐2) would increase bone ingrowth in an in vivo model of tissue differentiation in the rabbit tibia in the presence of phagocytosable polyethylene particles. A drug test chamber (DTC) was implanted in the proximal medial tibial metaphysis of mature rabbits unilaterally. The chamber contained a 1× 1 × 5‐mm tunnel for tissue ingrowth, and was connected to an osmotic diffusion pump. FGF‐2 was infused at dosages of 0, 0.5, 5, 50, or 500 ng/day for a 3‐week period, with subsequent harvesting of the ingrown tissue after each 3‐week treatment. The effects of ultrahigh molecular weight polyethylene particles (0.5‐μm diameter) on tissue ingrowth were determined by adding particles to the chamber at concentrations of 5.8 × 1011 (low dose) or 1.7 × 1012 (high dose) particles/mL, with and without infusion of 50 ng/day of FGF for 3 weeks. The tissue forming in the chamber was harvested after each treatment for histologic processing and morphometric analysis of bone ingrowth. Statistical analysis was performed using parametric tests (ANOVA), nonparametric tests (Kruskal–Wallis test) and post hoc tests. In the absence of particles, infusion of 50 ng FGF‐2 per day yielded the greatest amount of bone ingrowth. The high dose of particles suppressed bone ingrowth into the chamber, but the low dose particles did not (p = 0.0002, 95% confidence limits = 9.19–18.80). Infusion of 50 ng FGF‐2 per day significantly increased net bone formation in the presence of high‐dose UHMWPE particles (p = 0.039, 95% confidence limits = 1.41–6.79). There was a trend for decreased numbers of vitronectin‐receptor positive (osteoclast‐like) cells with the addition of FGF‐2, compared to particles alone (p = 0.08). Local delivery of FGF‐2 may prove useful in mitigating the adverse effects of wear debris (e.g., in treating early osteolytic lesions), and facilitating osseointegration of revision total joint replacements in situations where the bone bed is suboptimal and residual particles and granulomatous tissue are present. © 2003 Wiley Periodicals, Inc. J Biomed Mater Res 65A: 454–461, 2003
Vitamin D enhances mitogenesis mediated by keratinocyte growth factor receptor in keratinocytes
Authors: Gamady Anat , Koren Ruth , Ron Dina , Liberman Uri A. , Ravid Amiram
Published: May 2003

The hormonally active vitamin D metabolite, 1,25‐dihydroxyvitamin D3 (1,25(OH)2D3), and keratinocyte growth factor (KGF) belong to the network of autocrine and paracrine mediators in the skin. Both were shown to modulate keratinocyte proliferation, to reverse epidermal atrophy, to increase wound healing, and to reduce chemotherapy‐induced alopecia. The overlap between their activities may suggest that vitamin D exerts some of its actions by modulation of KGF activities in the skin. This notion was examined by using HaCaT keratinocytes cultured in serum‐free medium in the absence of exogenous growth factors and in the presence of the EGF receptor tyrosine kinase inhibitor AG 1478 that blocks their autonomous proliferation. These cells could be stimulated to proliferate by different fibroblast growth factors (FGFs). The relative mitogenic efficacy of basic FGF, acidic FGF, or KGF was in correlation with their affinities for the KGF receptor (KGFR). Forty‐eight hour co‐treatment with 1,25(OH)2D3 enhanced KGFR‐mediated cell proliferation in a dose dependent manner. Both ERK1/2 and c‐Jun N‐terminal kinase (JNK) were activated by the FGFs. Treatment with 1,25(OH)2D3 increased the activation of ERK but reduced the activation of JNK. Treatment with 1,25(OH)2D3 increased the levels of KGFR in the presence but not in the absence of KGF, probably due to inhibition of ligand‐induced receptor degradation. Inhibition of protein kinase C with bisindolylmaleimide did not interfere with the effect of 1,25(OH)2D3 on KGFR‐mediated ERK activation. Our results support the notion that the paracrine KGF–KGFR system in the skin can act in concert with the autocrine vitamin D system in keratinocytes to promote keratinocyte proliferation and survival under situations of stress and injury. J. Cell. Biochem. 89: 440–449, 2003. © 2003 Wiley‐Liss, Inc.
Retaining of the assembly capability of vimentin phosphorylated by mitogen‐activated protein kinase‐activated protein kinase‐2
Authors: Cheng Ting‐Jen , Tseng Yu‐Fang , Chang Whei‐meih , Chang Margaret Dah‐Tsyr , Lai Yiu‐Kay
Published: May 2003

Intermediate filament (IF) networks can be regulated by phosphorylation of unit proteins, such as vimentin, by specific kinases leading to reorganization of the IF filamentous structure. Recently, we identified mitogen‐activated protein kinase‐activated protein kinase‐2 (MAPKAP kinase‐2) as a vimentin kinase (Cheng and Lai [1998] J. Cell. Biochem. 71:169–181). Herein we describe the results of further in vitro studies investigating the effects of MAPKAP kinase‐2 phosphorylation on vimentin and the effects of the phosphorylation on the filamentous structure. We show that MAPKAP kinase‐2 mainly phosphorylates vimentin at Ser‐38, Ser‐50, Ser‐55, and Ser‐82, residues all located in the head domain of the protein. Surprisingly, and in stark contrast to phosphorylation by most other kinases, phosphorylation of vimentin by MAPKAP kinase‐2 has no discernable effect on its assembly. It suggested that structure disassembly is not the only obligated consequence of phosphorylated vimentin as regulated by other kinases. Finally, a mutational analysis of each of the phosphorylated serine residues in vimentin suggested that no single serine site was primarily responsible for structure maintenance, implying that the retention of filamentous structure may be the result of the coordinated action of several phosphorylated serine sites. This also shed new lights on the functional task(s) of vimentin that is intermediate filament proteins might provide a phosphate reservoir to accommodate the phosphate surge without any structural changes. J. Cell. Biochem. 89: 589–602, 2003. © 2003 Wiley‐Liss, Inc.
DcR3/TR6 modulates immune cell interactions
Authors: Wan Xiaochun , Shi Guixiu , Semenuk Mark , Zhang Jun , Wu Jiangping
Published: May 2003

DcR3/TR6, a secreted protein, is a member of TNF receptor family. Its ligands include FasL, LIGHT, and TL1A, all TNF family members. TR6 can interfere with FasL‐ or LTβR‐mediated apoptosis; it can also inhibit T‐cell costimulation by blocking the two‐way signaling between TR2 and LIGHT, and the one‐way signaling from TL1A to DR3. In this study, we discovered that TR6 was secreted by peripheral blood mononuclear cells (PBMC) stimulated by T‐cell mitogens. It inhibited actin polymerization of T cells upon mitogen stimulation, and repress T‐cell pseudopodium formation, which is known to be important for cell–cell interaction. As a consequence, T‐cell aggregation stimulated by alloantigens, anti‐CD3 or PHA was suppressed by either soluble or solid phase TR6‐Fc. This result suggests that TR6 might regulate T‐cell interaction with other cells such as antigen‐presenting cells (APC) or their fellow T cells by preventing them from forming inseparable cell clusters, which are undesirable for the progression of immune responses. © 2003 Wiley‐Liss, Inc.
Phospholipase C–protein kinase C mediated phospholipase D activation pathway is involved in tamoxifen induced apoptosis
Authors: Ahn Soo‐Jung , Yoon Mee‐Sup , Hyuk Shin , Han Wonshik , Yoon Yong‐Dal , Han Joong‐Soo , Noh Dong‐Young
Published: May 2003

Tamoxifen (TAM) is the endocrine therapeutic agent the most widely used in the treatment of breast cancer, and it operates primarily through the induction of apoptosis. In this study, we attempted to elucidate the non‐ER mediated mechanism behind TAM treatment, involving the phospholipase C–protein kinase C (PLC–PKC) mediated phospholipase D (PLD) activation pathway, using multimodality methods. In TAM treated MCF7 cells, the PLC and PLD protein and mRNA levels increased. Phosphatidylethanol (PEt) and diacylglycerol (DAG) generation also increased, showing increased activity of PLD and PLCγ1. Translocation of PKCα, from cytosol to membrane, was observed in TAM treated cells. By showing that both PKC and PLC inhibitors could reduce the effects of TAM‐induced PLD activation, we confirmed the role of PKC and PLC as upstream regulators of PLD. Finally, we demonstrated that TAM treatment reduced the viability of MCF7 cells and brought about rapid cell death. From these results, we confirmed the hypothesis that TAM induces apoptosis in breast cancer cells, and that the signal transduction pathway, involving PLD, PLC, and PKC, constitutes one of the possible mechanisms underlying the non‐ER mediated effects associated with TAM. © 2003 Wiley‐Liss, Inc.
The sphingosine 1‐phosphate receptor S1P4 regulates cell shape and motility via coupling to Gi and G12/13
Authors: Gräler Markus H. , Grosse Robert , Kusch Angelika , Kremmer Elisabeth , Gudermann Thomas , Lipp Martin
Published: May 2003

Sphingosine 1‐phosphate (S1P) receptors represent a novel subfamily of G‐protein‐coupled receptors binding S1P specifically and with high affinity. Although their in vivo functions remain largely unknown, in vitro extracellular application of S1P induces distinct S1P receptor‐dependent cellular responses including proliferation, differentiation, and migration. We have analyzed signaling pathways engaged by S1P4, which is highly expressed in the lymphoid system. Here we show that S1P4 couples directly to Gαi and even more effectively to Gα12/13‐subunits of trimeric G‐proteins, but not to Gαq unlike other S1P receptors. Consequently, CHO‐K1 cells ectopically expressing S1P4 potently activate the small GTPase Rho and undergo cytoskeletal rearrangements, inducing peripheral stress fiber formation and cell rounding, upon S1P stimulation. Overexpression of S1P4 in Jurkat T cells induces pertussis toxin‐sensitive cell motility even in the absence of exogenously added S1P. In addition, S1P4 is internalized upon binding of S1P. The capacity of S1P4 to mediate cellular responses, such as motility and shape change through Gαi‐ and Gα12/13‐coupled signaling pathways may be important for its in vivo function which is currently under investigation. © 2003 Wiley‐Liss, Inc.
Understanding of lubrication with neutrons
Authors: Wolff M. , Magerl A. , Frick B. , Zabel H.
Published: May 2003

Lubrication is an old but nevertheless barely understood phenomenon. Insufficient lubrication leads to permanent wear, causing an estimated damage in the USA of about 6 % of the GNP [1]. Already the Egyptian civilisation was aware of the usefulness of lubricants. The painting from the grotto at El‐Bershed dated about 1880 B.C. shows the use of a lubricant to spare the power of men [2]. In recent years new sophisticated experimental techniques like surface force apparatus [3, 4], friction force microscopy [3, 5] and quartz‐crystal microbalance [6] have been employed to test theories and computer simulations [3, 7] of friction mechanism. However, these methods cannot provide in situ information of the sliding action and the dynamical response on a molecular level. The large penetration power of thermal neutrons allow non‐destructive studies of samples in complex environments. Combining this with an outstanding energy resolution makes it possible to study simultaneously the macroscopic flow and the microscopic diffusion of flowing liquids. For a commercial motor oil we found surface slip. In addition, under shear the microscopic diffusion becomes anisotrop.
Adult primary extragonadal germ cell tumors: Treatment results and long‐term follow‐up
Authors: Andac Argon , Mert Basaran , Sevil Bavbek , Burak Sakar , Haluk Onat
Published: May 2003

Background

Primary extragonadal germ cell tumors (PEGCT) are rare neoplasms. They have a poor prognosis, different behavior, and natural course compared to their gonadal counterparts. Both primary and salvage treatment of these tumors constitute a challenge. We retrospectively evaluated the clinicopathologic status, therapeutic implications, and outcome of our patients with PEGCT.

Procedure

Between 1991 and 2000, 18 patients with PEGCT (median age 31 years; range 17–63), diagnosed with tru‐cut biopsy and treated with cisplatin‐based chemotherapy, were evaluated in respect to treatment response and outcome.

Results

Cisplatin‐based chemotherapy achieved a complete response rate of 27.8% and a partial response rate of 55.5%. Overall response rate was 83.3%. Only three patients were unresponsive to chemotherapy; an additional six patients with residual mass underwent surgical resection and were rendered disease‐free by surgery. The 5‐year actuarial event‐free and overall survival were 63.4 and 71.3%, respectively.

Conclusions

The outcomes of our patients with extragonadal primaries including mediastinal localization appear to be slightly better than those previously reported. Multimodality therapy is essential for these patients and given the relatively poor prognosis, prospective trials with large sample sizes, and new treatment approaches to improve outcome are required. Med Pediatr Oncol 2003;41:49–53. © 2003 Wiley‐Liss, Inc.
Amplitude modulation of nonlinear waves in a fluid‐filled tapered elastic tube
Authors: Demiray H.
Published: May 2003

In the present work, treating the arteries as a tapered, thin walled, long, and circularly conical prestressed elastic tube and using the reductive perturbation method, we have studied the amplitude modulation of nonlinear waves in such a fluid‐filled elastic tube. By considering the blood as an incompressible non‐viscous fluid, the evolution equation is obtained as the nonlinear Schrödinger equation with variable coefficients. It is shown that this type of equations admit a solitary wave type of solution with a variable wave speed. It is observed that the wave speed increases with distance for narrowing tubes while it decreases for expanding tubes.
Diffraction of plane waves by an acoustically penetrable strip located between two soft/hard half‐planes
Authors: Çınar G. , Büyükaksoy A.
Published: May 2003

Uniform asymptotic high‐frequency solution is developed for the diffraction of plane waves by an acoustically transmissive strip located between two half‐planes which are soft at the top and hard at the bottom. After simulating the partially transmissive strip by “resistive type” boundary conditions, the three‐part boundary value problem is formulated as a “Modified Matrix Wiener‐Hopf” equation. By performing the Wiener‐Hopf factorization of the kernel matrix through the Daniele‐Khrapkov method, the modified matrix Wiener‐Hopf equation is first reduced to a pair of coupled Fredholm integral equations of the second kind and then solved by iterations. Diffracted field expressions are derived up to the third order terms which include the surface wave field effects in a uniform manner.
Stress intensity factors for a penny‐shaped crack in a transversely isotropic layer bonded between two isotropic half spaces
Authors: Ataberk N.
Published: May 2003

In this paper, a problem in an elastic‐perfectly plastic dissimilar layered medium is considered. It is assumed that a transversely isotropic layer is sandwiched between two isotropic semi‐infinite half spaces, and contains a penny‐shaped crack located in its mid‐plane. The problem is formulated by using integral transform technique under uniform load and reduced to a singular integral equation. This integral equation is solved numerically by using Gaussian Quadrature Formulae. Stress intensity factors are given for various penny‐shaped crack sizes and transversely isotropic materials.
Interfacial edge crack between two bonded dissimilar orthotropic strips under antiplane point loading
Authors: Wu X.‐F. , Dzenis Y.A. , Zou W.‐S.
Published: May 2003

A closed‐form solution is obtained for the interfacial edge crack between two bonded dissimilar orthotropic strips loaded by antiplane point loading in form of screw dislocation or line force. Conformal mapping and existing dislocation solutions are utilized for constructing the fundamental solution of the problem. The stress intensity factor (SIF) and the energy release rate (ERR) are obtained explicitly.
Suppression of antiproliferative effects of tumor necrosis factor by transfection of cells with human platelet‐derived growth factor B/c‐sis gene
Authors: Aggarwal Bharat B. , Pocsik Eva , Totpal Klara , Ali-Osman Francis
Published: May 2003

The growth of cells is determined by the balance between growth‐stimulatory and growth‐inhibitory signals. In the present study, we demonstrate that the transfection of NIH 3T3 cells with a platelet‐derived growth factor (PDGF‐B/c‐sis) gene induces resistance to the anticellular effects of tumor necrosis factor (TNF). Human tumor cell lines that express elevated levels of c‐sis (e.g. epidermoid carcinoma, A‐431) are also TNF resistant, whereas those that express no significant levels of this gene (e.g. breast adenocarcinoma, MCF‐7) are TNF sensitive. Transfection of cells with the c‐sis gene leads to down‐modulation of TNF receptors and also a decrease in intracellular glutathione levels. Thus, our results demonstrate that over‐expression of PDGF‐B/c‐sis by certain tumor cells can lead to their protection from the anticellular effects of TNF.
Heat denaturation of pepsinogen in a water‐ethanol mixture
Authors: Makarov A.A. , Protasevich I.I. , Bazhulina N.P. , Esipova N.G.
Published: May 2003

The effect of ethanol and pH on thermodynamic parameters and cooperativity of pepsinogen heat denaturation was studied by scanning microcalorimetry. Addition of 20% ethanol decreases the protein denaturation temperature by 10.7°C at pH 6.4 and 15.8°C at pH 8.0. It also decreases the denaturation heat capacity increment from 5.8 to 4.2 kcal/K·mol. The dependences of calorimetric denaturation enthalpy on denaturation temperature both in water and 20% ethanol are linear and intersect at about 95°C. In 20% ethanol the pH shift from 5.9 to 8.0 results in a decreased number of cooperative domains in pepsinogen. This process causes no changes either in the secondary structure or in the local surroundings of aromatic amino acids. It is concluded that ethanol addition does not affect the cooperativity of pepsinogen denaturation substantially until the pH change provokes redistribution of charges in the protein molecule.
Phosphate regulation of biosynthesis of extracellular RNases of endospore‐forming bacteria
Authors: Znamenskaya L.V. , Gabdrakhmanova L.A. , Chernokalskaya E.B. , Leshchinskaya I.B. , Hartley R.W.
Published: May 2003

The gene for the extracellular ribonuclease of B. pumilus KMM62 (RNase Bp) has been cloned and sequenced. The structural gene for this enzyme is similar to those of the extracellular ribonucleases of B. intermedius 7P (binase) and B. amyloliquefaciens H2 (barnase), as are the regulatory regions of binase and RNase Bp. The regulatory region of the barnase gene, however, is quite different from the other two. In the promoter of the genes for binase and RNase Bp, but not in that for barnase, is a region similar to the Pho box of E. coli. We have established that inorganic phosphate suppresses the synthesis of the binase and RNase Bp, but does not effect the synthesis of barnase.
A common topology of proteins catalyzing ATP‐triggered reactions
Authors: Yoshida Masasuke , Amano Toyoki
Published: May 2003

A protein fold, six parallel β strands surrounding the central α helix, is likely to be a common structure in protein families known to have a typical set of nucleotide binding consensus sequenced motifs A and B and to catalyze ATP‐triggered reactions. According to this ATP‐triggered protein fold, the conserved Glu (or Asp), which acts as a general base to activate a water molecule for an in‐line attack of the γ‐phosphate, is at the exit of the second β strand. The fifth β strand may be involved in propagation of conformational change triggered by ATP hydrolysis.
HOQNO interaction with cytochrome b in succinate:menaquinone oxidoreductase from Bacillus subtilis
Authors: Smirnova Irina A. , Hägerhäll Cecilia , Konstantinov Alexandre A. , Hederstedt Lars
Published: May 2003

2‐n‐Heptyl4‐hydroxyquinoline‐N‐oxide (HOQNO) inhibits the succinate:quinone oxidoreductase activity of isolated and membrane‐bound succinate:menaquinone oxidoreductase of B. subtilis. The inhibition pattern resembles closely that observed for α‐thenoyltrifluoroacetone and carboxins in the mitochondrial succinate:ubiquinone oxidoreductase: ca. 90% of the activity is highly sensitive to HOQNO (K

i
ca. 0.2 μM for the isolated enzyme) whereas the rest 10% proves to be resistant to the inhibitor. HOQNO binding is shown to perturb the absorption spectrum of the ferrous di‐heme cytochrome b of the B. subtilis succinate:quinone oxidoreductase both in the α and Soret bands. In addition, the inhibitor is shown to bring about a negative shift of E
m of the low‐potential heme b. It is suggested that HOQNO interacts with a menasemiquinone binding site near the low‐potential heme and suppresses the MQ.−‐to‐MQH2 step of the quinone reductase reaction but allows partly for the MQ‐to‐MQ.− transition to occur; dismutation of MQ. formed in the latter reaction to MQ and MQH2 may account for the 10% of the enzyme activity insensitive to HOQNO.
Rapid kinetics of membrane potential generation by cytochrome c oxidase with the photoactive Ru(II)‐tris‐bipyridyl derivative of cytochrome c as electron donor
Authors: Zaslavsky D.L. , Smirnova I.A. , Siletsky S.A. , Kaulen A.D. , Millett F. , Konstantinov A.A.
Published: May 2003

Yeast iso‐1‐cytochrome c covalently modified at cysteine‐102 with (4‐bromomethyl‐4′‐methylbipyridine)[bis(bi‐pyridine)]Ru2+ (Ru‐102‐Cyt c) has been used as a photoactive electron donor to mitochondrial cytochrome c oxidase (COX) reconstituted into phospholipid vesicles. Rapid kinetics of membrane potential generation by the enzyme following flash‐induced photoreduction of Ru‐102‐Cyt c heme has been measured and compared to photovoltaic responses observed with Ru(II)(bipy‐ridyl)3 (RuBpy) as the photoreductant [D.L. Zaslavsky et al. (1993) FEBS Lett. 336, 389–393]. At low ionic strength, when Ru‐102‐Cyt c forms a tight electrostatic complex with COX, flash‐activation results in a polyphasic electrogenic response corresponding to transfer of a negative charge to the interior of the vesicles. The initial rapid phase is virtually identical to the 50 μs transient observed in the presence of RuBpy as the photoactive electron donor which originates from electrogenic reduction of heme a by CuA. CuA reduction by Ru‐102‐Cyt c turns out to be not electrogenic in agreement with the peripheral location of visible copper in the enzyme. A millisecond phase (τ ca. 4 ms) following the 50 μs initial part of the response and associated with vectorial translocation of protons linked to oxygen intermediate interconversion in the binuclear centre, can be resolved both with RuBpy and Ru‐102‐Cyt c as electron donors; however, this phase is small in the absence of added H2O2. In addition to these two transients, the flash‐induced electrogenic response in the presence of Ru‐102‐Cyt c reveals a large slow phase of Δψ generation not observed with RuBpy. This phase is completely quenched upon inclusion of 100 μM ferricyanide in the medium and originates from a second order reaction of COX with the excess Ru‐102‐Cyt c
2+ generated by the flash in a solution.
Quinacrine mustard and lipophilic cations inhibitory to both vacuolar H+‐ATPase and F0F1‐ATP synthase
Authors: Moriyama Yoshinori , Patel Vikram , Futai Masamitsu
Published: May 2003

Various lipophilic cations, such as quinacrine mustard and dequalinium, which are known to inhibit mitochondrial F1‐ATPase, strongly inhibited vacuolar H+‐ATPase purified from bovine adrenal chromaffin granules. Quinacrine mustard bound irreversibly to vacuolar H+‐ATPase subunit A, and the 115 kDa accessory polypeptide and dithiothreitol had no effect. The binding was competitively inhibited by chlorpromazine and quinacrine, and these compounds specifically reduced the amount of labeling of subunit A. Quinacrine mustard also prevented the binding of [α‐32P]ATP to subunit A but had no effect on the binding of to either subunit A or the 115 kDa accessory polypeptide. These results suggest that the binding site of quinacrine mustard in subunit A is not related to the N‐ethylmaleimide‐binding site(s), which is important for activity.
DNA‐binding sperm proteins with oligo‐arginine clusters function as potent activators for egg CK‐II
Authors: Ohtsuki Kenzo , Nishikawa Yukihiro , Saito Hitoshi , Munakata Hiroshi , Kato Toyoki
Published: May 2003

The stimulatory effect of DNA‐binding sperm proteins (histone and protamine) on the phosphorylation of p98 (ERp99/GRp94, one of the Hsp‐90 family of proteins) by egg casein kinase II (CK‐II) was investigated in vitro. It was found that (i) phosphorylation of p98 by egg CK‐II in vitro is greatly stimulated by poly‐Arg, but not by poly‐Lys; and (ii) similar stimulation is observed with sperm histones H2B2 and H2B3 (sea urchin) and fish protamines, such as salmine A1 (salmon) and protamine 3a (rainbow trout). These findings suggest that these DNA‐binding sperm proteins function as potent activators for CK‐II in fertilized eggs. All of these DNA‐binding sperm proteins contain at least an oligo‐Arg cluster as a common feature, which can interact with an acidic amino acid cluster of the regulatory β‐subunit CK‐II.
Identification of a novel protein containing two C2 domains selectively expressed in the rat brain and kidney
Authors: Kwon Oh-Joo , Gainer Harold , Wray Susan , Chin Hemin
Published: May 2003

We have isolated and characterized a rat brain cDNA clone which encodes a new protein of 474 amino acids in length which contains two C2 domains structurally homologous to those present in synaptotagmins. The overall amino acid identity in C2 domains between this protein and the synaptotagmins is 36–44%. This protein also contains 3 putative consensus sequences for phosphorylation by cAMP‐dependent protein kinase. RNA blot hybridization revealed a 3.0 kb transcript abundantly expressed only in the rat brain and the kidney. Thus, we called this brain/kidney protein (B/K). In situ hybridization and Northern blot analyses showed that the B/K transcript was found in forebrain including the olfactory bulb, cerebral cortex, hippocampus, and hypothalamus. In the kidney, high levels of B/K transcript were expressed in the papillary region of the inner medulla, the inner stripe of the outer medulla and the cortex. The selective expression in forebrain and kidney suggests that B/K may be involved in similar cAMP‐dependent processes at these very different sites.