bluuebunny/crossref_metadata_2025 huggingface dataset:
Glucocorticoids modulate amino acid-induced translation initiation in human skeletal muscle
Authors: Liu Zhenqi , Li Guolian , Kimball Scot R. , Jahn Linda A. , Barrett Eugene J.
Published: March 2004

Amino acids are unique anabolic agents in that they nutritively signal to mRNA translation initiation and serve as substrates for protein synthesis in skeletal muscle. Glucocorticoid excess antagonizes the anabolic action of amino acids on protein synthesis in laboratory animals. To examine whether excessive glucocorticoids modulate mixed amino acid-signaled translation initiation in human skeletal muscle, we infused an amino acid mixture (10% Travasol) systemically to 16 young healthy male volunteers for 6 h in the absence ( n = 8) or presence ( n = 8) of glucocorticoid excess (dexamethasone 2 mg orally every 6 h for 3 days). Vastus lateralis muscles were biopsied before and after amino acid infusion, and the phosphorylation of eukaryotic initiation factor (eIF) 4E-binding protein 1 (4E-BP1), ribosomal protein S6 kinase (p70S6K), and eIF2α and the guanine nucleotide exchange activity of eIF2B were measured. Systemic infusion of mixed amino acids significantly stimulated the phosphorylation of 4E-BP1 ( P < 0.04) and p70S6K( P < 0.001) and the dephosphorylation of eIF2α ( P < 0.003) in the control group. Dexamethasone treatment did not alter the basal phosphorylation state of 4E-BP1, p70S6K, or eIF2α; however, it abrogated the stimulatory effect of amino acid infusion on the phosphorylation of 4E-BP1 ( P = 0.31) without affecting amino acid-induced phosphorylation of p70S6K( P = 0.002) or dephosphorylation of eIF2α ( P = 0.003). Neither amino acid nor dexamethasone treatment altered the guanine nucleotide exchange activity of eIF2B. We conclude that changes of amino acid concentrations within the physiological range stimulate mRNA translation by enhancing the binding of mRNA to the 43S preinitiation complex, and the activity of p70S6Kand glucocorticoid excess blocks the former action in vivo in human skeletal muscle.
Protection against puromycin aminonucleoside-induced chronic renal disease in the Wistar-Furth rat
Authors: Erdely Aaron , Freshour Gary , Smith Cheryl , Engels Kevin , Olson Jean L. , Baylis Chris
Published: March 2004

The Wistar-Furth (WF) rat is protected against chronic renal disease (CRD) following 5/6th ablation/infarction vs. the Sprague-Dawley (SD) rat, and protection was associated with preserved renal nitric oxide (NO) production. This study examined CRD induced with repeated administration of puromycin aminonucleoside (PAN). SD PAN developed nephrotic range proteinuria (>1 g/24 h), and at 15 wk severe renal injury developed and the glomerular filtration rate (GFR) was reduced to ∼10% of sham. Total NO production, renal NO synthase (NOS) activity, and renal neuronal (n) and medullary endothelial (e)NOS abundance were reduced in the SD PAN. WF PAN exhibited less severe initial proteinuria (>400 mg/24 h), which abated within weeks, whereas GFR was normal and injury was minimal at 15 wk. Total NO production and renal NOS activity and abundance were significantly elevated compared with SD PAN. NOS mRNA (nNOS, eNOS, and inducible NOS) was not altered in WF, whereas SD showed significant increases in NOS gene expression with PAN. In conclusion, WF showed resistance to a second model of CRD with maintained renal NOS activity compared with SD.
Finite element analysis of active Eustachian tube function
Authors: Ghadiali Samir N. , Banks Julie , Swarts J. Douglas
Published: March 2004

The inability to open the collapsible Eustachian tube (ET) has been related to the development of chronic otitis media. Although ET dysfunction may be due to anatomic and/or mechanical abnormalities, the precise mechanisms by which these structural properties alter ET opening phenomena have not been investigated. Previous investigations could only speculate on how these structural properties influence the tissue deformation processes responsible for ET opening. We have, therefore, developed a computational technique that can quantify these structure-function relationships. Cross-sectional histological images were obtained from eight normal adult human subjects, who had no history of middle ear disease. A midcartilaginous image from each subject was used to create two-dimensional finite element models of the soft tissue structures of the ET. ET opening phenomena were simulated by applying muscle forces on soft tissue surfaces in the appropriate direction and were quantified by calculating the resistance to flow (Rv) in the opened lumen. A sensitivity analysis was conducted to determine the relative importance of muscle forces and soft-tissue elastic properties. Muscle contraction resulted in a medial-superior rotation of the medial lamina, stretching deformation in the Ostmann's fatty tissue, and lumen dilation. Variability in baseline Rv values correlated with tissue size, whereas the functional relationship between Rv and a given mechanical parameter was consistent in all subjects. ET opening was found to be highly sensitive to the applied muscle forces and relatively insensitive to cartilage elastic properties. These computational models have, therefore, identified how different tissue elements alter ET opening phenomena, which elements should be targeted for treatment, and the optimal mechanical properties of these tissue constructs.
Group I Metabotropic Glutamate Receptor Activation Produces a Direct Excitation of Identified Septohippocampal Cholinergic Neurons
Authors: Wu Min , Hajszan Tibor , Xu Changqing , Leranth Csaba , Alreja Meenakshi
Published: March 2004

Septohippocampal cholinergic neurons innervate the hippocampus and provide it with almost its entire acetylcholine. Axon collaterals of these neurons also release acetylcholine within the septum and thereby maintain the firing activity of septohippocampal GABAergic neurons. A loss of septohippocampal cholinergic neurons occurs in various neurodegenerative disorders associated with cognitive dysfunctions. group I metabotropic glutamate receptors have been implicated in septohippocampal-dependent learning and memory tasks. In the present study, we examined the physiological and pharmacological effects of a potent and selective group I metabotropic glutamate receptor (mGluR) agonist S-3,5-dihydroxyphenylglycine (DHPG) on rat septohippocampal cholinergic neurons that were identified in brain slices using a selective fluorescent marker. In whole cell recordings, DHPG produced a reversible, reproducible and a direct postsynaptic and concentration-dependent excitation in 100% of septohippocampal cholinergic neurons tested with an EC50 of 2.1 μM. Pharmacologically, the effects of DHPG were partially/completely reduced by the mGluR1 antagonists, 7-hydrox-iminocyclopropan[b]chromen-1a-carboxylic acid ethyl ester and (+)-2-methyl-4-carboxyphenylglycine. Addition of the mGluR5 antagonist, 2-methyl-6-(phenylethnyl)pyridine hydrochloride, reduced the remaining response to DHPG, suggesting involvement of both receptor subtypes in a subpopulation of septohippocampal cholinergic neurons. In double-immunolabeling studies, 74% of septohippocampal cholinergic neurons co-localized mGluR1α-immunoreactivity and 35% co-localized mGluR5-immunoreactivity. Double-immunolabeling studies at the light and electron-microscopic levels showed that vesicular glutamate transporter 2 terminals make asymmetric synaptic contacts with septohippocampal cholinergic neurons. These findings may be of significance in treatment of cognitive deficits associated with neurodegenerative disorders as a group I mGluR-mediated activation of septohippocampal cholinergic neurons would enhance the release of acetylcholine both in the hippocampus and in the septum.
A numerical study of effects of the axial stress on unsteadyliquid pipeline flows
Authors: Watanabe Masaji , Kono Yukio , Suito Hiroshi
Published: March 2004

We study the effects of the axial component of the shear stress on
unsteady pipeline flows. We show that the axial component of the
shear stress should be introduced in the modeling of unsteady
flows, and as a numerical model, we propose a one‐dimensional
momentum equation in which a term containing the second
derivative of the velocity with respect to space is
introduced. The momentum equation and the continuity equation are
converted into a system suitable for the application of upstream
difference approximations. Numerical results are presented, and
their correspondence with experimental results is examined to see
how our model captures phenomena observed experimentally.
Cleavage of Focal Adhesion Kinase in Vascular Smooth Muscle Cells Overexpressing Membrane-Type Matrix Metalloproteinases
Authors: Shofuda Tomoko , Shofuda Ken-ichi , Ferri Nicola , Kenagy Richard D. , Raines Elaine W. , Clowes Alexander W.
Published: March 2004

Background—

Membrane-type matrix metalloproteinases (MT-MMPs) were initially identified as cell surface activators of MMP-2 (gelatinase A). We have reported that MT1-MMPs and MT3-MMPs are expressed by activated vascular smooth muscle cells (SMCs) and play a role in the regulation of cell function. Overexpression of MT-MMPs results in cell rounding, decreased adherence, and increased migration. Because integrin-mediated cell adhesion regulates these events, we have investigated the functional relationship between MT-MMPs and focal adhesion assembly.

Methods and Results—

Using adenoviral vectors we show that overexpression of MT-MMPs reduces the number of focal contacts, whereas the cell surface expression of integrin subunits remains unchanged. The 125-kDa focal adhesion kinase (FAK) is cleaved resulting in a 90-kDa fragment under these conditions, and paxillin is partially dissociated from FAK after its cleavage. Pretreatment of cells with BB94, a synthetic MMP inhibitor, rescues cell adhesion and prevents changes in focal adhesions, supporting a potential role for MT-MMP enzymatic activities.

Conclusions—

This study provides the first evidence that MT-MMPs are not only important in matrix degradation but also may affect the function of focal adhesions through FAK cleavage.
C-Reactive Protein Does Not Directly Induce Tissue Factor in Human Monocytes
Authors: Paffen Elaine , Vos Hans L. , Bertina Rogier M.
Published: March 2004

Objective—It is generally assumed that C-reactive protein (CRP) induces synthesis of tissue factor (TF) in monocytic cells, thereby potentially initiating intravascular blood coagulation. We aimed to elucidate the mechanism of CRP-induced TF expression in monocytes and monocyte-derived macrophages (MDMs) in vitro.

Methods and Results—Monocytes were isolated from the blood of healthy donors and cultured with or without CRP or lipopolysaccharide (LPS) to study the time course of TF antigen and TF mRNA expression. Addition of 100 μg/mL CRP did not result in a significant increase in TF antigen (range: 9 to 163 pg/106cells, n=11) and TF mRNA (relative number of TF transcripts; NTF=0.01 to 0.33), when compared with nonstimulated cells (TF antigen 7 to 46 pg/106cells, NTF=0.01 to 0.13). Variation of CRP concentration and exposure time did not affect the TF response. Similar results were obtained in monocytes cultured in suspension and in MDMs. In contrast, TF was strongly induced by 10 μg/mL LPS (TF antigen 1125 to 6120 pg/106cells, NTF=5.94 to 23.43). Cultured monocytes did express FcRγII, a putative CRP receptor, and addition of CRP induced a 7-fold increase in the production of monocyte chemoattractant protein-1 (MCP-1). Interestingly, CRP addition to peripheral blood mononuclear cells (PBMCs) did result in TF expression on monocytic cells.

Conclusions—The absence of TF induction after incubation of purified monocytes with CRP indicates that CRP is unable to induce TF expression in monocytes and MDMs directly. The presence of CRP-induced TF expression in PBMCs suggests that CRP can induce TF indirectly, probably through cross-talk between cells.
Clinical Usefulness of Very High and Very Low Levels of C-Reactive Protein Across the Full Range of Framingham Risk Scores
Authors: Ridker Paul M , Cook Nancy
Published: March 2004

Background—

High-sensitivity C-reactive protein (hsCRP) is a strong independent risk factor for cardiovascular events, and levels of hsCRP of <1, 1 to <3, and ≥3 mg/L have been suggested to define low-, moderate-, and high-risk groups. However, the positive predictive value of very low (<0.5 mg/L) and very high levels of hsCRP (>10.0 mg/L) is uncertain.

Methods and Results—

Baseline levels of hsCRP were evaluated among 27 939 apparently healthy women who were followed up for myocardial infarction, stroke, coronary revascularization, or cardiovascular death. Crude and Framingham Risk Score (FRS)–adjusted relative risks (RRs) of incident cardiovascular events were calculated across a full range of hsCRP levels. Cardiovascular risks increased linearly from the very lowest (referent) to the very highest levels of hsCRP. Crude RRs for those with baseline hsCRP levels of <0.5, 0.5 to <1.0, 1.0 to <2.0, 2.0 to <3.0, 3.0 to <4.0, 4.0 to <5.0, 5.0 to <10.0, 10.0 to <20.0, and ≥20.0 mg/L were 1.0, 2.2, 2.5, 3.1, 3.7, 4.2, 4.9, 6.3, and 7.6, respectively (
P
for trend <0.001). After adjustment for FRS, these risks were 1.0, 1.6, 1.6, 1.7, 1.9, 2.2, 2.3, 2.8, and 3.1 (
P
for trend <0.001). All risk estimates remained significant in analyses stratified by FRS and after control for diabetes. Of the total cohort, 15.1% had hsCRP <0.50 mg/L, and 5.4% had hsCRP >10.0 mg/L.

Conclusions—

Both very low (<0.5 mg/L) and very high (>10 mg/L) levels of hsCRP provide important prognostic information on cardiovascular risk. hsCRP is clinically useful for risk prediction across a full range of values and across a full range of FRS.
Sphingosine 1-Phosphate Transactivates the Platelet-Derived Growth Factor β Receptor and Epidermal Growth Factor Receptor in Vascular Smooth Muscle Cells
Authors: Tanimoto Tatsuo , Lungu Andreea O. , Berk Bradford C.
Published: March 2004

Sphingosine 1-phosphate (S1P) is a bioactive lipid generated during vascular injury that regulates cell growth, differentiation, survival, and motility via endothelial differentiation gene (EDG) family G protein–coupled receptors. Although several G protein–coupled receptor ligands transactivate receptor tyrosine kinases, such as the epidermal growth factor receptor (EGFR), S1P-stimulated receptor tyrosine kinase transactivation has not been well studied. We show that platelet-derived growth factor β receptor (PDGFβR) and EGFR are tyrosine phosphorylated in response to S1P in rat aortic vascular smooth muscle cells (VSMCs). S1P-stimulated transactivation of PDGFβR and EGFR was mediated via Gi-coupled EDG receptors. S1P-stimulated transactivation of EGFR and PDGFβR was dependent on Src, reactive oxygen species, and cholesterol-rich membranes. A phosphoinositide 3-kinase–Akt pathway was activated by S1P and blocked by AG1296 and AG1478. Activation of extracellular signal–regulated kinase (ERK) 1 and ERK2 pathway by S1P was blocked only by AG1478. In Chinese hamster ovary cells that expressed exogenous EDG-1, activation of Akt and ERK1/2 in response to S1P was observed and was enhanced by coexpression of PDGFβR or EGFR. S1P-mediated VSMC proliferation was shown to be secondary to transactivation, because it was suppressed by AG1296 and AG1478. These data establish S1P as an important stimulus for EGFR and PDGFβR activation in VSMCs that may have important implications for the vessel response to injury.
Cell-Demanded Liberation of VEGF
121
From Fibrin Implants Induces Local and Controlled Blood Vessel Growth
Authors: Ehrbar Martin , Djonov Valentin G. , Schnell Christian , Tschanz Stefan A. , Martiny-Baron Georg , Schenk Ursula , Wood Jeanette , Burri Peter H. , Hubbell Jeffrey A. , Zisch Andreas H.
Published: March 2004

Although vascular endothelial growth factor (VEGF) has been described as a potent angiogenic stimulus, its application in therapy remains difficult: blood vessels formed by exposure to VEGF tend to be malformed and leaky. In nature, the principal form of VEGF possesses a binding site for ECM components that maintain it in the immobilized state until released by local cellular enzymatic activity. In this study, we present an engineered variant form of VEGF, α
2
PI
1–8
-VEGF
121
, that mimics this concept of matrix-binding and cell-mediated release by local cell-associated enzymatic activity, working in the surgically-relevant biological matrix fibrin. We show that matrix-conjugated α
2
PI
1–8
-VEGF
121
is protected from clearance, contrary to native VEGF
121
mixed into fibrin, which was completely released as a passive diffusive burst. Grafting studies on the embryonic chicken chorioallantoic membrane (CAM) and in adult mice were performed to assess and compare the quantity and quality of neovasculature induced in response to fibrin implants formulated with matrix-bound α
2
PI
1–8
-VEGF
121
or native diffusible VEGF
121
. Our CAM measurements demonstrated that cell-demanded release of α
2
PI
1–8
-VEGF
121
increases the formation of new arterial and venous branches, whereas exposure to passively released wild-type VEGF
121
primarily induced chaotic changes within the capillary plexus. Specifically, our analyses at several levels, from endothelial cell morphology and endothelial interactions with periendothelial cells, to vessel branching and network organization, revealed that α
2
PI
1–8
-VEGF
121
induces vessel formation more potently than native VEGF
121
and that those vessels possess more normal morphologies at the light microscopic and ultrastructural level. Permeability studies in mice validated that vessels induced by α
2
PI
1–8
-VEGF
121
do not leak. In conclusion, cell-demanded release of engineered VEGF
121
from fibrin implants may present a therapeutically safe and practical modality to induce local angiogenesis.
Urokinase-Type Plasminogen Activator Receptor Is Involved in Mediating the Apoptotic Effect of Cleaved High Molecular Weight Kininogen in Human Endothelial Cells
Authors: Cao Dian J. , Guo Yan-Lin , Colman Robert W.
Published: March 2004

Cleaved high molecular weight kininogen (HKa) has been shown to inhibit in vivo neovascularization and induce apoptosis of endothelial cells. We have shown that HKa-induced apoptosis correlated with its antiadhesive effect and was regulated by extracellular matrix (ECM) proteins. In this study, we identified the urokinase-type plasminogen activator receptor (uPAR) as a target of HKa activity at the endothelial cell surface. Anti-uPAR antibodies blocked the apoptotic effect of HKa. Further studies revealed that uPAR formed a signaling complex containing integrin α
v
β
3
or α
5
β
1
, caveolin, and Src kinase Yes in endothelial cells. HKa physically disrupted the formation of this complex in a manner that paralleled its apoptotic effect. For the first time, our results provide a mechanistic explanation for the previous observation that HKa selectively induces apoptosis of endothelial cells grown on vitronectin, but not cells grown on fibronectin. These data also resolve the controversial role of uPAR in mediating the apoptotic and antiadhesive activities of HKa.
Rapid chemokine secretion from endothelial cells originates from 2 distinct compartments
Authors: Øynebråten Inger , Bakke Oddmund , Brandtzaeg Per , Johansen Finn-Eirik , Haraldsen Guttorm
Published: March 2004

The neutrophil-attracting chemokine interleukin 8 (IL-8) is stored in the Weibel-Palade body (WPB) of endothelial cells (ECs) from which it can be rapidly released after exposure to the secretagogues histamine or thrombin. In this manner, IL-8 may enable rapid recruitment of leukocytes to inflammatory sites. To explore the possible storage of EC-derived chemokines that may attract other subsets of leukocytes, we examined the intracellular localization and secretagogue responsiveness of growth-related oncogene α (GROα), monocyte chemoattractant protein-1 (MCP-1), eotaxin-3, interferon-γ-inducible protein 10 (IP-10), and regulated on activation, normal T-cell expressed and secreted (RANTES). While eotaxin-3, GROα, and MCP-1 were rapidly released from ECs, the release of the T-cell attractors RANTES and IP-10 was not sensitive to the secretagogues. Moreover, of the 3 former chemokines, only eotaxin-3 was stored in WPBs. GROα and MCP-1 resided mainly in smaller vesicles compatible with sorting to a different, histamine-responsive compartment, which has been described in ECs although not reported to contain chemokines. In conclusion, we propose that rapid release of chemokines is restricted to those primarily recruiting leukocytes of the innate immune system, and that their storage in ECs is not restricted to the WPB compartment. (Blood. 2004;104:314-320)
Direct evidence of leukocyte adhesion in arterioles by angiotensin II
Authors: Álvarez Ángeles , Cerdá-Nicolás Miguel , Naim Abu Nabah Yafa , Mata Manuel , Issekutz Andrew C. , Panés Julián , Lobb Roy R. , Sanz Maria-Jesus
Published: March 2004

Although leukocytes adhere in arteries in various vascular diseases, to date no endogenous proinflammatory molecule has been identified to initiate leukocyte adhesion in the arterial vasculature. This study was undertaken to assess angiotensin II (Ang II)-induced leukocyte adhesion in arterioles in vivo. Rats received intraperitoneal injections of Ang II; 4 hours later, leukocyte recruitment in mesenteric microcirculation was examined using intravital microscopy. Ang II (1 nM) produced significant arteriolar leukocyte adhesion of mononuclear cells. Using function-blocking monoclonal antibodies (mAbs) against different rat cell adhesion molecules (CAMs), we discovered that this effect was dependent on P-selectin and β2-integrin. In postcapillary venules, Ang II also induced leukocyte infiltration, which was reduced by P-selectin and by β2- and α4-integrin blockade. Interestingly, neutrophils were the primary cells recruited in venules. Although β2-integrin expression in peripheral leukocytes of Ang II-treated animals was not altered, it was increased in peritoneal cells. Immunohistochemical studies revealed increased P-selectin, E-selectin, intercellular cell adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) expression in response to Ang II in arterioles and venules. These findings provide the first evidence that Ang II causes leukocyte adhesion to the arterial endothelium in vivo at physiologically relevant doses. Therefore, Ang II may be a key molecule in cardiovascular diseases in which leukocyte adhesion to the arteries is a characteristic feature. (Blood. 2004;104:402-408)
Constitutive STAT6 activation in primary mediastinal large B-cell lymphoma
Authors: Guiter Chrystelle , Dusanter-Fourt Isabelle , Copie-Bergman Christiane , Boulland Marie-Laure , le Gouvello Sabine , Gaulard Philippe , Leroy Karen , Castellano Flavia
Published: March 2004

Primary mediastinal large B-cell lymphoma (PMBL), currently recognized as a diffuse large B-cell lymphoma (DLBCL) subtype, shows increased expression of interleukin 4 (IL-4)/IL-13 signaling effectors and targets, suggesting constitutive activation of these pathways. We therefore investigated the functional state of the signal transducer and activator of transcription 6 (STAT6), mediating IL-4/IL-13 transcriptional effects. Constitutive STAT6 phosphorylation and DNA-binding activity were detected in PMBL cell lines but not DLBCL cell lines. Moreover, immunohistochemical analysis revealed nuclear phosphorylated STAT6 (P-STAT6) in 8 of 11 PMBL, compared with 1 of 10 DLBCL primary tumors (P = .01). IL-4 and IL-13 transcripts were absent in PMBL cell lines and expressed at low levels in tumors, indicating that, contrary to classical Hodgkin lymphoma (cHL), STAT6 activation is not due to an autocrine IL-4/IL-13 secretion. We demonstrated an amplification of the JAK2 gene in 2 of 6 PMBL cases, and showed higher JAK2 mRNA levels in PMBL compared with DLBCL (P = .005). The Janus kinase 2 (JAK2) was constitutively phosphorylated in the PMBL MedB1 cell line. MedB1 treatment with JAK2 inhibitor AG490 partially decreased STAT6 phosphorylation, suggesting that JAK2 is partially involved in STAT6 activation in these cells. Our findings highlight phosphorylated STAT6 as a characteristic distinguishing PMBL from DLBCL, but a common feature to PMBL and cHL, supporting the hypothesis of common pathogenic events in these 2 lymphomas. (Blood. 2004;104: 543-549)
Infection of C57BL/10ScCr and C57BL/10ScNCr mice withLeishmania majorreveals a role for Toll-like receptor 4 in the control of parasite replication
Authors: Kropf P , Freudenberg N , Kalis C , Modolell M , Herath S , Galanos C , Freudenberg M , Müller I
Published: March 2004

The innate immune system is essential for host defense; it senses the presence of potentially pathogenic-invading microorganisms, and the contribution of Toll-like receptors (TLRs) to this response is increasingly recognized. In the present study, we investigated the contribution of TLR4 to the course of cutaneous leishmaniasis in vivo. We used C57BL/10ScNCr (TLR40/0) and C57BL/10ScCr [TLR4/interleukin-12 (IL-12)Rβ20/0] mice and compared the course of Leishmania major infection, parasite load, cell recruitment, and cytokine profile with those of wild-type C57BL/10ScSn mice. Our results confirm the importance of IL-12 receptor-mediated signaling in resistance to L. major infections. Importantly, we show that the lack of TLR4 results in an increased permissiveness for parasite growth during the innate and adaptive phase of the immune response and in delayed healing of the cutaneous lesions. The use of the tlr4 transgenic mouse strain TCr5 demonstrated unequivocally that TLR4 contributes to the efficient control of Leishmania growth in vivo.
Oxygen radical-induced natural killer cell dysfunction: role of myeloperoxidase and regulation by serotonin
Authors: Betten Åsa , Dahlgren Claes , Mellqvist Ulf-Henrik , Hermodsson Svante , Hellstrand Kristoffer
Published: March 2004

Natural killer (NK) cells are functionally suppressed and induced to apoptosis by reactive oxygen species (ROS) produced by mononuclear phagocytes (MPs). These inhibitory events are reversed by the biogenic amine serotonin. MPs generate hydrogen peroxide (H2O2), which is processed further by myeloperoxidase (MPO) to even more toxic compounds. Earlier studies suggest that serotonin scavenges MP-derived oxygen radicals generated by the MPO-H2O2 system. These findings led us to explore the capability of MPO-deficient MPs to induce NK cell dysfunction. We show that MPs recovered from subjects with MPO deficiency trigger inhibition of NK cells. In addition, MPs recovered from healthy subjects conveyed suppression of NK cells in the presence of the MPO inhibitor ceruloplasmin. We conclude that ROS-dependent inhibition of NK cell function is unrestricted by the availability of MPO-derived oxygen radicals and that the protecting properties of serotonin may operate in the absence of functional MPO. Our data suggest a complex mechanism of MP-induced NK cell inhibition, which comprises the generation of interchangeable oxygen radicals.
Morphine-induced macrophage apoptosis: oxidative stress and strategies for modulation
Authors: Bhat Rajani S , Bhaskaran Madhu , Mongia Anil , Hitosugi Naoko , Singhal Pravin C
Published: March 2004

Occurrence of macrophage apoptosis has been implicated for the altered immune function found in an opiate milieu. In the present study, we evaluated the role of oxidative stress in morphine-induced macrophage apoptosis. Morphine promoted the apoptosis of macrophages. This effect of morphine was associated with the production of superoxide and nitric oxide (NO). Antioxidants provided protection against morphine-induced macrophage injury. In addition, diphenyleneiodonium chloride, an inhibitor of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation, attenuated the proapoptotic effect of morphine. Antitransforming growth factor-β (anti-TGF-β) antibody and propranolol (an inhibitor of the phospholipase D pathway) inhibited morphine-induced superoxide generation as well as apoptosis. N′-Tetraacetic acid tetra (acetoxymethyl) ester, a calcium-chelating agent, inhibited morphine-induced apoptosis, whereas thapsigargin (a calcium agonist) stimulated macrophage apoptosis under basal as well as morphine-stimulated states. These studies suggest that morphine-induced macrophage apoptosis is mediated through downstream signaling involving TGF-β and NO production. Moreover, there is NADPH oxidation activation involving phospholipase D and Ca2+, leading to the generation of superoxide. In in vivo studies, administration of N-acetyl cysteine and preinduction of heme oxygenase activity and epoetin α prevented morphine-induced peritoneal macrophage apoptosis, thus further confirming the role of oxidative stress in morphine-induced macrophage apoptosis.
Abbreviated Course of Radiation Therapy in Older Patients With Glioblastoma Multiforme: A Prospective Randomized Clinical Trial
Authors: Roa W. , Brasher P.M.A. , Bauman G. , Anthes M. , Bruera E. , Chan A. , Fisher B. , Fulton D. , Gulavita S. , Hao C. , Husain S. , Murtha A. , Petruk K. , Stewart D. , Tai P. , Urtasun R. , Cairncross J.G. , Forsyth P.
Published: March 2004

Purpose

To prospectively compare standard radiation therapy (RT) with an abbreviated course of RT in older patients with glioblastoma multiforme (GBM).

Patients and Methods

One hundred patients with GBM, age 60 years or older, were randomly assigned after surgery to receive either standard RT (60 Gy in 30 fractions over 6 weeks) or a shorter course of RT (40 Gy in 15 fractions over 3 weeks). The primary end point was overall survival. The secondary end points were proportionate survival at 6 months, health-related quality of life (HRQoL), and corticosteroid requirement. HRQoL was assessed using the Karnofsky performance status (KPS) and Functional Assessment of Cancer Therapy-Brain (FACT-Br).

Results

All patients had died at the time of analysis. Overall survival times measured from randomization were similar at 5.1 months for standard RT versus 5.6 months for the shorter course (log-rank test, P = .57). The survival probabilities at 6 months were also similar at 44.7% for standard RT versus 41.7% for the shorter course (lower-bound 95% CI, −13.7). KPS scores varied markedly but were not significantly different between the two groups (Wilcoxon test, P = .63). Low completion rates of the FACT-Br (45%) precluded meaningful comparisons between the two groups. Of patients completing RT as planned, 49% of patients (standard RT) versus 23% required an increase in posttreatment corticosteroid dosage (χ2 test, P = .02).

Conclusion

There is no difference in survival between patients receiving standard RT or short-course RT. In view of the similar KPS scores, decreased increment in corticosteroid requirement, and reduced treatment time, the abbreviated course of RT seems to be a reasonable treatment option for older patients with GBM.
Glucose Homeostasis and Tissue Transcript Content of Insulin Signaling Intermediates in Four Inbred Strains of Mice: C57BL/6, C57BLKS/6, DBA/2, and 129X1
Authors: Goren H. Joseph , Kulkarni Rohit N. , Kahn C. Ronald
Published: March 2004

Transgenic mice phenotypes generally depend on the background strains used in their creation. To examine the effects of genetic background on insulin signaling, we analyzed glucose homeostasis in four inbred strains of mice [C57BL/6 (B6), C57BLKS/6 (KLS), DBA/2 (DBA), and 129X1] and quantitated mRNA content of insulin receptor (IR) and its substrates in insulin-responsive tissues. At 2 months, the male B6 mouse is the least glucose-tolerant despite exhibiting similar insulin sensitivity and first-phase insulin secretion as the other strains. The 129X1 male mouse islet contains less insulin and exhibits a higher threshold for glucose-stimulated first-phase insulin secretion than the other strains. Female mice generally manifest better glucose tolerance than males, which is likely due to greater insulin sensitivity in liver and adipose tissue, a robust first-phase insulin secretion in B6 and KLS females, and improved insulin sensitivity in muscle in DBA and 129X1 females. At 6 months, although males exhibit improved first-phase insulin secretion, their physiology was relatively unchanged, whereas female B6 and KLS mice became less insulin sensitive. Gene expression of insulin signaling intermediates in insulin-responsive tissues was generally not strain dependent with the cell content of IR mRNA being highest. IR substrate (IRS)-1 and IRS-2 mRNA are ubiquitously expressed and IRS-3 and IRS-4 mRNA were detected in significant amounts in fat and brain tissues, respectively. These data indicate strain-, gender-, and age-dependent tissue sensitivity to insulin that is generally not associated with transcript content of IR or its substrates and should be taken into consideration during phenotypic characterization of transgenic mice.
Minireview: Characterization of Influence of Central Nociceptin/Orphanin FQ on Consummatory Behavior
Authors: Olszewski Pawel K. , Levine Allen S.
Published: March 2004

Nociceptin/orphanin FQ (N/OFQ), a peptide closely related to dynorphin A, is the endogenous agonist of the NOP receptor that moderately increases food intake under various conditions. Its orexigenic properties are mediated by the brain circuitry. In the present review, we focus on discussing the nature of hyperphagic effects of N/OFQ with special emphasis on its function within feeding-related neural networks. Although some of N/OFQ’s orexigenic effects resemble those induced by opioids, reward-dependent feeding appears to be affected in a different manner by agonists of the NOP and classical opioid receptors. Also, data suggest that N/OFQ may not only promote feeding initiation, but rather its role may be to inhibit signaling responsible for inhibition of consummatory behavior. Central systems involved in termination of feeding that seem to be influenced by N/OFQ encompass oxytocin, α-MSH, and CRH.
Expression of Progesterone Receptors A and B in the Mouse Ovary during the Estrous Cycle
Authors: Gava Natalie , Clarke Christine L. , Byth Karen , Arnett-Mansfield Rebecca L. , deFazio Anna
Published: March 2004

Progesterone plays a central role in the regulation of ovarian function. The progesterone receptor (PR) has been shown to be essential for ovulation because mice lacking PR fail to ovulate and are infertile. PR is expressed as two isoforms, PRA and PRB, which have been shown to have different functional activities. In this study, we investigated the cellular distribution of PRA and PRB in the ovaries and oviducts of cycling mice using immunohistochemistry with isoform-specific monoclonal antibodies. In the ovary, on the evening of proestrus before ovulation, both the granulosa and theca cells of the preovulatory follicles expressed both PR isoforms. PRA and PRB staining was also observed in the theca cells of preantral and antral follicles, whereas only PRB was observed in the granulosa cells of primary, preantral, and antral follicles and in the corpus luteum. In the oviduct, PRA was the predominant isoform observed, expressed in both the epithelial and stromal cells, whereas PRB was only detected in the epithelial cells. The differences in PRA and PRB localization in the ovary and oviduct may reflect diverse functions for PRA and PRB in reproductive tissues and may have important implications in understanding the mechanisms of progesterone action.
Regulation of transcription of meiotic cell cycle and terminal differentiation genes by the testis-specific Zn-finger proteinmatotopetli
Authors: Perezgasga Lucia , Jiang JianQiao , Bolival Benjamin , Hiller Mark , Benson Elizabeth , Fuller Margaret T. , White-Cooper Helen
Published: March 2004

A robust developmentally regulated and cell type specific transcriptional programme is activated in primary spermatocytes in preparation for differentiation of the male gametes during spermatogenesis. Work in Drosophila is beginning to reveal the genetic networks that regulate this gene expression. The Drosophila aly-class meiotic arrest loci are essential for activation of transcription of many differentiation-specific genes, as well as several genes important for meiotic cell cycle progression,thus linking meiotic cell cycle progression to cellular differentiation during spermatogenesis. The three previously described aly-class proteins(aly, comr and achi/vis) form a complex and are associated with chromatin in primary spermatocytes. We identify, clone and characterize a new aly-class meiotic arrest gene, matotopetli (topi), which encodes a testis-specific Zn-finger protein that physically interacts with Comr. The topimutant phenotype is most like achi/vis in that topi function is not required for the nuclear localization of Aly or Comr, but is required for their accumulation on chromatin. Most target genes in the transcriptional programme depend on both topi and achi/vis; however, a small subset of target genes are differentially sensitive to loss of topior achi/vis, suggesting that these aly-class predicted DNA binding proteins can act independently in some contexts.
Development of primitive and definitive hematopoiesis from nonhuman primate embryonic stem cells in vitro
Authors: Umeda Katsutsugu , Heike Toshio , Yoshimoto Momoko , Shiota Mitsutaka , Suemori Hirofumi , Luo Hong Yuan , Chui David H. K. , Torii Ryuzo , Shibuya Masabumi , Nakatsuji Norio , Nakahata Tatsutoshi
Published: March 2004

Although information about the development of primitive and definitive hematopoiesis has been elucidated in murine embryos and embryonic stem (ES)cells, there have been few in vitro studies of these processes in primates. In this study, we investigated hematopoietic differentiation from cynomolgus monkey ES cells grown on OP9, a stromal cell line deficient in macrophage colony-stimulating factor. Primitive erythrocytes (EryP) and definitive erythrocytes (EryD) developed sequentially from ES cells in the culture system; this was confirmed by immunostaining and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of embryonic, fetal and adult globin genes. EryP were detected on day 8 without exogenous erythropoietin (EPO), whereas EryD appeared on day 16 and had an indispensable requirement for exogenous EPO. RT-PCR analysis of the cultures revealed a sequential expression of genes associated with primitive and definitive hematopoietic development that was equivalent to that seen during primate ontogeny in vivo. Vascular endothelial growth factor (VEGF) increased, in a dose-dependent manner, not only the number of floating hematopoietic cells,but also the number of adherent hematopoietic cell clusters containing CD34-positive immature progenitors. In colony assays, exogenous VEGF also had a dose-dependent stimulatory effect on the generation of primitive erythroid colonies. More efficient primitive and definitive erythropoiesis was induced by re-plating sorted CD34-positive cells. Thus, this system reproduces early hematopoietic development in vitro and can serve as a model for analyzing the mechanisms of hematopoietic development in primates.
Palmitoylation controls trafficking of GAD65 from Golgi membranes to axon-specific endosomes and a Rab5a-dependent pathway to presynaptic clusters
Authors: Kanaani Jamil , Diacovo Maria Julia , El-Husseini Alaa El-Din , Bredt David S. , Baekkeskov Steinunn
Published: March 2004

The GABA-synthesizing enzyme GAD65 is synthesized as a soluble cytosolic protein but undergoes post-translational modification(s) to become anchored to the cytosolic face of Golgi membranes before targeting to synaptic vesicle membranes in neuroendocrine cells. Palmitoylation of cysteines 30 and 45 in GAD65 is not required for targeting to Golgi membranes but is crucial for post-Golgi trafficking to presynaptic clusters in neurons. Here, we show that palmitoylated GAD65 colocalizes with the small GTP-binding protein Rab5a in Golgi membranes and in axons but not in dendrites. In the presence of the constitutively positive mutant Rab5(Q79L) palmitoylation resulted in polarized targeting of GAD65 to giant Rab5a-positive axonal endosomes, characterized by the absence of the Rab5a-effector molecule EEA1 and the transferrin receptor. By contrast, Rab5a-positive/EEA1-positive somatodendritic giant endosomes containing the transferrin receptor were devoid of GAD65. Palmitoylation-deficient GAD65 was excluded from endosomal compartments. A dominant negative mutant of Rab5a, Rab5a(S34N), specifically blocked axonal trafficking and presynaptic clustering of palmitoylated GAD65, but did not affect axonal trafficking of mutants of GAD65 that fail to traffic to giant axonal endosomes containing Rab5a(Q79L). Two transmembrane synaptic vesicle proteins, VAMP2 and VGAT also localized to the axonal giant endosomes, and their axonal trafficking and presynaptic clustering was blocked by Rab5a(S34N). The results suggest that palmitoylation of GAD65 regulates the trafficking of the protein from Golgi membranes to an endosomal trafficking pathway in axons that is dependent on Rab5a and is required for the targeting of several synaptic vesicle proteins to presynaptic clusters.
Smad7 is required for TGF-β-induced activation of the small GTPase Cdc42
Authors: Edlund Sofia , Landström Maréne , Heldin Carl-Henrik , Aspenström Pontus
Published: March 2004

Transforming growth factor β (TGF-β) is a potent regulator of cell growth and differentiation in many cell types. The Smad signaling pathway constitutes a main signal transduction route downstream of TGF-β receptors. The inhibitory Smads, Smad6 and Smad7, are considered to function as negative regulators of the TGF-β/Smad signaling cascade. In a previous study, we found that TGF-β induces rearrangements of the actin filament system in human prostate carcinoma cells and that this response requires the small GTPases Cdc42 and RhoA. On the basis of the current view on the function of Smad7 in TGF-β signaling, we hypothesized that Smad7 would function as a negative regulator of the TGF-β-induced activation of Cdc42 and RhoA, but instead we found that the reverse is the case; Smad7 is required for the TGF-β-induced activation of Cdc42 and the concomitant reorganization of the actin filament system. These observations propose a novel role for Smad7 in TGF-β-dependent activation of Rho GTPases.
Glucose induces de novo lipogenesis in rat muscle satellite cells through a sterol-regulatory-element-binding-protein-1c-dependent pathway
Authors: Guillet-Deniau Isabelle , Pichard Anne-Lise , Koné Aminata , Esnous Catherine , Nieruchalski Myriam , Girard Jean , Prip-Buus Carina
Published: March 2004

We previously reported that sterol-regulatory-element-binding-protein-1c (SREBP-1c) mediates insulin upregulation of genes encoding glycolytic and lipogenic enzymes in rat skeletal muscle. Here, we assessed whether glucose could regulate gene expression in contracting myotubes deriving from cultured muscle satellite cells. Glucose uptake increased twofold after a 30 minute treatment with a high glucose concentration, suggesting an acute glucose-stimulated glucose uptake. Time-course experiments showed that, within 3 hours, glucose stimulated the expression of hexokinase II, fatty acid synthase and acetyl-CoA-carboxylase-2 proteins, leading to an increased lipogenic flux and intracellular lipid accumulation in contracting myotubes. Furthermore, kinetic experiments indicated that glucose upregulated SREBP-1c precursor and nuclear proteins within 30 minutes, SREBP-1c nuclear translocation being confirmed using immunocytochemistry. In addition, the knockdown of SREBP-1 mRNA using a RNA-interference technique totally abrogated the glucose-induced upregulation of lipogenic enzymes, indicating that SREBP-1c mediates the action of glucose on these genes in rat skeletal muscle. Finally, we found that glucose rapidly stimulated SREBP-1c maturation through a Jak/STAT dependent pathway. We propose that increased intramuscular lipid accumulation associated with muscle insulin resistance in obesity or type-2 diabetes could arise partly from de novo fatty acid synthesis in skeletal muscle.
Temperature Dependence of the Structure of Alkyl Monolayers on Si(111) Surface via Si–C Bond by ATR-FT-IR Spectroscopy
Authors: Yamada Ryo , Ara Masato , Tada Hirokazu
Published: March 2004

The temperature dependence of C–H stretching modes of alkyl monolayer formed on Si(111) surface was investigated by an attenuated total reflection Fourier transform infrared spectroscopy from room temperature up to 540 K. Continuous disordering of the monolayer was indicated from the gradual peak shifts toward higher frequency in C–H stretch modes upon heating. The irreversible conformational disorder was introduced in the monolayer above 440 K.
Iodo-cyclization of N-Homoallyl Thioamides Leading to 2,4-Diaryl-5,6-dihydro-4H-1,3-thiazines
Authors: Murai Toshiaki , Niwa Hisayuki , Kimura Tsutomu , Shibahara Fumitoshi
Published: March 2004

Iodo-cyclization of N-homoallyl thioamides was carried out in the presence of Et3N to form 2,4-diaryl-5,6-dihydro-4H-1,3-thiazines in good to high yields. The reaction of thioamides bearing 1-naphthyl or 2-methoxyphenyl group at the carbon atom adjacent to the nitrogen atom showed high diastereoselectivity. The relative stereochemistry was confirmed by X-ray molecular structure analyses. The obtained thiazines were converted to 6-alkylidene-1,3-thiazines by treating with pyrrolidine.
A study of behavioural responses of non-human primates to air transport and re-housing
Authors: Honess P. E. , Johnson P. J. , Wolfensohn S. E.
Published: March 2004

More long-tailed macaques ( Macaca fascicularis) than any other primate are imported into the UK for research, and journey times may be of up to 58 h. Whilst a number of studies have examined the stress associated with transport, these have typically involved laboratory rodents and livestock, and little is known of its effect on non-human primates. This paper reports the results of a study of behavioural changes in a group of long-tailed macaques transported by air from standard breeding conditions and then re-housed in standard laboratory primate conditions. The animals were studied prior to their departure, immediately after their arrival, and 3 weeks after that. Data were collected on individual time budgets using focal animal sampling and on hierarchy using a feeding trial. The data were analysed for changes in behavioural repertoires and for social perturbation that would be reflected in hierarchical changes. Changes in behaviour occurred which reflected heightened levels of stress in the study group. It was also clear that although there was some adjustment of behaviour, after an initial change on arrival at the new establishment, there was no return to levels observed at the breeding facility within the first month. This study demonstrates that, as a whole, the process of international air transport and re-housing in laboratory conditions may result in the compromising of the welfare of the study animals.