bluuebunny/crossref_metadata_2025 huggingface dataset:
Intensity Modulated Radiation Therapy for Retroperitoneal Sarcoma:A Case for Dose Escalation and Organ at Risk Toxicity Reduction
Authors: Koshy Mary , Landry Jerome C. , Lawson Joshua D. , Staley Charles A. , Esiashvili Natia , Howell Rebecca , Ghavidel Shahram , Davis Lawrence W.
Published: December 2003

Purpose: Radiation therapy for retroperitoneal sarcoma remains challenging because of proximity to surrounding organs at
risk (OAR). We report the use of intensity modulated radiation therapy (IMRT) in the treatment of retroperitoneal sarcomas
to minimize dose to OAR while concurrently optimizing tumor dose coverage.

Patients and methods: From January 2000 to October 2002, 10 patients (average age 56 years) with retroperitoneal sarcoma
and one with inguinal sarcoma were treated with radiation at Emory University. Prescription dose to the planning treatment
volume (PTV) was commonly 50.4 at 1.8 Gy/fraction. CT simulation was used in each patient, three patients were treated
with 3D‐conformal treatment (3D‐CRT), and the remaining eight received multi‐leaf collimator‐based (MLC) IMRT.
IMRT treatment fields ranged from eight to 11 and average volume treated was 3498 cc. Optimal 3D‐CRT plans were
generated and compared with IMRT with respect to tumor coverage and OAR dose toxicity. Dose volume histograms were
compared for both the 3D‐CRT and IMRT plans.

Results: Mean dose to small bowel decreased from 36 Gy with 3D‐CRT to 27 Gy using IMRT, and tumor coverage (V95)
increased from 95.3% with 3D‐CRT to 98.6% using IMRT. Maximum and minimum doses delivered to the PTV were
significantly increased by 6 and 22%, respectively (P = 0.011, P = 0.055). Volume of small bowel receiving > 30Gy was
significantly decreased from 63.5 to 43.1% with IMRT compared with conventional treatment (P = 0.043). Seven patients
developed grade 2 nausea, three developed grade 2 diarrhea, one had grade 2 skin toxicity, and one patient developed grade
3 liver toxicity (RTOG toxicity scale). No other delayed toxicities related to radiation were observed. At a median follow‐up
of 58 weeks, there were no local recurrences and only one patient developed disease progression with distant metastasis in
the liver.

Conclusions: IMRT for retroperitoneal sarcoma allowed enhanced tumor coverage and better sparing of dose to
critical normal structures such as small bowel, liver, and kidney.
Escalation of dose has a positive impact on local control for retroperitoneal sarcoma;
IMRT may be an effective method to achieve this goal. We are evaluating preoperative dose escalation to 59.4 Gy.
Premonitory Pain Preceding Swelling: A Distinctive ClinicalPresentation of Synovial Sarcoma which may Prompt Early Detection
Authors: De Silva M. V. Chandu , Barrett Ann , Reid Robin
Published: December 2003

Purpose: The aim of this paper is to document the unusual presentation of long‐standing pain at the tumour site before
development of a swelling in patients with synovial sarcoma.

Patients/methods and results: The clinical presentation of 53 patients with synovial sarcoma was compared with 56 randomly
selected patients with other sarcomas of the trunk and extremities. The two groups were similar with regard to age
(P = 0.980), sex (P = 0.784) duration of symptoms
(P = 0.697), size (P = 0.931) and site of tumour (P = 0.288). Sixteen
(30.2%) patients with synovial sarcoma had pain before development of a swelling compared to two (3.6%) patients with
other sarcomas (P < 0.001, odds ratio = 11.68, 95% confidence interval 2.53, 53.83). The mean duration of such pain was
37 months (median 24, range 6–120 months). The nature of the pain was variable. Eight patients had sharply localised
tenderness. Calcification seen in the X‐rays of four patients was initially misdiagnosed as benign lesions. A swelling was
ultimately detected by MRI, CT, ultrasound or at physical examination. The mean duration from first presentation with pain
till diagnosis of synovial sarcoma was 20 months. In three patients, at explorative surgery there was friable, vascular or
necrotic tissue in the absence of a well‐defined tumour mass.

Discussion: The occurrence of long‐standing pain at the tumour site prior to development of a swelling is significantly more
common with synovial sarcomas than with other sarcomas. Awareness of this unusual presentation and appropriate
investigation may enable detection of synovial sarcoma at a prognostically favourable early stage.
Pelvic Girdle Reconstruction Based on Spinal Fusion and IschialScrew Fixation in a Case of Aneurysmal Bone Cyst
Authors: Honl Matthias , Westphal Florian , Carrero Volker , Morlock Michael , Schwieger Karsten , Hille Ekkehard , Delling G.
Published: December 2003

A case of lytic lesion of the pelvis in a 23‐year‐old woman is presented. A biopsy led to the diagnosis aneurysmal bone cyst
(ABC). Due to the histologically very aggressive growth of the tumor, a low malignant osteosarcoma could not be excluded.
In an initial operation the tumour, affecting the sacrum, the iliac crest and the lower lumbar spine was resected. Temporary
restabilisation of the pelvic ring was achieved by a titanium plate. The histological examination of the entire tumour
confirmed the diagnosis ABC. After 6 months, the MRI showed no recurrence. The observed tilt of the spine to the operated
side on the sacral base prompted a second surgical procedure: a transpedicular fixation of L5 and L4 was connected via bent
titanium stems to the ischium, where the fixation was achieved by two screws. This construction allowed the correction of the
base angle and yielded a stable closure of the pelvic ring. The patient has now been followed for 6 years: the bone grafts have
been incorporated and, in spite of radiological signs of screw loosening in the ischium, the patient is fully rehabilitated and
free of symptoms. Pedicle screws in the lower spine can be recommended for fixation of a pelvic ring discontinuity.
Angiosarcoma of the Scalp Presenting in Association WithBorderline Malignant Phyllodes Tumour of the Breast
Authors: Harden Susan V. , Ball Richard Y. , Harnett Adrian N.
Published: December 2003

Phyllodes tumours and angiosarcoma are both rare mesenchymal tumours. There are no reports of their coexistence in the
literature except in families with germline p53 mutations. Here we report a case of an elderly woman who developed an
extensive angiosarcoma of the scalp nearly 4 years after surgical removal of a borderline malignant phyllodes tumour of the
breast. The scalp lesion was initially thought more likely to be a metastasis of her first rare tumour than a second equally rare
primary tumour, but histologically this was not the case. The case and the literature are discussed.
Comparative intervention study among Danish daycare children: the effect on illness of time spent outdoors
Authors: Mygind Ole , Rønne Tove , Søe Anne-Lise , Wachmann Carsten Henrik , Ricks Philip
Published: December 2003

Aims: The purpose of the study was to examine the correlation between illnesses, and in particular contagious illnesses, among daycare children and the amount of time spent outdoors, and whether through the intervention of increasing the amount of time spent outdoors it is possible to lower the incidence of illness. Methods: The study was conducted among eight daycare centres within the same county. A total of 326 children, 17 homeroom groups, were divided into two study groups. Each of the two study groups had a two-month period in which the percentage of time spent outdoors per day was almost double that of the other study group. Results: The overall frequency of sick days was 4%. There was no significant effect of spending more time outdoors on the number of illness episodes, length of the episode, or cause of illness. Conclusion: Our negative findings are in accordance with the theory that illness among daycare children is largely a result of contact among children and the hygiene standards of the institution, and thus these are the factors that may need to be addressed to reduce the level of illness among daycare children.
Causes of death between 1911 - 1950 in a Swedish province with a population characterized by longevity: Effects on life expectancy
Authors: Baigi Amir , Högstedt Benkt , Isacsson Sven-Olof , Odén Anders , Herrström Per
Published: December 2003

Aims: Life expectancy in Sweden is among the highest in the world, and the province of Halland has the highest life expectancy in Sweden today. In an earlier paper the authors reported that life expectancy in the province of Halland in the south-west of the country was approx. 3.5 years above the national average between 1911 and 1950. The aim of this study was to explore the influence of different causes of death on life expectancy in Sweden and the distribution of these causes of death in Halland compared with Sweden as a whole during the same period of time. Method: Causes of death between 1911 and 1950 in the whole of Sweden and in Halland were obtained from the archives of Statistics Sweden. A trend analysis was performed on the impact of the various causes of death on life expectancy in Sweden. Calendar year, age, and sex were controlled for in a Poisson model. The distribution and incidence of the most frequent causes of death were compared between Halland and Sweden as a whole. Results: The decreasing mortality risk due to infectious diseases and the simultaneous increase in the risk of mortality from tumours and circulatory diseases contributed most to the change in life expectancy in Sweden. In Halland there was a lower mortality risk in the seven most important causes of death, which accounted for approx 80% of all deaths during the study period. Conclusions: The lower mortality risk from infectious diseases mostly favoured the improvement in life expectancy in Halland up to the mid-1930s. Thereafter, a low mortality risk from tumours and particularly circulatory diseases gained increasing importance although there was always a difference in favour of Halland from the beginning of the study period. Thus, the positive trend in life expectancy that favours Halland today seems to have existed for a long period of time.
Effect of living conditions in the parental home and youth paths on the social class differences in mortality among women
Authors: Pensola Tiina H. , Martikainen Pekka
Published: December 2003

Aims: A longitudinal study was undertaken to assess the effects of parental home and youth paths on the adult social class differences in mortality among women. Methods: The study used population registration data on all Finnish women aged 30 - 34 in 1990 for whom information on their childhood characteristics and youth paths were available from the 1970, 1975, 1980, 1985, and 1990 censuses. Cause of death follow-up was for the period 1991-98 (1,185 deaths). Results: Adjusting for parental social class, family type, and number of siblings attenuated the effects of adult social class on cardiovascular disease mortality by 27%; for all external causes this attenuation was negligible. Educational, marital, and employment paths accounted for a substantial part (30 - 85%) of the social class differences in mortality for all specific causes of death. Conclusions: Although living conditions in the parental home were associated with mediating life trajectories in youth their effect on adult social class differences in mortality was moderate. Youth paths have a pervasive influence on mortality risks and social class differences in mortality in middle adulthood.
Pathway Illuminated: Visualizing Protein Kinase C Signaling
Authors: Violin Jonathan D. , Newton Alexandra C.
Published: December 2003

Protein kinase C has been at the center of cell signaling since the discovery 25 years ago that it transduces signals that promote phospholipid hydrolysis. In recent years, the use of genetically encoded fluorescent reporters has enabled studies of the regulation of protein kinase C signaling in living cells. Advances in imaging techniques have unveiled unprecedented detail of the signal processing mechanics of protein kinase C, from the second messengers calcium and diacylglycerol that regulate protein kinase C activity, to the locations and kinetics of different protein kinase C isozymes, to the spatial and temporal dynamics of substrate phosphorylation by this key enzyme. This review discusses how fluorescence imaging studies have illuminated the fidelity with which protein kinase C transduces rapidly changing extracellular information into intracellular phosphorylation signals. IUBMB Life, 55: 653‐660, 2003
Competitive, Reversible, Physiological? Inhibition of Mitochondrial Cytochrome Oxidase by Nitric Oxide
Authors: Cooper Chris
Published: December 2003

In the mid 1990s a number of research groups recognized that mitochondrial oxygen consumption could be reversibly inhibited by nitric oxide at the level of the enzyme cytochrome c oxidase. The inhibition was apparently competitive with respect to the oxygen concentration. This review critically assesses the present state of knowledge as regards the hypothesis that nitric oxide is a competitive, reversible, physiological inhibitor of cytochrome oxidase. IUBMB Life, 55: 591‐597, 2003
The Contribution of Salvador Moncada to Our Understanding of the Biology of Nitric Oxide
Authors: Clementi Emilio
Published: December 2003

The observation, by Furchgott and Zawadzki, that a factor of short average life, released by endothelial cells accounted for vasodilation was the beginning of one of the most fascinating adventures in the recent history of science. The discovery that this released factor was nitric oxide had tremendous implications for our understanding not only of the homeostasis of the vascular tissue, but also of a variety of other biological processes ranging from synaptic plasticity to regulation of immune responses. This review article will lead the reader through the landmark events in this adventure, highlighting the fundamental role played by Salvador Moncada and his team. IUBMB Life, 55: 563‐565, 2003
Peroxynitrous Acid ‐ Where is the Hydroxyl Radical?
Authors: Kissner Reinhard , Nauser Thomas , Kurz Christophe , Koppenol Willem
Published: December 2003

Peroxynitrite is an inorganic toxin of physiological interest, formed from the diffusion‐controlled reaction of superoxide and nitrogen monoxide with a rate constant of (1.6 ± 0.3) × 1010 M ‐ 1 s ‐ 1. On the basis of three experiments we conclude that homolysis of the O‐O bond in peroxynitrous acid is unlikely: (1) the yield of nitrite from the decomposition of peroxynitrite shows a dependence on the peroxynitrite concentration and is lower than expected for homolysis; (2) the yield of [15N]nitrate from the reaction of [15N]nitrite with peroxynitrous acid predicted by homolysis does not correspond to that found experimentally, and (3) the reaction of peroxynitrous acid with monohydroascorbate does not yield ascorbyl radicals. Activation volumes determined from high‐pressure kinetic studies are inconclusive. IUBMB Life, 55: 567‐572, 2003
Truncated Hemoglobins and Nitric Oxide Action
Authors: Milani Mario , Pesce Alessandra , Ouellet Hugues , Guertin Michel , Bolognesi Martino
Published: December 2003

Truncated hemoglobins (trHbs) build a separate subfamily within the hemoglobin superfamily; they are scarcely related by sequence similarity to (non‐)vertebrate hemoglobins, displaying amino acid sequences in the 115 ‐ 130 residue range. The trHb tertiary structure is based on a 2‐on‐2 α‐helical sandwich, which hosts a unique hydrophobic cavity/tunnel system, traversing the protein matrix, from the molecular surface to the heme distal site. Such a protein matrix system may provide a path for diffusion of ligands to the heme. In Mycobacterium tuberculosis trHbN the heme‐bound oxygen molecule is part of an extended hydrogen bond network including the heme distal residues TyrB10 and GlnE11. In vitro experiments have shown that M. tuberculosis trHbN supports efficiently nitric oxide dioxygenation, yielding nitrate. Such a reaction would provide a defense barrier against the nitrosative stress raised by host macrophages during lung infection. It is proposed that the whole protein architecture, the heme distal site hydrogen bonded network, and the unique protein matrix tunnel, are optimally designed to support the pseudo‐catalytic role of trHbN in converting the reactive NO species into the harmless NO3‐. IUBMB Life, 55: 623‐627, 2003
The Anti‐Parasitic Effects of Nitric Oxide
Authors: Ascenzi Paolo , Bocedi Alessio , Gradoni Luigi
Published: December 2003

Endogenous and exogenous nitric oxide (NO) possesses anti‐parasitic effects on both Protozoa and Metazoa. However, NO production requires a tight control to limit cytotoxic damage to the host's own cells. The best known parasitic macromolecular targets for NO(‐donors) are cysteine proteases, which are relevant in several aspects of the parasite life cycle and parasite‐host relationships, and appear as promising targets for anti‐parasitic chemotherapy. IUBMB Life, 55: 573‐578, 2003
Nitric Oxide Signaling in Plant‐Pathogen Interactions
Authors: Romero‐Puertas María , Delledonne Massimo
Published: December 2003

Nitric oxide (NO), first characterized as an endothelium‐derived relaxation factor, is involved in diverse cellular processes including neuronal signaling, blood pressure homeostasis, and immune response. Recent studies have also revealed a role for NO as a signaling molecule in plants. As a developmental regulator, NO promotes germination, leaf extension and root growth, and delays leaf senescence and fruit maturation. Moreover, NO acts as a key signal in plant resistance to incompatible pathogens by triggering resistance‐associated hypersensitive cell death. In addition, NO activates the expression of several defense genes (e.g. pathogenesis‐related genes, phenylalanine ammonialyase, chalcone synthase) and could play a role in pathways leading to systemic acquired resistance. IUBMB Life, 55: 579‐583, 2003
Mitochondria as Targets of Apoptosis Regulation by Nitric Oxide
Authors: Vieira Helena , Kroemer Guido
Published: December 2003

In addition to their vital role as the cell's power stations, mitochondria exert an important function in apoptosis. In response to most if not all apoptosis inducers, mitochondrial membranes are permeabilized, leading to the release of potentially toxic proteins, mostly from the intermembrane space to the rest of the cells. Such pro‐apoptotic intermembrane proteins include the caspase‐independent death effector AIF, as well as cytochrome c, which can trigger the activation of caspases, once it has reached the cytosol. The mitochondrial permeabilization process can be induced by a variety of different xenobiotics, via a direct effect on mitochondrial membranes. Alternatively, mitochondrial permeabilization can be induced by endogenous second messengers, which are elicited in response to stress. The permeabilization process is controlled by the mitochondrial permeability transition pore complex (PTPC), by proteins of the Bcl‐2/Bax family, as well as by lipids and metabolites. Nitric oxide (NO) is one of the second messengers that can trigger apoptosis by inducing mitochondrial membrane permeabilization. This effect may involve a direct effect on the PTPC and/or indirect effects secondary to the NO‐mediated inhibition of oxidative phosphorylation. This has far‐reaching implications for the pathophysiology of NO. IUBMB Life, 55: 613‐616, 2003
Trichosanthin Induces Leakage and Membrane Fusion of Liposome
Authors: Xia Xiao‐feng , Zhang Fan , Shaw Pang‐Chui , Sui Sen‐Fang
Published: December 2003

Trichosanthin (TCS) is a ribosome inactivating protein with multiple pharmacological properties. Here the interaction between TCS and a phospholipid bilayer is investigated to provide evidence for membrane translocation mechanism of TCS. The results show that TCS can destabilize liposomes made by phospholipids with negatively charged head group. The destabilization effect is pH‐dependent and happens only under acidic conditions. Membrane fusion is also seen to accompany the destabilizing process. The interaction between a phospholipid bilayer and C7, a mutant of TCS with 7 residues at its C‐terminus deleted, has been investigated. Deleting the C‐terminus almost completely abolishes the destabilizing effect of TCS on the phospholipid bilayer, which implicates the C‐terminus in the interaction between trichosanthin and the membrane. IUBMB Life, 55: 681‐687, 2003
Mechanistic Insights Revealed Through Characterization of a Novel Chromophore in Selenophosphate Synthetase from Escherichia coli
Authors: Wolfe Matt D.
Published: December 2003

The incorporation of selenium into specific proteins and tRNAs requires selenophosphate (SePO3), whose formation is catalyzed by selenophosphate synthetase. In a Mg/ATP‐dependent reaction, selenophosphate synthetase catalyzes the phosphorylation of selenide to yield AMP, inorganic phosphate, and SePO3. In this report, a previously unrecognized chromophore covalently attached to selenophosphate synthetase is characterized. The UV/Vis spectrum of selenophosphate synthetase has a feature centered at 315 nm that is irreversibly destroyed by alkylation. Moreover, addition of Zn2 +, which is known to inhibit selenophosphate synthetase, reversibly quenches the 315 nm absorption. Since Zn2 + is known to bind to Cys17, these data strongly suggest that this residue participates in the 315 nm absorption. Upon incubation with both Mg2 + and ATP, the λmax of the chromophore shifts to 340 nm, and it is shown that the shift requires binding of nucleotide having a hydrolyzable γ‐phosphoryl group. These data indicate that either the chromophore is directly involved in phosphoryl transfer or indirectly reflects a phosphorylation‐dependent conformational change in selenophosphate synthetase. This work provides the first spectroscopic handle on catalytic steps associated with SePO3 synthesis, which will be used to study the molecular structure of the chromophore and its role in the catalytic mechanism of selenophosphate synthetase. IUBMB Life, 55: 689‐693, 2003
Stress-induced transcription of satellite III repeats
Authors: Jolly Caroline , Metz Alexandra , Govin Jérôme , Vigneron Marc , Turner Bryan M. , Khochbin Saadi , Vourc'h Claire
Published: December 2003

Exposure of mammalian cells to stress induces the activation of heat shock transcription factor 1 (HSF1) and the subsequent transcription of heat shock genes. Activation of the heat shock response also correlates with a rapid relocalization of HSF1 within a few nuclear structures termed nuclear stress granules. These stress-induced structures, which form primarily on the 9q12 region in humans through direct binding of HSF1 to satellite III repeats, do not colocalize with transcription sites of known hsp genes. In this paper, we show that nuclear stress granules correspond to RNA polymerase II transcription factories where satellite III repeats are transcribed into large and stable RNAs that remain associated with the 9q12 region, even throughout mitosis. This work not only reveals the existence of a new major heat-induced transcript in human cells that may play a role in chromatin structure, but also provides evidence for a transcriptional activity within a locus considered so far as heterochromatic and silent.
i-SNAREs
Authors: Varlamov Oleg , Volchuk Allen , Rahimian Vahid , Doege Claudia A. , Paumet Fabienne , Eng William S. , Arango Nancy , Parlati Francesco , Ravazzola Mariella , Orci Lelio , Söllner Thomas H. , Rothman James E.
Published: December 2003

A new functional class of SNAREs, designated inhibitory SNAREs (i-SNAREs), is described here. An i-SNARE inhibits fusion by substituting for or binding to a subunit of a fusogenic SNAREpin to form a nonfusogenic complex. Golgi-localized SNAREs were tested for i-SNARE activity by adding them as a fifth SNARE together with four other SNAREs that mediate Golgi fusion reactions. A striking pattern emerges in which certain subunits of the cis-Golgi SNAREpin function as i-SNAREs that inhibit fusion mediated by the trans-Golgi SNAREpin, and vice versa. Although the opposing distributions of the cis- and trans-Golgi SNAREs themselves could provide for a countercurrent fusion pattern in the Golgi stack, the gradients involved would be strongly sharpened by the complementary countercurrent distributions of the i-SNAREs.
Impaired Development of CD4+ CD25+ Regulatory T Cells in the Absence of STAT1
Authors: Nishibori Takeaki , Tanabe Yoshinari , Su Leon , David Michael
Published: December 2003

Type I and II interferons (IFNs) exert opposing effects on the progression of multiple sclerosis, even though both IFNs use the signal transducer and activator of transcription 1 (STAT1) as a signaling mediator. Here we report that STAT1-deficient mice expressing a transgenic T cell receptor against myelin basic protein spontaneously develop experimental autoimmune encephalomyelitis with dramatically increased frequency. The heightened susceptibility to this autoimmune disease appears to be triggered by a reduced number as well as a functional impairment of the CD4+ CD25+ regulatory T cells in STAT1-deficient animals. Adoptive transfer of wild-type regulatory T cells into STAT1-deficient hosts is sufficient to prevent the development of autoimmune disease. These results demonstrate an essential role of STAT1 in the maintenance of immunological self-tolerance.
BH3-only Protein Noxa Is a Mediator of Hypoxic Cell Death Induced by Hypoxia-inducible Factor 1α
Authors: Kim Jee-Youn , Ahn Hyun-Jong , Ryu Jong-Hoon , Suk Kyoungho , Park Jae-Hoon
Published: December 2003

Hypoxia is a common cause of cell death and is implicated in many disease processes including stroke and chronic degenerative disorders. In response to hypoxia, cells express a variety of genes, which allow adaptation to altered metabolic demands, decreased oxygen demands, and the removal of irreversibly damaged cells. Using polymerase chain reaction–based suppression subtractive hybridization to find genes that are differentially expressed in hypoxia, we identified the BH3-only Bcl-2 family protein Noxa. Noxa is a candidate molecule mediating p53-induced apoptosis. We show that Noxa promoter responds directly to hypoxia via hypoxia-inducible factor (HIF)-1α. Suppression of Noxa expression by antisense oligonucleotides rescued cells from hypoxia-induced cell death and decreased infarction volumes in an animal model of ischemia. Further, we show that reactive oxygen species and resultant cytochrome c release participate in Noxa-mediated hypoxic cell death. Altogether, our results show that Noxa is induced by HIF-1α and mediates hypoxic cell death.
Toll-like Receptors Induce a Phagocytic Gene Program through p38
Authors: Doyle Sean E. , O'Connell Ryan M. , Miranda Gustavo A. , Vaidya Sagar A. , Chow Edward K. , Liu Philip T. , Suzuki Shinobu , Suzuki Nobutaka , Modlin Robert L. , Yeh Wen-Chen , Lane Timothy F. , Cheng Genhong
Published: December 2003

Toll-like receptor (TLR) signaling and phagocytosis are hallmarks of macrophage-mediated innate immune responses to bacterial infection. However, the relationship between these two processes is not well established. Our data indicate that TLR ligands specifically promote bacterial phagocytosis, in both murine and human cells, through induction of a phagocytic gene program. Importantly, TLR-induced phagocytosis of bacteria was found to be reliant on myeloid differentiation factor 88–dependent signaling through interleukin-1 receptor–associated kinase-4 and p38 leading to the up-regulation of scavenger receptors. Interestingly, individual TLRs promote phagocytosis to varying degrees with TLR9 being the strongest and TLR3 being the weakest inducer of this process. We also demonstrate that TLR ligands not only amplify the percentage of phagocytes uptaking Escherichia coli, but also increase the number of bacteria phagocytosed by individual macrophages. Taken together, our data describe an evolutionarily conserved mechanism by which TLRs can specifically promote phagocytic clearance of bacteria during infection.
Cytomegalovirus Misleads Its Host by Priming of CD8 T Cells Specific for an Epitope Not Presented in Infected Tissues
Authors: Holtappels Rafaela , Podlech Jürgen , Pahl-Seibert Marcus-Folker , Jülch Markus , Thomas Doris , Simon Christian O. , Wagner Markus , Reddehase Matthias J.
Published: December 2003

Cytomegaloviruses (CMVs) code for several proteins that inhibit the presentation of antigenic peptides to CD8 T cells. Although the molecular mechanisms of CMV interference with the major histocompatibility complex class I pathway are long understood, surprisingly little evidence exists to support a role in vivo. Here we document the first example of the presentation of an antigenic peptide being blocked by a CMV immune evasion protein in organs relevant to CMV disease. Although this Db-restricted peptide, which is derived from the antiapoptotic protein M45 of murine CMV (mCMV), is classified as an immunodominant peptide based on response magnitude and long-term memory, adoptive transfer of M45 epitope-specific CD8 T cells did not protect against infection with wild-type mCMV. Notably, the same cells protected C57BL/6 mice infected with an mCMV mutant in which immune evasion protein m152/gp40 is deleted. These data indicate that direct presentation or cross-presentation of an antigenic peptide by professional antigen-presenting cells can efficiently prime CD8 T cells that fail in protection against CMV organ disease because m152/gp40 prevents presentation of this peptide in pathogenetically relevant tissue cells.
Essential Role of Survivin, an Inhibitor of Apoptosis Protein, in T Cell Development, Maturation, and Homeostasis
Authors: Xing Zheng , Conway Edward M. , Kang Chulho , Winoto Astar
Published: December 2003

Survivin is an inhibitor of apoptosis protein that also functions during mitosis. It is expressed in all common tumors and tissues with proliferating cells, including thymus. To examine its role in apoptosis and proliferation, we generated two T cell–specific survivin-deficient mouse lines with deletion occurring at different developmental stages. Analysis of early deleting survivin mice showed arrest at the pre–T cell receptor proliferating checkpoint. Loss of survivin at a later stage resulted in normal thymic development, but peripheral T cells were immature and significantly reduced in number. In contrast to in vitro studies, loss of survivin does not lead to increased apoptosis. However, newborn thymocyte homeostatic and mitogen-induced proliferation of survivin-deficient T cells were greatly impaired. These data suggest that survivin is not essential for T cell apoptosis but is crucial for T cell maturation and proliferation, and survivin-mediated homeostatic expansion is an important physiological process of T cell development.
Nonmuscle Myosin IIB Is Involved in the Guidance of Fibroblast Migration
Authors: Lo Chun-Min , Buxton Denis B. , Chua Gregory C.H. , Dembo Micah , Adelstein Robert S. , Wang Yu-Li
Published: December 2003

Although myosin II is known to play an important role in cell migration, little is known about its specific functions. We have addressed the function of one of the isoforms of myosin II, myosin IIB, by analyzing the movement and mechanical characteristics of fibroblasts where this protein has been ablated by gene disruption. Myosin IIB null cells displayed multiple unstable and disorganized protrusions, although they were still able to generate a large fraction of traction forces when cultured on flexible polyacrylamide substrates. However, the traction forces were highly disorganized relative to the direction of cell migration. Analysis of cell migration patterns indicated an increase in speed and decrease in persistence, which were likely responsible for the defects in directional movements as demonstrated with Boyden chambers. In addition, unlike control cells, mutant cells failed to respond to mechanical signals such as compressing forces and changes in substrate rigidity. Immunofluorescence staining indicated that myosin IIB was localized preferentially along stress fibers in the interior region of the cell. Our results suggest that myosin IIB is involved not in propelling but in directing the cell movement, by coordinating protrusive activities and stabilizing the cell polarity.
Ago1 and Dcr1, Two Core Components of the RNA Interference Pathway, Functionally Diverge from Rdp1 in Regulating Cell Cycle Events inSchizosaccharomyces pombe
Authors: Carmichael Jon B. , Provost Patrick , Ekwall Karl , Hobman Tom C.
Published: December 2003

In the fission yeast Schizosaccharomyces pombe, three genes that function in the RNA interference (RNAi) pathway, ago1+, dcr1+, and rdp1+, have recently been shown to be important for timely formation of heterochromatin and accurate chromosome segregation. In the present study, we present evidence that null mutants for ago1+and dcr1+but not rdp1+, exhibit abnormal cytokinesis, cell cycle arrest deficiencies, and mating defects. Subsequent analyses showed that ago1+and dcr1+are required for regulated hyperphosphorylation of Cdc2 when encountering genotoxic insults. Because rdp1+is dispensable for this process, the functions of ago1+and dcr1+in this pathway are presumably independent of their roles in RNAi-mediated heterochromatin formation and chromosome segregation. This was further supported by the finding that ago1+is a multicopy suppressor of the S-M checkpoint deficiency and cytokinesis defects associated with loss of Dcr1 function, but not for the chromosome segregation defects of this mutant. Accordingly, we conclude that Dcr1-dependent production of small interfering RNAs is not required for enactment and/or maintenance of certain cell cycle checkpoints and that Ago1 and Dcr1 functionally diverge from Rdp1 to control cell cycle events in fission yeast. Finally, exogenous expression of hGERp95/EIF2C2/hAgo2, a human Ago1 homolog implicated in posttranscriptional gene silencing, compensated for the loss of ago1+function in S. pombe. This suggests that PPD proteins may also be important for regulation of cell cycle events in higher eukaryotes.
Interaction with Smad4 Is Indispensable for Suppression of BMP Signaling by c-Ski
Authors: Takeda Masafumi , Mizuide Masafumi , Oka Masako , Watabe Tetsuro , Inoue Hirofumi , Suzuki Hiroyuki , Fujita Toshiro , Imamura Takeshi , Miyazono Kohei , Miyazawa Keiji
Published: December 2003

c-Ski is a transcriptional corepressor that interacts strongly with Smad2, Smad3, and Smad4 but only weakly with Smad1 and Smad5. Through binding to Smad proteins, c-Ski suppresses signaling of transforming growth factor-β (TGF-β) as well as bone morphogenetic proteins (BMPs). In the present study, we found that a mutant of c-Ski, termed c-Ski (ARPG) inhibited TGF-β/activin signaling but not BMP signaling. Selectivity was confirmed in luciferase reporter assays and by determination of cellular responses in mammalian cells (BMP-induced osteoblastic differentiation of C2C12 cells and TGF-β–induced epithelial-to-mesenchymal transdifferentiation of NMuMG cells) and Xenopus embryos. The ARPG mutant recruited histone deacetylases 1 (HDAC1) to the Smad3-Smad4 complex but not to the Smad1/5-Smad4 complex. c-Ski (ARPG) was unable to interact with Smad4, and the selective loss of suppression of BMP signaling by c-Ski (ARPG) was attributed to the lack of Smad4 binding. We also found that c-Ski interacted with Smad3 or Smad4 without disrupting Smad3-Smad4 heteromer formation. c-Ski (ARPG) would be useful for selectively suppressing TGF-β/activin signaling.
Tumor Necrosis Factor-α Promotes Survival of Opossum Kidney Cells via Cdc42-induced Phospholipase C-γ1 Activation and Actin Filament Redistribution
Authors: Papakonstanti Evangelia A. , Stournaras Christos
Published: December 2003

Although the renal proximal tubular epithelial cells are targeted in a variety of inflammatory diseases of the kidney, the signaling mechanism by which tumor necrosis factor (TNF)-α exerts its effects in these cells remains unclear. Here, we report that TNF-α elicits antiapoptotic effects in opossum kidney cells and that this response is mediated via actin redistribution through a novel signaling mechanism. More specifically, we show that TNF-α prevents apoptosis by inhibiting the activity of caspase-3 and this effect depends on actin polymerization state and nuclear factor-κB activity. We also demonstrate that the signaling cascade triggered by TNF-α is governed by the phosphatidylinositol-3 kinase, Cdc42/Rac1, and phospholipase (PLC)-γ1. In this signaling cascade, Cdc42 was found to be selectively essential for PLC-γ1 activation, whereas phosphatidylinositol-3,4,5-triphosphate alone is not sufficient to activate the phospholipase. Moreover, PLC-γ1 was found to associate in vivo with the small GTPase(s). Interestingly, PLC-γ1 was observed to associate with constitutively active (CA) Cdc42V12, but not with CA Rac1V12, whereas no interaction was detected with Cdc42(T17N). The inactive Cdc42(T17N) and the PLC-γ1 inhibitor U73122 prevented actin redistribution and depolymerization, confirming that both signaling molecules are responsible for the reorganization of actin. Additionally, the actin filament stabilizer phallacidin potently blocked the nuclear translocation of nuclear factor-κB and its binding activity, resulting in abrogation of the TNF-α-induced inhibition of caspase-3. To conclude, our findings suggest that actin may play a pivotal role in the response of opossum kidney cells to TNF-α and implicate Cdc42 in directly regulating PLC-γ1 activity.
A Single Amino Acid Change (Asp 53→ Ala53) Converts Survivin from Anti-apoptotic to Pro-apoptotic
Authors: Song Zhiyin , Liu Shixin , He He , Hoti Naser , Wang Yi , Feng Shanshan , Wu Mian
Published: December 2003

Survivin is a member of the inhibitor of apoptosis protein (IAP) family that has been implicated in both apoptosis inhibition and cell cycle control. Recently, Survivin has attracted growing attention because of its tumor-specific expression and potential applications in tumor therapy. However, its inhibitory mechanism and subcellular localization remain controversial. Here, we report a novel Survivin mutant Surv-D53A, which displays a function opposite to Survivin and a distinctive subcellular distribution compared with its wild-type counterpart. Surv-D53A was shown to induce apoptosis in a p53-independent manner, indicating that tumor suppressor p53 is not involved in its apoptosis pathway. Surv-D53A was shown to markedly sensitize apoptosis induced by TRAIL, doxorubicin, and RIP3. We also demonstrated that similar to wild-type Survivin, Surv-D53A was localized in cytoplasm in interphase and to midbody at telophase. However, it fails to colocalize in chromosomes with Aurora-B in metaphase as wt-Survivin. Surv-D53A mutant is less stable than wt-Survivin and is degraded more rapidly by ubiquitin-proteasome pathway. Additionally, we found that Surv-D53A interacts with wt-Survivin to form heterodimer or with itself to form mutant homodimer, which may account for the loss of its antiapoptotic function. Finally, unlike Survivin*Survivin, neither Surv-D53A*Survivin nor Surv-D53A*Surv-D53A is able to bind to Smac/DIABLO, which may explain the underlying mechanism for its abolishment of antiapoptotic activity of Survivin.
Positive Role of IQGAP1, an Effector of Rac1, in Actin-Meshwork Formation at Sites of Cell-Cell Contact
Authors: Noritake Jun , Fukata Masaki , Sato Kazumasa , Nakagawa Masato , Watanabe Takashi , Izumi Nanae , Wang Shujie , Fukata Yuko , Kaibuchi Kozo
Published: December 2003

The small guanosine triphosphatase Rac1 is activated by E-cadherin-mediated cell-cell adhesion and is required for the accumulation of actin filaments, E-cadherin, and β-catenin at sites of cell-cell contact. However, the modes of activation and action of Rac1 remain to be clarified. We here found that suppression of IQGAP1, an actin-binding protein and an effector of Rac1, by small interfering RNA apparently reduced the accumulation of actin filaments, E-cadherin, and β-catenin at sites of cell-cell contact in Madin-Darby canine kidney II epithelial cells under the conditions in which knockdown of Rac1 reduced them. Knockdown of Rac1 did not affect the localization of these junctional components in cells expressing a constitutively active IQGAP1 mutant defective in Rac1/Cdc42 binding. Knockdown of either Rac1 or IQGAP1 accelerated the 12-O-tetradecanoylphorbol-13-acetate-induced cell-cell dissociation. The basal Rac1 activity, which was maintained by E-cadherin-mediated cell-cell adhesion, was inhibited in the IQGAP1-knocked down cells, whereas the Rac1 activity was increased in the cells overexpressing IQGAP1. Together, these results indicate that Rac1 enhances the accumulation of actin filaments, E-cadherin, and β-catenin by acting on IQGAP1 and suggest that there exists a positive feedback loop comprised of “E-cadherin-mediated cell-cell adhesion→Rac1 activation→actin-meshwork formation by IQGAP1→increasing E-cadherin-mediated cell-cell adhesion.”
A Cell-Specific Transgenic Approach inXenopusReveals the Importance of a Functional p24 System for a Secretory Cell
Authors: Bouw Gerrit , Van Huizen Rick , Jansen Eric J.R. , Martens Gerard J.M.
Published: December 2003

The p24α, -β, -γ, and -δ proteins are major multimeric constituents of cycling endoplasmic reticulum-Golgi transport vesicles and are thought to be involved in protein transport through the early secretory pathway. In this study, we targeted transgene overexpression of p24δ2specifically to the Xenopus intermediate pituitary melanotrope cell that is involved in background adaptation of the animal and produces high levels of its major secretory cargo proopiomelanocortin (POMC). The transgene product effectively displaced the endogenous p24 proteins, resulting in a melanotrope cell p24 system that consisted predominantly of the transgene p24δ2protein. Despite the severely distorted p24 machinery, the subcellular structures as well as the level of POMC synthesis were normal in these cells. However, the number and pigment content of skin melanophores were reduced, impairing the ability of the transgenic animal to fully adapt to a black background. This physiological effect was likely caused by the affected profile of POMC-derived peptides observed in the transgenic melanotrope cells. Together, our results suggest that in the early secretory pathway an intact p24 system is essential for efficient secretory cargo transport or for supplying cargo carriers with the correct protein machinery to allow proper secretory protein processing.
Coactivation of Rac1 and Cdc42 at Lamellipodia and Membrane Ruffles Induced by Epidermal Growth Factor
Authors: Kurokawa Kazuo , Itoh Reina E. , Yoshizaki Hisayoshi , Nakamura Yusuke Ohba Takeshi , Matsuda Michiyuki
Published: December 2003

A major function of Rho-family GTPases is to regulate the organization of the actin cytoskeleton; filopodia, lamellipodia, and stress fiber are regarded as typical phenotypes of the activated Cdc42, Rac, and Rho, respectively. Using probes based on fluorescent resonance energy transfer, we report on the spatiotemporal regulation of Rac1 and Cdc42 at lamellipodia and membrane ruffles. In epidermal growth factor (EGF)-stimulated Cos1 and A431 cells, both Rac1 and Cdc42 were activated diffusely at the plasma membrane, followed by lamellipodial protrusion and membrane ruffling. Although Rac1 activity subsided rapidly, Cdc42 activity was sustained at lamellipodia. A critical role of Cdc42 in these EGF-induced morphological changes was demonstrated as follows. First, phorbol 12-myristate 13-acetate, which activated Rac1 but not Cdc42, could not induce full-grown lamellipodia in Cos1 cells. Second, a GTPase-activating protein for Cdc42, KIAA1204/CdGAP, inhibited lamellipodial protrusion and membrane ruffling without interfering with Rac1 activation. Third, expression of the Cdc42-binding domain of N-WASP inhibited the EGF-induced morphological changes. Therefore, Rac1 and Cdc42 seem to synergistically induce lamellipodia and membrane ruffles in EGF-stimulated Cos1 cells and A431 cells.
Methionine Sulfoxide Reduction in Mammals: Characterization of Methionine-R-Sulfoxide Reductases
Authors: Kim Hwa-Young , Gladyshev Vadim N.
Published: December 2003

Methionine residues in proteins are susceptible to oxidation by reactive oxygen species, but can be repaired via reduction of the resulting methionine sulfoxides by methionine-S-sulfoxide reductase (MsrA) and methionine-R-sulfoxide reductase (MsrB). However, the identity of all methionine sulfoxide reductases involved, their cellular locations and relative contributions to the overall pathway are poorly understood. Here, we describe a methionine-R-sulfoxide reduction system in mammals, in which two MsrB homologues were previously described. We found that human and mouse genomes possess three MsrB genes and characterized their protein products, designated MsrB1, MsrB2, and MsrB3. MsrB1 (Selenoprotein R) was present in the cytosol and nucleus and exhibited the highest methionine-R-sulfoxide reductase activity because of the presence of selenocysteine (Sec) in its active site. Other mammalian MsrBs contained cysteine in place of Sec and were less catalytically efficient. MsrB2 (CBS-1) resided in mitochondria. It had high affinity for methionine-R-sulfoxide, but was inhibited by higher concentrations of the substrate. The human MsrB3 gene gave rise to two protein forms, MsrB3A and MsrB3B. These were generated by alternative splicing that introduced contrasting N-terminal and C-terminal signals, such that MsrB3A was targeted to the endoplasmic reticulum and MsrB3B to mitochondria. We found that only mitochondrial forms of mammalian MsrBs (MsrB2 and MsrB3B) could compensate for MsrA and MsrB deficiency in yeast. All mammalian MsrBs belonged to a group of zinc-containing proteins. The multiplicity of MsrBs contrasted with the presence of a single mammalian MsrA gene as well as with the occurrence of single MsrA and MsrB genes in yeast, fruit flies, and nematodes. The data suggested that different cellular compartments in mammals maintain a system for repair of oxidized methionine residues and that this function is tuned in enzyme- and stereo-specific manner.
Repression of Na,K-ATPase β1-Subunit by the Transcription Factor Snail in Carcinoma
Authors: Espineda Cromwell E. , Chang Jay H. , Twiss Jeffery , Rajasekaran Sigrid A. , Rajasekaran Ayyappan K.
Published: December 2003

The Na,K-ATPase consists of two essential α- and β-subunits and regulates the intracellular Na+and K+homeostasis. Although the α-subunit contains the catalytic activity, it is not active without functional β-subunit. Here, we report that poorly differentiated carcinoma cell lines derived from colon, breast, kidney, and pancreas show reduced expression of the Na,K-ATPase β1-subunit. Decreased expression of β1-subunit in poorly differentiated carcinoma cell lines correlated with increased expression of the transcription factor Snail known to down-regulate E-cadherin. Ectopic expression of Snail in well-differentiated epithelial cell lines reduced the protein levels of E-cadherin and β1-subunit and induced a mesenchymal phenotype. Reduction of Snail expression in a poorly differentiated carcinoma cell line by RNA interference increased the levels of Na,K-ATPase β1-subunit. Furthermore, Snail binds to a noncanonical E-box in the Na,K-ATPase β1-subunit promoter and suppresses its promoter activity. These results suggest that down-regulation of Na,K-ATPase β1-subunit and E-cadherin by Snail are associated with events leading to epithelial to mesenchymal transition.