bluuebunny/crossref_metadata_2025 huggingface dataset:
Efficacy of topical phenol decontamination strategies on severity of acute phenol chemical burns and dermal absorption: in vitro and in vivo studies in pig skin
Authors: Monteiro-Riviere Nancy A , Inman Alfred O , Jackson Hilary , Dunn Brendan , Dimond Stephen
Published: November 2002

Pure phenol is colorless and used in the manufacture of phenolic resins, plastics, explosives, fertilizers, paints, rubber, textiles, adhesives, pharmaceuticals, paper, soap, and wood preservatives. The purpose of this study was to compare the efficacy of several phenol decontamination strategies following dermal exposure using the pig as a model for human exposure, and then assess the effect of the two best treatments on phenol absorption in the isolated perfused porcine skin flap (IPPSF). Six anesthetized Yorkshire pigs were exposed to 89% aqueous phenol for 1 min using Hilltop chambers (10 skin sites/pig; 400 μl/site). Exposure to phenol was followed by one of 10 different decontamination procedures: 1-, 5-, 15-, and 30-min water wash; Ivory1 soap solution; polyethylene glycol (PEG 400); PEG 400/industrial methylated spirits (IMS); PEG 400/ethanol (EtOH); polyvinyl pyrrolidone (PVP)/70% isopropanol (IPA); and 70% IPA. For each of the last five strategies, 1-min treatment washes were repeatedly alternated with 1-min water washes for a total of 15 min. Evaluation was based on scoring of erythema, edema, and histological parameters such as intracellular and intercellular epidermal edema, papillary dermal edema, perivascular infiltrates, pyknotic stratum basale cells, and epidermal-dermal separation. It was concluded that PEG 400 and 70% IPA were superior to the other treatments investigated and equally efficacious in the reduction of phenol-induced skin damage. In addition, phenol absorption was assessed utilizing the two most effective in vivo treatments in the IPPSF. The assessment of percutaneous absorption of phenol found the PEG 400, 70% IPA, and 15-min water treatments significantly (P < 0.05) reduced phenol absorption relative to no treatment.
Practice patterns of antiphospholipid syndrome at a tertiary teaching hospital in Lebanon
Authors: Major S , Pashayan N , Atweh S , Chidiac-Tannoury R , Shaar S , Uthman I , Masri A , Arayssi T
Published: November 2002

The objective of the study was to describe the practice patterns of the antiphospholipid syndrome (APS) as compared with consensus guidelines for diagnosis and to determine whether practice patterns correlate with patient demographics and physician specialty.

A retrospective medical chart review was conducted at the American University Hospital, in Beirut, Lebanon. All adult and pediatric patients admitted to the hospital between 1 January and 31 December 1998 who underwent either anticardiolipinantibodies (aCL) or lupus anticoagulant(LA) testing were included in the study.

Work-up of APS syndrome was compared with: (a) the consensus guidelines for clinical diagnosis; (b) physician specialty; and (c) patient demographics (age, gender, ethnicity, health insurance status).

Eighty-seven patients fulfilled at least one clinical criterion for APS; 92% were for work-up of thrombosis and 8% for pregnancy morbidities. Fifty-one percent underwent both aCL and LA. Overall 38% (33) of patients had an abnormal test result, however only 18% (6) underwent retesting, of whom only two satisfied a minimum of 6 weeks between test and retest. The APS diagnostic work-up was requested by 11 different specialties. Rheumatologists were the most consistent in asking for both tests.

APS is seen and diagnosed by a variety of medical specialties. Practice patterns as compared with the latest consensus are sub-optimal, and need to be improved. Interventions to help improve this have been discussed and are being implemented.
The autoimmune response to chromatin antigens in systemic lupus erythematosus: autoantibodies against histone H1 are a highly specific marker for SLE associated with increased disease activity
Authors: Schett G , Smolen J , Zimmermann C , Hiesberger H , Hoefler E , Fournel S , Muller S , Rubin R L , Steiner G
Published: November 2002

This study investigates specificity, sensitivity and concomitant presence of antibodies against histone H1 (H1), nucleosomes (NUC), chromatin (CHR) and dsDNA in patients with systemic lupus erythematosus (SLE), analyses their association with SLE disease activity and characterizes the immunodominant epitope reactivity of anti-H1 antibodies and its relation to SLE disease activity. In a cross-sectional study 394 sera of patients with various rheumatic diseases and healthy subjects were analysed by ELISA for antibodies against H1, NUC, CHR and dsDNA. In addition, a longitudinal analysis was performed that included 121 sequential serum samples derived from 16 SLE patients to assess the relation of these antibodies as well as antibodies to histone H2B to SLE disease activity. To assess epitope reactivity of anti-H1 antibodies overlapping synthetic peptides covering the entire H1 sequence were used. Anti-H1 antibodies yielded a sensitivity of approximately 45% and a specificity of over 98% for SLE, which was comparable to that found for anti-dsDNA antibodies. Anti-CHR and anti-NUC antibodies were of similar sensitivity but slightly (anti-CHR) or considerably (anti-NUC) less specific for SLE (95 and 85%, respectively). The sequential analysis revealed a strong correlation of anti-H1 antibodies with SLE disease activity that was better than the correlation of anti-dsDNA and anti-NUC antibodies, while only weak correlation was found for anti-CHR and anti-H2B antibodies. The immunodominant epitope for anti-H1 was localised between amino acids 204 and 218 (pp204–218) and immune reactivity to this epitope also correlated with disease activity. Anti-H1 is a highly specific marker for SLE with a diagnostic value comparable to anti-dsDNA. A positive testing for anti-H1 indicates increased disease activity, as does the appearance of antibodies to its immunodominant epitope pp204–218.
Gender and age differences in systemic lupus erythematosus. A study of 489 Greek patients with a review of the literature
Authors: Voulgari P V , Katsimbri P , Alamanos Y , Drosos A A
Published: November 2002

We investigated whether gender and age influence the clinical course and outcome in systemic lupus erythematosus (SLE) patients. Thus, we analyzed the clinical and laboratory data of 489 SLE patients at presentation and during follow-up. In addition, disease activity score (using the European Consensus Lupus Activity Measure, ECLAM) and organ damage index (using the Systemic Lupus International Collaboration Clinics/American College of Rheumatology Damage Index, DI) were measured. Furthermore, data from both sexes were analyzed according to the following age groups: < 55 years (younger group) and > 55 years (older group). There were 68 men and 421 women, giving a ratio of 1:7. We found no differences in the mean age, mean age at diagnosis, disease duration as well as duration of follow-up between men and women. Young men presented more frequently with serositis and discoid lesions, while women presented with Raynaud's phenomenon (RP) and malar rash. Regarding the laboratory findings, young women presented more often with anti-Ro(SSA) and anti-La(SSB) antibodies, while increased levels of erythrocyte sedimentation rate (ESR) were found in old women. During follow-up, men had serositis and renal disease more frequently, while the women's group were found to complain of RP, photosensitivity and mucosal ulcers more frequently, especially in young women. Anemia, leukopenia, thrombocytopenia and elevated levels of ESR were also found more frequently in young women during follow-up. However, there were no significant differences concerning ECLAM and DI scores between the two gender groups. Using multiple logistic regression analysis, a statistically significant association of malar rash, discoid lesions, serositis, RP, anti-Ro(SSA)= La(SSB) and increased ESR with sex was found independently of age, while only malar rash showed a statistically significant association with age independentlyof sex. Thus, we conclude that gender influences the clinical expression of the disease independentlyof age, while both gender and age do not affect the overall damage score.
Clinical significance of acquired activated protein C resistance in patients with systemic lupus erythematosus
Authors: Muñoz-Rodríguez F-J , Reverter J-C , Font J , Tàssies D , Espinosa G , Cervera R , Carmona F , Balasch J , Ingelmo M , Ordinas A
Published: November 2002

Antiphospholipid antibodies (aPL) may induce acquired activated protein C resistance (acquired APCR). The role of acquired APCR in patients with systemic lupus erythematosus (SLE) is not well known. To evaluate the prevalenceof acquiredAPCR and its associationwith clinical manifestations we studied 103 consecutive SLE patients and 103 matched controls. APCR in the undiluted test and after dilution in factor V deficient plasma, factor V Leiden, protein C and S, lupus anticoagulant,and anti-cardiolipin,anti-β2-glycoprotein I and anti-prothrombin antibodies were determined. Factor V Leiden was found in 4% in both patients and controls.The prevalenceof acquiredAPCR was 22% for the undiluted assay and 17% in the diluted test. In SLE patients,acquired APCR was associated with aPL (39 vs 13% in undiluted assay, P=0.007; and 33 vs 7% in the diluted test, P=0.001). Arterial thromboses were found in 24% of patients with acquired APCR and in 6% of patients without (P=0.04). However, no relationship was found with venous thrombosis. Acquired APCR was also associated with pregnancy losses: miscarriages in 70% of women with acquired APCR vs 32% in those without (P=0.03). Thus, in SLE patients acquired APCR seems to be associated with increased prevalence of arterial thrombosis and pregnancy losses.
Algorithms for calculation of daylight factors in streets
Authors: Matusiak B , Aschehoug Ø
Published: November 2002

This paper presents algorithms for calculation of sky components and daylight factors in linear building structures using the solid angle projection method. In order to estimate the precision of calculations two sky models; the uniform sky and CIE overcast sky, both giving equal illuminance on the horizontal plane, are used. Daylight factors calculated for CIE overcast sky are also compared to daylight factors measured in a physical scale model of a linear atrium in an artificial sky of the mirror-box type.
Direct illuminance caching: a way to enhance the performance of RADIANCE
Authors: Bauer Carsten
Published: November 2002

The RADIANCE lighting simulation software suite has proven itself a valuable means of producing visualizations and enabling analysis of all kinds of lighting tasks. One of its key features is the accurate calculation of the indirect light component by hemisphere sampling throughout the scene. Currently, there is a certain amount of redundancy within the algorithm. The performance reduction caused by this increases strongly with the number of light sources and becomes significant when more than five sources are present. A method for circumventing this redundancy is presented and its potential for considerably reducing rendering time is shown. The limitations of the new method are discussed and guidelines for its use are given, followed by some remarks on image generation for presentation purposes. The source code of the additional module is provided for downloading.
Quantification of parallax errors in sky simulator domes for clear sky conditions
Authors: Mardaljevic J
Published: November 2002

Scale model illuminance measurements in sky simulator domes are inherently subject to parallax errors. The magnitude of these errors under a number of Commission Internationale de l’É clairage (CIE) clear sky configurations is quantified using computer simulation techniques. In practical operation of a sky simulator dome, a second parallax error in the normalization measurements for horizontal illuminance is likely to compound the parallax error in the other illuminance measurements. This additional parallax error is accounted for in the simulations. The concept of a parallax-bounded volume is introduced. This is the volume of the dome which, on the basis of parallax alone, must contain a scale model if it is not to be subject to errors in the measurement of illuminance beyond a given tolerance. The findings indicate that, on the basis of a credible design goal for the sky simulator dome, high accuracy illuminance predictions (610%) are practically unattainable.
Prognostic Factors for Relapse in Stage I Seminoma Managed by Surveillance: A Pooled Analysis
Authors: Warde Padraig , Specht Lena , Horwich Alan , Oliver Tim , Panzarella Tony , Gospodarowicz Mary , von der Maase Hans
Published: November 2002

PURPOSE: Several management options are available to patients with stage I seminoma, including adjuvant radiotherapy, surveillance, and adjuvant chemotherapy. We performed a pooled analysis of patients from the four largest surveillance studies to better delineate prognostic factors associated with disease progression.

PATIENTS AND METHODS: Individual patient data were obtained from each center (Princess Margaret Hospital, Danish Testicular Cancer Study Group, Royal Marsden Hospital, and Royal London Hospital) for 638 patients. Tumor characteristics (size, histologic subtype, invasion of rete testis, and tumor invasion into small vessels [SVI]) as well as age at diagnosis were analyzed for prognostic importance for relapse.

RESULTS: With a median follow-up of 7.0 years (range, 0.02 to 17.5 years), 121 relapses were observed for an actuarial 5-year relapse-free rate (RFR) of 82.3%. On univariate analysis, tumor size (RFR: ≤ 4 cm, 87%; > 4 cm, 76%; P = .003), rete testis invasion (RFR: 86% [absent] v 77% [present], P = .003), and the presence of SVI (RFR: 86% [absent] v 77% [present], P = .038) were predictive of relapse. On multivariate analysis, tumor size (≤ 4 cm v > 4 cm, hazard ratio 2.0; 95% confidence interval [CI], 1.3 to 3.2) and invasion of the rete testis (hazard ratio 1.7; 95% CI, 1.1 to 2.6) remained as important predictors for relapse.

CONCLUSION: We have identified size of primary tumor and rete testis invasion as important prognostic factors for relapse in patients with stage I seminoma managed with surveillance. This information will allow patients and clinicians to choose management based on a more accurate assessment of an individual patient’s risk of relapse. In addition, it will allow clinicians to tailor follow-up protocols based on risk of occult disease.
Prospective, Randomized, Double-Blind, Placebo-Controlled Trial of Marimastat After Response to First-Line Chemotherapy in Patients With Small-Cell Lung Cancer: A Trial of the National Cancer Institute of Canada-Clinical Trials Group and the European Organization for Research and Treatment of Cancer
Authors: Shepherd Frances A. , Giaccone Giuseppe , Seymour Lesley , Debruyne Channa , Bezjak Andrea , Hirsh Vera , Smylie Michael , Rubin Sheldon , Martins Heidi , Lamont Alan , Krzakowski Maciej , Sadura Anna , Zee Benny
Published: November 2002

PURPOSE: Increased expression of metalloproteinases is associated with poor prognosis in small-cell lung cancer (SCLC). This trial was undertaken to determine whether adjuvant treatment with the metalloproteinase inhibitor marimastat could prolong survival in responding patients with SCLC after chemotherapy.

PATIENTS AND METHODS: SCLC patients in complete or partial remission were eligible. They were stratified by radiotherapy (early, late, or none), stage (extensive or limited), response (complete or partial), and cooperative group (National Cancer Institute of Canada-Clinical Trials Group or European Organization for Research and Treatment of Cancer). They were randomized to receive marimastat 10 mg or placebo orally bid for up to 2 years.

RESULTS: There were 532 eligible patients (266 marimastat and 266 placebo). Stage was limited for 279 patients (52%) and extensive for 253 (48%). Best response to induction therapy was complete remission for 176 patients (33%), partial remission for 341 (64%), and 15 patients (3%) had undergone surgical resection. The median time to progression for marimastat patients was 4.3 months compared with 4.4 months for placebo patients (P = .81). Median survivals for marimastat and placebo patients were 9.3 months and 9.7 months, respectively (P = .90) Toxicity was generally limited to musculoskeletal symptoms (18% grade 3/4 for marimastat). Dose modifications for musculoskeletal toxicity were required in 90 patients (33%) on the marimastat arm, and 87 (32%) permanently stopped marimastat because of toxicity. Patients on marimastat had significantly poorer quality of life at 3 and 6 months.

CONCLUSION: Treatment with marimastat after induction therapy for SCLC did not result in improved survival and had a negative impact on quality of life.
Efficacy and Safety of Carbon Ion Radiotherapy in Bone and Soft Tissue Sarcomas
Authors: Kamada Tadashi , Tsujii Hirohiko , Tsuji Hiroshi , Yanagi Tsuyoshi , Mizoe Jun-etsu , Miyamoto Tadaaki , Kato Hirotoshi , Yamada Shigeru , Morita Shinroku , Yoshikawa Kyousan , Kandatsu Susumu , Tateishi Akio
Published: November 2002

PURPOSE: To evaluate the tolerance for and effectiveness of carbon ion radiotherapy in patients with unresectable bone and soft tissue sarcomas.

PATIENTS AND METHODS: We conducted a phase I/II dose escalation study of carbon ion radiotherapy. Fifty-seven patients with 64 sites of bone and soft tissue sarcomas not suited for resection received carbon ion radiotherapy. Tumors involved the spine or paraspinous soft tissues in 19 patients, pelvis in 32 patients, and extremities in six patients. The total dose ranged from 52.8 to 73.6 gray equivalent (GyE) and was administered in 16 fixed fractions over 4 weeks (3.3 to 4.6 GyE/fraction). The median tumor size was 559 cm3 (range, 20 to 2,290 cm3). The minimum follow-up was 18 months.

RESULTS: Seven of 17 patients treated with the highest total dose of 73.6 GyE experienced Radiation Therapy Oncology Group grade 3 acute skin reactions. Dose escalation was then halted at this level. No other severe acute reactions (grade > 3) were observed in this series. The overall local control rates were 88% and 73% at 1 year and 3 years of follow-up, respectively. The median survival time was 31 months (range, 2 to 60 months), and the 1- and 3-year overall survival rates were 82% and 46%, respectively.

CONCLUSION: Carbon ion radiotherapy seems to be a safe and effective modality in the management of bone and soft tissue sarcomas not eligible for surgical resection, providing good local control and offering a survival advantage without unacceptable morbidity.
Efficacy of Fluorine-18-Deoxyglucose Positron Emission Tomography in Detecting Tumor Recurrence After Local Ablative Therapy for Liver Metastases: A Prospective Study
Authors: Langenhoff B.S. , Oyen W.J.G. , Jager G.J. , Strijk S.P. , Wobbes Th. , Corstens F.H.M. , Ruers T.J.M.
Published: November 2002

PURPOSE: The aims of this prospective study were to investigate the potential role of fluorine-18-deoxyglucose (FDG) positron emission tomography (PET) in determining the efficacy of the local tumor ablative process and to determine the added value of FDG-PET in the detection of tumor recurrence during follow-up.

PATIENTS AND METHODS: Twenty-three patients with unresectable colorectal liver metastases were followed up after local ablative therapy consisting of a standard protocol including FDG-PET scanning, computed tomography (CT) scanning, and carcinoembryonic antigen measurements. The mean follow-up period was 16 months (range, 10 to 21 months).

RESULTS: Ninety-six lesions was treated, 56 by local ablative treatment. Within 3 weeks after local ablative treatment, 51 lesions became photopenic on FDG-PET, while five lesions (in five patients) showed persistent activity on FDG-PET. In four of five FDG-PET–positive lesions, a local recurrence developed during follow-up; one FDG-PET–positive lesion turned out to be an abscess. None of the FDG-PET–negative lesions developed a local recurrence during a mean follow-up period of 16 months. During follow-up, 11 patients showed recurrence in the liver outside of the treated area. In all cases, previously negative FDG-PET scans became positive. Extrahepatic recurrence was encountered in nine patients during follow-up; FDG-PET showed all nine cases of tumor recurrence. There was one false-positive FDG-PET caused by an intra-abdominal abscess. In all patients, the time point of detection of recurrence by FDG-PET was considerably earlier than the detection by CT.

CONCLUSION: FDG-PET seems to have a significant impact in measuring treatment efficacy directly after local ablative therapy. Furthermore, FDG-PET has an added value in patient follow-up because it reveals recurrences earlier than conventional diagnostic modalities.
Domains of type 1 protein phosphatase inhibitor-2 required for nuclear and cytoplasmic localization in response to cell-cell contact
Authors: Leach Craig , Eto Masumi , Brautigan David L.
Published: November 2002

Inhibitor-2 of type 1 protein phosphatase is a phosphoprotein conserved among all eukaryotes, and it appears in both the nucleus and cytoplasm of tissue culture cells. We discovered that endogenous inhibitor-2 is concentrated in the nucleus of cells cultured at low density, whereas cells growing at high density excluded inhibitor-2 from the nucleus. There was rapid redistribution of inhibitor-2 when cells were replated at low or high density. Localization of myc-tagged forms of inhibitor-2 showed that residues 119-197 were required for nuclear accumulation in low-density cells and residues 78-119 were required for cytoplasmic localization in high-density cells. Fusion of inhibitor-2 residues 78-119 to green fluorescent protein was sufficient to produce cytoplasmic retention. Inhibitor-2 fused to triple tandem green fluorescent protein (100 kDa) was imported into the nucleus of low-density cells but was not excluded from the nucleus when cells reached high density, implying that inhibitor-2 was actively imported into the nucleus but exited by passive diffusion instead of active export. We conclude that inhibitor-2 contains two separate domains that control its localization in the nucleus or cytoplasm. This change in inhibitor-2 localization may direct inhibitor-2 to different forms of protein phosphatase 1 or change the localization of protein phosphatase, as part of the cellular response to cell-cell contacts at high density.
Hair follicle dermal cells repopulate the mouse haematopoietic system
Authors: Lako Majlinda , Armstrong Lyle , Cairns Paul M. , Harris Sue , Hole Nicholas , Jahoda Colin A. B.
Published: November 2002

Skin and hair follicle stem cell biology is the focus of increasing interest, not least because the adult hair follicle has well defined dermal and epithelial populations that display distinct developmental properties. Recent evidence suggests that a number of adult cell populations have much broader stem cell capabilities than previously thought. To examine whether this applied to the hair follicle, and with a view to developing the follicle as a stem cell model system we investigated whether adult hair follicles were capable of demonstrating haematopoietic stem cell activity. To investigate haematopoietic activity in hair follicles we first used in vitro haematopoietic colony assays. This demonstrated that rodent hair follicle end bulbs as well as micro-dissected dermal papilla and dermal sheath cells actively produced cells of erythroid and myeloid lineages but that follicle epithelial cells did not. As a more stringent test, we then transplanted cultured dermal papilla or dermal sheath cells from transgenically marked donor mice into lethally irradiated recipient mice and observed multi-lineage haematopoietic reconstitution when assayed at intervals of up to one year. Colony assays from bone marrow of primary recipients revealed that over 70% of clonogenic precursors were derived from donor hair follicle cells. When bone marrow from primary mice was harvested and used to repopulate secondary myeloablated recipients, multi-lineage haematopoietic engraftment was observed. Our data show that dermal but not epidermal compartments of the adult hair follicle have much broader stem cell activities than previously described. Although the treatment for many forms of blood disorder, such as leukemia, often requires transplantation of haematopoietic stem cells (HSC),their availability can be rate limiting. Given its easy accessibility, our identification of the hair follicle as a source of extramedullary haematopoietic stem cell activity makes it an attractive potential source for blood stem cell therapeutics and highlights its value as a model system in adult stem cell biology.
Binding of protein kinase B to the plakin family member periplakin
Authors: van den Heuvel A. Pieter J. , de Vries-Smits Alida M. M. , van Weeren Pascale C. , Dijkers Pascale F. , de Bruyn Kim M. T. , Riedl Jürgen A. , Burgering Boudewijn M. T.
Published: November 2002

The serine/threonine kinase protein kinase B (PKB/c-Akt) acts downstream of the lipid kinase phosphoinositide 3-kinase (PI3K) and functions as an essential mediator in many growth-factor-induced cellular responses such as cell cycle regulation, cell survival and transcriptional regulation. PI3K activation generates 3′-phosphorylated phosphatidylinositol lipids(PtdIns3P) and PKB activation requires PtdIns3P-dependent membrane translocation and phosphorylation by upstream kinases. However PKB activation and function is also regulated by interaction with other proteins. Here we show binding of PKB to periplakin, a member of the plakin family of cytolinker proteins. Interaction between PKB and periplakin was mapped to part of the pleckstrin homology (PH) domain of PKB, which is probably not involved in lipid binding, and indeed binding to periplakin did not affect PKB activation. We therefore investigated the possibility that periplakin may act as a scaffold or localization signal for PKB. In cells endogenous periplakin localizes to different cellular compartments, including plasma membrane,intermediate filament structures, the nucleus and mitochondria. Overexpression of the C-terminal part of periplakin, encompassing the PKB binding region,results in predominant intermediate filament localization and little nuclear staining. This also resulted in inhibition of nuclear PKB signalling as indicated by inhibition of PKB-dependent Forkhead transcription factor regulation. These results suggest a possible role for periplakin as a localization signal in PKB-mediated signalling.
Src family tyrosine kinases regulate adhesion-dependent tyrosine phosphorylation of 5′-inositol phosphatase SHIP2 during cell attachment and spreading on collagen I
Authors: Prasad Nagendra , Topping Robert S. , Decker Stuart J.
Published: November 2002

Inositol phosphatases play an important role in regulation of cellular levels of lipid second messengers. Recently we have reported a novel function for SHIP2 in cell adhesion and spreading. In this study, we further characterize the adhesion-dependent tyrosine phosphorylation of SHIP2 and examine the role of Src family tyrosine kinases in the regulation of SHIP2 function. SHIP2 was tyrosine phosphorylated during cell attachment and spreading on collagen I, but not on fibronectin, collagen IV, laminin or poly-L-lysine. SHIP2 tyrosine phosphorylation, induced by plating on a collagen-I-coated surface but not by epidermal growth factor or insulin treatment of cells, was completely blocked by small molecule inhibitors of Src family kinases. SHIP2 could be phosphorylated in vitro by recombinant Src kinase and tyrosines 986-987 in the NPXY motif of SHIP2 appear to be the major sites of phosphorylation for Src both in vitro and in vivo. An activated form of Src induced strong tyrosine phosphorylation of SHIP2 while a dominant-negative form decreased collagen-I-dependent SHIP2 phosphorylation. SHIP2 associated with the adapter protein Shc via its NPXY motif during cell spreading on collagen I in a Src activity-dependent manner. Expression of SHIP2 with mutated NPXY motif caused deregulation of lamellipodia formation during spreading on collagen I. These observations indicate that SHIP2 is regulated by Src family kinases during cell attachment and spreading on collagen I and suggest an important role for SHIP2 as a part of a signaling pathway that regulates actin cytoskeleton remodeling.
Role of β3-endonexin in the regulation of NF-κB-dependent expression of urokinase-type plasminogen activator receptor
Authors: Besta Felicitas , Massberg Steffen , Brand Korbinian , Müller Elke , Page Sharon , Grüner Sabine , Lorenz Michael , Sadoul Karin , Kolanus Waldemar , Lengyel Ernst , Gawaz Meinrad
Published: November 2002

Endothelial migration on extracellular matrix is regulated by integrins and proteolysis. Previous studies showed that β3-integrins regulate expression of the urokinase-type plasminogen activator receptor(uPAR) through outside-in signalling involving the cytoplasmic domain. Here we show that overexpression of the integrin-binding proteinβ 3-endonexin decreased uPAR promoter (-398 base-pair fragment)activity that is constitutively active in endothelial cells. Mutation of the NF-κB promoter binding site (-45 bp) impaired the ability ofβ 3-endonexin to downregulate uPAR promoter activity. Immunoprecipitation studies showed that β3-endonexin interacts directly with the p50/p65 transactivation complex and thereby inhibits binding of κB oligonucleotides to the p50/p65 complex. Moreover, binding ofβ 3-endonexin to p50 was inhibited in the presence of κB but not mutated κB oligonucleotides, suggesting a sterical competition between β3-endonexin and κB DNA for the p50/p65 complex. We therefore propose that β3-endonexin acts as regulator of uPAR expression in β3-integrin-mediated endothelial cell migration through direct interaction with p50/p65. Since NF-κB regulates the expression of matrix degrading enzymes, the present results define a role of β3-endonexin in regulatingβ 3-integrin-mediated adhesion and pericellular proteolysis.
JNK1 modulates osteoclastogenesis through both c-Jun phosphorylation-dependent and -independent mechanisms
Authors: David Jean-Pierre , Sabapathy Kanaga , Hoffmann Oskar , Idarraga Maria H. , Wagner Erwin F.
Published: November 2002

Phosphorylation of the N-terminal domain of Jun by the Jun kinases (JNKs) modulates the transcriptional activity of AP-1, a dimeric transcription factor typically composed of c-Jun and c-Fos, the latter being essential for osteoclast differentiation. Using mice lacking JNK1 or JNK2, we demonstrate that JNK1, but not JNK2, is specifically activated by the osteoclast-differentiating factor RANKL. Activation of JNK1, but not JNK2, is required for efficient osteoclastogenesis from bone marrow monocytes (BMMs). JNK1 protects BMMs from RANKL-induced apoptosis during differentiation. In addition, BMMs from mice carrying a mutant of c-Jun phosphorylation sites (JunAA/JunAA), as well as cells lacking either c-Jun or JunD, which is another JNK substrate, revealed that c-Jun phosphorylation and c-Jun itself, but not JunD, are essential for efficient osteoclastogenesis. Moreover, JNK1-dependent c-Jun phosphorylation in response to RANKL is not involved in the anti-apoptotic function of JNK1. Thus, these data provide genetic evidence that JNK1 activation modulates osteoclastogenesis through both c-Jun-phosphorylation-dependent and -independent mechanisms.
The spatio-temporal organization of DNA replication sites is identical in primary, immortalized and transformed mammalian cells
Authors: Dimitrova Daniela S. , Berezney Ronald
Published: November 2002

We investigated the organization of DNA replication sites in primary (young or presenescent), immortalized and transformed mammalian cells. Four different methods were used to visualize replication sites: in vivo pulse-labeling with 5-bromo-2′-deoxyuridine (BrdU), followed by either acid depurination, or incubation in nuclease cocktail to expose single-stranded BrdU-substituted DNA regions for immunolabeling; biotin-dUTP labeling of nascent DNA by run-on replication within intact nuclei and staining with fluorescent streptavidin;and, finally, immunolabeling of the replication fork proteins PCNA and RPA. All methods produced identical results, demonstrating no fundamental differences in the spatio-temporal organization of replication patterns between primary, immortal or transformed mammalian cells. In addition, we did not detect a spatial coincidence between the early firing replicons and nuclear lamin proteins, the retinoblastoma protein or the nucleolus in primary human and rodent cells. The retinoblastoma protein does not colocalize in vivo with members of the Mcm family of proteins (Mcm2, 3 and 7) at any point of the cell cycle and neither in the chromatin-bound nor in the soluble nucleoplasmic fraction. These results argue against a direct role for the retinoblastoma or nuclear lamin proteins in mammalian DNA synthesis under normal physiological conditions.
The mitotic-spindle-associated protein astrin is essential for progression through mitosis
Authors: Gruber Jens , Harborth Jens , Schnabel Jörg , Weber Klaus , Hatzfeld Mechthild
Published: November 2002

Astrin is a mitotic-spindle-associated protein expressed in most human cell lines and tissues. However, its functions in spindle organization and mitosis have not yet been determined. Sequence analysis revealed that astrin has an N-terminal globular domain and an extended coiled-coil domain. Recombinant astrin was purified and characterized by CD spectroscopy and electron microscopy. Astrin showed parallel dimers with head-stalk structures reminiscent of motor proteins, although no sequence similarities to known motor proteins were found. In physiological buffers, astrin dimers oligomerized via their globular head domains and formed aster-like structures. Silencing of astrin in HeLa cells by RNA interference resulted in growth arrest, with formation of multipolar and highly disordered spindles. Chromosomes did not congress to the spindle equator and remained dispersed. Cells depleted of astrin were normal during interphase but were unable to progress through mitosis and finally ended in apoptotic cell death. Possible functions of astrin in mitotic spindle organization are discussed.
Transforming growth factor-β and epidermal growth factor synergistically stimulate epithelial to mesenchymal transition (EMT) through a MEK-dependent mechanism in primary cultured pig thyrocytes
Authors: Grände Mats , Franzen Åsa , Karlsson Jan-Olof , Ericson Lars E. , Heldin Nils-Erik , Nilsson Mikael
Published: November 2002

Enhancement of tumor cell growth and invasiveness by transforming growth factor-β (TGF-β) requires constitutive activation of the ras/MAPK pathway. Here we have investigated how MEK activation by epidermal growth factor (EGF) influences the response of fully differentiated and growth-arrested pig thyroid epithelial cells in primary culture to TGF-β1. The epithelial tightness was maintained after single stimulation with EGF or TGF-β1 (both 10 ng/ml) for 48 hours. In contrast, co-stimulation abolished the transepithelial resistance and increased the paracellular flux of [3H]inulin within 24 hours. Reduced levels of the tight junction proteins claudin-1 and occludin accompanied the loss of barrier function. N-cadherin, expressed only in few cells of untreated or single-stimulated cultures, was at the same time increased 30-fold and co-localised with E-cadherin at adherens junctions in all cells. After 48 hours of co-stimulation, both E- and N-cadherin were downregulated and the cells attained a fibroblast-like morphology and formed multilayers. TGF-β1 only partially inhibited EGF-induced Erk phosphorylation. The MEK inhibitor U0126 prevented residual Erk phosphorylation and abrogated the synergistic responses to TGF-β1 and EGF. The observations indicate that concomitant growth factor-induced MEK activation is necessary for TGF-β1 to convert normal thyroid epithelial cells to a mesenchymal phenotype.
In vivo characterization of diatom multipartite plastid targeting signals
Authors: Apt Kirk E. , Zaslavkaia Lioudmila , Lippmeier J. Casey , Lang Markus , Kilian Oliver , Wetherbee Rick , Grossman Arthur R. , Kroth Peter G.
Published: November 2002

Plastids of diatoms and related algae are delineated by four membranes: the outermost membrane (CER) is continuous with the endoplasmic reticulum while the inner two membranes are homologous to plastid envelope membranes of vascular plants and green algae. Proteins are transported into these plastids by pre-sequences that have two recognizable domains. To characterize targeting of polypeptides within diatom cells, we generated constructs encoding green fluorecent protein (GFP) fused to leader sequences. A fusion of GFP to the pre-sequence of BiP [an endoplasmic reticulum (ER)-localized chaperone]resulted in accumulation of GFP within the ER; a construct encoding the pre-sequence of a plastid protein fused to GFP was directed into the plastids. Additional constructs demonstrated that the N-terminal region of the bipartite plastid targeting pre-sequence was necessary for transport of polypeptides to the lumen of the ER, while the C-terminal region was shown to enable the proteins to traverse the plastid double envelope membrane. Our data strongly support the hypothesis of a multi-step plastid targeting process in chromophytic algae and raises questions about the continuity of the ER and CER and the function of the latter in polypeptide trafficking.
Nuclear organization in differentiating oligodendrocytes
Authors: Nielsen Joseph A. , Hudson Lynn D. , Armstrong Regina C.
Published: November 2002

Many studies have suggested that the 3D organization of chromatin and proteins within the nucleus contributes to the regulation of gene expression. We tested multiple aspects of this nuclear organization model within a primary cell culture system. Oligodendrocyte lineage cells were examined to facilitate analysis of nuclear organization relative to a highly expressed tissue-specific gene, proteolipid protein (PLP), which exhibits transcriptional upregulation during differentiation from the immature progenitor stage to the mature oligodendrocyte stage. Oligodendrocyte lineage cells were isolated from brains of neonatal male rodents, and differentiation from oligodendrocyte progenitors to mature oligodendrocytes was controlled with culture conditions. Genomic in situ hybridization was used to detect the single copy of the X-linked PLP gene within each interphase nucleus. The PLP gene was not randomly distributed within the nucleus, but was consistently associated with the nuclear periphery in both progenitors and differentiated oligodendrocytes. PLP and a second simultaneously upregulated gene, the myelin basic protein (MBP) gene, were spatially separated in both progenitors and differentiated oligodendrocytes. Increased transcriptional activity of the PLP gene in differentiated oligodendrocytes corresponded with local accumulation of SC35 splicing factors. Differentiation did not alter the frequency of association of the PLP gene with domains of myelin transcription factor 1 (Myt1), which binds the PLP promoter. In addition to our specific findings related to the PLP gene, these data obtained from primary oligodendrocyte lineage cells support a nuclear organization model in which (1) nuclear proteins and genes can exhibit specific patterns of distribution within nuclei, and (2) activation of tissue-specific genes is associated with changes in local protein distribution rather than spatial clustering of coordinately regulated genes. This nuclear organization may be critical for complex nucleic-acid—protein interactions controlling normal cell development,and may be an important factor in aberrant regulation of cell differentiation and gene expression in transformed cells.
The UCS family of myosin chaperones
Authors: Hutagalung Alex H. , Landsverk Megan L. , Price Maureen G. , Epstein Henry F.
Published: November 2002

The canonical UCS (UNC-45/Cro1/She4p) protein, Caenorhabditis elegans UNC-45, was one of the earliest molecules to be shown genetically to be necessary for sarcomere assembly. Genetic analyses of homologues in several fungal species indicate that the conserved UCS domain functionally interacts with conventional type II and unconventional type V myosins. In C. elegans and other invertebrate species, UNC-45 and its orthologues interact with both sarcomeric and non-sarcomeric myosins whereas, in vertebrates, there are two UNC-45 isoforms: a general cell (GC) and a striated muscle (SM) isoform. Although the mechanism of action of UCS proteins is unknown, recent biochemical studies suggest that they may act as molecular chaperones that facilitate the folding and/or maturation of myosin.
Two mammalian UNC-45 isoforms are related to distinct cytoskeletal and muscle-specific functions
Authors: Price Maureen G. , Landsverk Megan L. , Barral Jose M. , Epstein Henry F.
Published: November 2002

Previous studies have shown that the UNC-45 protein of C. elegansis required for normal thick filament assembly, binds Hsp90 and the myosin head, and shows molecular chaperone activity. We report here that mice and humans each have two genes that are located on different chromosomes, encode distinct UNC-45-like protein isoforms, and are expressed either in multiple tissues or only in cardiac and skeletal muscles. Their expression is regulated during muscle differentiation in vitro, with the striated muscle isoform mRNA appearing during myoblast fusion. Antisense experiments in C2C12 skeletal myogenic cells demonstrate that decreasing the general cell isoform mRNA reduces proliferation and fusion, while decreasing the striated muscle isoform mRNA affects fusion and sarcomere organization. These results suggest that the general cell UNC-45 isoform may have primarily cytoskeletal functions and that the striated muscle UNC-45 isoform may be restricted to roles in muscle-specific differentiation.
Insect lipoprotein follows a transferrin-like recycling pathway that is mediated by the insect LDL receptor homologue
Authors: Van Hoof Dennis , Rodenburg Kees W. , Van der Horst Dick J.
Published: November 2002

The lipoprotein of insects, high-density lipophorin (HDLp), is homologous to that of mammalian low-density lipoprotein (LDL) with respect to its apolipoprotein structure. Moreover, an endocytic receptor for HDLp has been identified (insect lipophorin receptor, iLR) that is homologus to the LDL receptor. We transfected LDL-receptor-expressing CHO cells with iLR cDNA to study the endocytic uptake and intracellular pathways of LDL and HDLp simultaneously. Our studies provide evidence that these mammalian and insect lipoproteins follow distinct intracellular routes after receptor-mediated endocytosis. Multicolour imaging and immunofluorescence was used to visualize the intracellular trafficking of fluorescently labeled ligands in these cells. Upon internalization, which can be completely inhibited by human receptor-associated protein (RAP), mammalian and insect lipoproteins share endocytic vesicles. Subsequently, however, HDLp evacuates the LDL-containing endosomes. In contrast to LDL, which is completely degraded in lysosomes after dissociating from its receptor, both HDLp and iLR converge in a nonlysosomal juxtanuclear compartment. Colocalization studies with transferrin identified this organelle as the endocytic recycling compartment via which iron-depleted transferrin exits the cell. Fluorescently labeled RAP is also transported to this recycling organelle upon receptor-mediated endocytosis by iLR. Internalized HDLp eventually exits the cell via the recycling compartment, a process that can be blocked by monensin, and is re-secreted with a t½of ∼13 minutes. From these observations, we conclude that HDLp is the first non-exchangeable apolipoprotein-containing lipoprotein that follows a transferrin-like recycling pathway despite the similarities between mammalian and insect lipoproteins and their receptors.
Lipid rafts and the local density of ErbB proteins influence the biological role of homo- and heteroassociations of ErbB2
Authors: Nagy Peter , Vereb György , Sebestyén Zsolt , Horváth Gábor , Lockett Stephen J. , Damjanovich Sándor , Park John W. , Jovin Thomas M. , Szöllősi János
Published: November 2002

The ErbB family of transmembrane receptor tyrosine kinases plays an important role in the pathogenesis of many cancers. The four members of the family, ErbB1-4, form various homo- and heterodimers during the course of signal transduction. A second hierarchical level of molecular associations involving 102-103 molecules, termed large-scale clustering, has also been identified, but the regulatory factors and biological consequences of such structures have not been systematically evaluated. In this report, we describe the states of association of ErbB2 and their relationship to local ErbB3 density and lipid rafts based on quantitative fluorescence microscopy of SKBR-3 breast cancer cells. Clusters of ErbB2 colocalized with lipid rafts identified by the GM1-binding B subunit of cholera toxin. Pixel-by-pixel analysis of fluorescence resonance energy transfer between labeled antibodies indicated that the homoassociation (homodimerization) of ErbB2 was proportional to the local density of ErbB2 and inversely proportional to that of ErbB3 and of the raft-specific lipid GM1. Crosslinking lipid rafts with the B subunit of cholera toxin caused dissociation of the rafts and ErbB2 clusters, an effect that was independent of the cytoskeletal anchoring of ErbB2. Crosslinking also decreased ErbB2-ErbB3 heteroassociation and the EGF- and heregulin-induced tyrosine phosphorylation of Shc. When cells were treated with the anti-ErbB2 monoclonal antibody 4D5 (parent murine version of Trastuzumab used in the immunotherapy of breast cancer), internalization of the antibody was inhibited by crosslinking of lipid rafts, but the antiproliferative activity of 4D5 was retained and even enhanced. We conclude that local densities of ErbB2 and ErbB3, as well as the lipid environment profoundly influence the association properties and biological function of ErbB2.
Move over protein kinase C, you've got company: alternative cellular effectors of diacylglycerol and phorbol esters
Authors: Brose Nils , Rosenmund Christian
Published: November 2002

Diacylglycerol is an essential second messenger in mammalian cells. The most prominent intracellular targets of diacylglycerol and of the functionally analogous phorbol esters belong to the protein kinase C (PKC) family. However,at least five alternative types of high-affinity diacylglycerol/phorbol-ester receptor are known: chimaerins, protein kinase D, RasGRPs, Munc13s and DAG kinase γ. Recent evidence indicates that these have functional roles in diacylglycerol second messenger signalling in vivo and that several cellular processes depend on these targets rather than protein kinase C isozymes. These findings contradict the still prevalent view according to which all diacylglycerol/phorbol-ester effects are caused by the activation of protein kinase C isozymes. RasGRP1 (in Ras/Raf/MEK/ERK signalling) and Munc13-1 (in neurotransmitter secretion) are examples of non-PKC diacylglycerol/phorbol-ester receptors that mediate diacylglycerol and phorbol-ester effects originally thought to be caused by PKC isozymes. In the future, pharmacological studies on PKC must be complemented with alternative experimental approaches to allow the separation of PKC-mediated effects from those caused by alternative targets of the diacylglycerol second messenger pathway. The examples of RasGRP1 and Munc13-1 show that detailed genetic analyses of C1-domain-containing non-PKC diacylglycerol/phorbol-ester receptors in mammals are ideally suited to achieve this goal.