bluuebunny/crossref_metadata_2025 huggingface dataset:
TLR3-induced activation of mast cells modulates CD8+ T-cell recruitment
Authors: Orinska Zane , Bulanova Elena , Budagian Vadim , Metz Martin , Maurer Marcus , Bulfone-Paus Silvia
Published: April 2005

Mast cells play an important role in host defense against various pathogens, but their role in viral infection has not been clarified in detail. dsRNA, synthesized by various types of viruses and mimicked by polyinosinic-polycytidylic acid (poly(I:C)) is recognized by Toll-like receptor 3 (TLR3). In this study, we demonstrate that poly(I:C) injection in vivo potently stimulates peritoneal mast cells to up-regulate a number of different costimulatory molecules. Therefore, we examined the expression and the functional significance of TLR3 activation in mast cells. Mast cells express TLR3 on the cell surface and intracellularly. After stimulation of mast cells with poly(I:C) and Newcastle disease virus (NDV), TLR3 is phosphorylated and the expression of key antiviral response cytokines (interferon β, ISG15) and chemokines (IP10, RANTES) is upregulated. Interestingly, mast cells activated via TLR3-poly(I:C) potently stimulate CD8+ T-cell recruitment. Indeed, mast-cell–deficient mice (KitW/KitW-v) given an intraperitoneal injection of poly(I:C) show a decreased CD8+ T-cell recruitment, whereas granulocytes normally migrate to the peritoneal cavity. Mast-cell reconstitution of KitW/KitW-v mice normalizes the CD8+ T-cell influx. Thus, mast cells stimulated through engagement of TLR3 are potent regulators of CD8+ T-cell activities in vitro and in vivo.
HFE cross-talks with the MHC class I antigen presentation pathway
Authors: de Almeida Sérgio F. , Carvalho Isabel F. , Cardoso Carla S. , Cordeiro João V. , Azevedo Jorge E. , Neefjes Jacques , de Sousa Maria
Published: April 2005

HFE is a protein known to be involved in iron metabolism; yet, other than its homology with major histocompatibility complex (MHC) class I molecules, it has not been described as having an immunologic function. Here we report that peripheral blood mononuclear cells (PBMCs) from patients with hereditary hemochromatosis (HH) carrying the C282Y mutation in HFE have reduced cell-surface expression of MHC class I due to an enhanced endocytosis rate of MHC class I molecules caused by premature peptide and β2-microglobulin dissociation. This faster turnover also leads to increased expression levels of cell-surface free class I heavy chains in mutant PBMCs. Biochemical analysis indicates an earlier peptide loading and endoplasmic reticulum maturation of MHC class I molecules in C282Y mutant cells. Thermostability assays further showed that in HFE mutants the MHC class I peptide loading gives rise to low-stability heterotrimers that dissociate prematurely during its intracellular traffic. The present results suggest the existence of an intriguing cross-talk between a particular HFE mutation and the classical MHC class I route. These findings constitute the first description of peptide presentation pathway abnormalities linked to HFE and provide additional evidence for the occurrence of immunologic defects in patients with HH.
Immunoglobulin gene analysis reveals 2 distinct cells of origin for EBV-positive and EBV-negative Burkitt lymphomas
Authors: Bellan Cristiana , Lazzi Stefano , Hummel Michael , Palummo Nazzareno , de Santi Margherita , Amato Teresa , Nyagol Joshua , Sabattini Elena , Lazure Thierry , Pileri Stefano A. , Raphael Martine , Stein Harald , Tosi Piero , Leoncini Lorenzo
Published: April 2005

The normal counterpart of the neoplastic B cells in Burkitt lymphoma (BL) is still unclear. Based on immunoglobulin gene rearrangement studies, some authors suggest an origin from germinal center cells and others from memory B cells. However, most of these studies rely on cell lines or on a small series of cases. To help clarify the cell of origin of BL, semi-nested polymerase chain reaction (PCR) was performed to amplify the VDJ rearrangements of the immunoglobulin heavy chain (VH) genes, and the resultant amplificates were sequenced for comparison with known germline VH segments. The results of this approach revealed that all cases (15 endemic BL [eBL], 10 sporadic BL [sBL], and 6 AIDS-related BL) harbor mutated VH genes, with different mutation ranges among the 3 types of BL. The eBL and AIDS-related forms showed considerably higher mutation rates than the sBL form (5.1%, 5.4%, and 1.5%, respectively). The mutations in eBL and AIDS-related BL also showed signs of antigen selection, whereas no signs of antigen selection were found in sBL. Finally, after subcloning the amplificates, sequence analysis revealed no signs of ongoing mutations in any of the cases analyzed. Given that one of the main differences between eBL and AIDS-related BL on the one hand and sBL on the other hand is the association with Epstein-Barr virus (EBV), we compared EBV-positive and EBV-negative BLs independently of their geographic origin and HIV status. The differences in the number of somatic mutations and antigen selection were even more evident when this approach was used. According to our molecular results, it appears that EBV-positive and EBV-negative BL may originate from 2 distinct subsets of B cells, pointing to a particular role for the germinal-center reaction in the pathogenesis of these tumors. The different types of C-MYC translocation reported in BL may also be related to the different stages of B-cell maturation.
Estimating the mean and variance from the median, range, and the size of a sample
Authors: Hozo Stela Pudar , Djulbegovic Benjamin , Hozo Iztok
Published: April 2005

Background

Usually the researchers performing meta-analysis of continuous outcomes from clinical trials need their mean value and the variance (or standard deviation) in order to pool data. However, sometimes the published reports of clinical trials only report the median, range and the size of the trial.

Methods

In this article we use simple and elementary inequalities and approximations in order to estimate the mean and the variance for such trials. Our estimation is distribution-free, i.e., it makes no assumption on the distribution of the underlying data.

Results

We found two simple formulas that estimate the mean using the values of the median (m), low and high end of the range (a and b, respectively), and n (the sample size). Using simulations, we show that median can be used to estimate mean when the sample size is larger than 25. For smaller samples our new formula, devised in this paper, should be used. We also estimated the variance of an unknown sample using the median, low and high end of the range, and the sample size. Our estimate is performing as the best estimate in our simulations for very small samples (n ≤ 15). For moderately sized samples (15  70), the formula range/6 gives the best estimator for the standard deviation (variance).

We also include an illustrative example of the potential value of our method using reports from the Cochrane review on the role of erythropoietin in anemia due to malignancy.

Conclusion

Using these formulas, we hope to help meta-analysts use clinical trials in their analysis even when not all of the information is available and/or reported.
Evaluation of the VP22 protein for enhancement of a DNA vaccine against anthrax
Authors: Perkins Stuart D , Flick-Smith Helen C , Garmory Helen S , Essex-Lopresti Angela E , Stevenson Freda K , Phillpotts Robert J
Published: April 2005

Background

Previously, antigens expressed from DNA vaccines have been fused to the VP22 protein from Herpes Simplex Virus type I in order to improve efficacy. However, the immune enhancing mechanism of VP22 is poorly understood and initial suggestions that VP22 can mediate intercellular spread have been questioned. Despite this, fusion of VP22 to antigens expressed from DNA vaccines has improved immune responses, particularly to non-secreted antigens.

Methods

In this study, we fused the gene for the VP22 protein to the gene for Protective Antigen (PA) from Bacillus anthracis, the causative agent of anthrax. Protective immunity against infection with B. anthracis is almost entirely based on a response to PA and we have generated two constructs, where VP22 is fused to either the N- or the C-terminus of the 63 kDa protease-cleaved fragment of PA (PA63).

Results

Following gene gun immunisation of A/J mice with these constructs, we observed no improvement in the anti-PA antibody response generated. Following an intraperitoneal challenge with 70 50% lethal doses of B. anthracis strain STI spores, no difference in protection was evident in groups immunised with the DNA vaccine expressing PA63 and the DNA vaccines expressing fusion proteins of PA63 with VP22.

Conclusion

VP22 fusion does not improve the protection of A/J mice against live spore challenge following immunisation of DNA vaccines expressing PA63.
The caveolae-mediated sv40 entry pathway bypasses the golgi complex en route to the endoplasmic reticulum
Authors: Norkin Leonard C , Kuksin Dmitry
Published: April 2005

Background

Simian virus 40 (SV40) enters cells via an atypical caveolae-mediated endocytic pathway, which delivers the virus to a new intermediary compartment, the caveosome. The virus then is believed to go directly from the caveosome to the endoplasmic reticulum. Cholera toxin likewise enters via caveolae and traffics to caveosomes. But, in contrast to SV40, cholera toxin is transported from caveosomes to the endoplasmic reticulum via the Golgi. For that reason, and because the caveosome and Golgi may have some common markers, we revisited the issue of whether SV40 might access the endoplasmic reticulum via the Golgi.

Results

We confirmed our earlier finding that SV40 co localizes with the Golgi marker β-COP. However, we show that the virus does not co localize with the more discriminating Golgi markers, golgin 97 and BODIPY-ceramide.

Conclusion

The caveolae-mediated SV40 entry pathway does not intersect the Golgi. SV40 is seen to co localize with β-COP because that protein is a marker for caveosomes as well as the Golgi. Moreover, these results are consistent with the likelihood that the caveosome is a sorting organelle. In addition, there are at least two distinct but related routes by which a ligand might traffic from the caveosome to the ER; one route involving transport through the Golgi, and another pathway that does not involve the Golgi.
EBV latent membrane protein 1 abundance correlates with patient age but not with metastatic behavior in north African nasopharyngeal carcinomas
Authors: Khabir Abdelmajid , Karray Hela , Rodriguez Sandrine , Rosé Mathieu , Daoud Jamel , Frikha Mounir , Boudawara Tahia , Middeldorp Jaap , Jlidi Rachid , Busson Pierre
Published: April 2005

Background

Undifferentiated nasopharyngeal carcinomas are rare in a majority of countries but they occur at a high incidence in South China and to a lesser extent in North Africa. They are constantly associated with the Epstein-Barr virus (EBV) regardless of patient geographic origin. In North Africa, the distribution of NPC cases according to patient age is bi-modal with a large group of patients being around 50 years old (80%) and a smaller group below 25 years old. We and others have previously shown that the juvenile form of NPC has distinct biological characteristics including a low amount of p53 and Bcl2 in the tumor tissue and a low level of anti-EBV IgG and IgA in the peripheral blood.

Results

To get more insight on potential oncogenic mechanisms specific of these two forms, LMP1 abundance was assessed in 82 NPC patients of both groups, using immuno-histochemistry and semi-quantitative evaluation of tissue staining. Serum levels of anti-EBV antibodies were simultaneously assessed. For LMP1 staining, we used the S12 antibody which has proven to be more sensitive than the common anti-LMP1 CS1-4 for analysis of tissue sections. In all NPC biopsies, at least a small fraction of cells was positively stained by S12. LMP1 abundance was strongly correlated to patient age, with higher amounts of the viral protein detected in specimens of the juvenile form. In contrast, LMP1 abundance was not correlated to the presence of lymph node or visceral metastases, nor to the risk of metastatic recurrence. It was also independent of the level of circulating anti-EBV antibodies.

Conclusion

The high amount of LMP1 recorded in tumors from young patients confirms that the juvenile form of NPC has specific features regarding not only cellular but also viral gene expression.
Microbial growth tests in anti-neoplastic injectable solutions
Authors: Paris Isabelle , Paci Angelo , Rey Jean-Baptiste , Bourget Philippe
Published: April 2005

The Institut Gustave-Roussy (IGR) Department of Clinical Pharmacy (DCP) ensures the annual preparation of about 30 000 therapeutic batches of anti-neoplastic agents.High performance thin-layer chromatography (HPTLC) allows postproduction quality control of these batches.Although the centralized chemotherapy manufacturing unit has been recently ISO 9001:2000 certified, it was considered to improve the quality level of manufactured batches even further.The viability of micro-organisms (bacteria and fungi) in appropriate sterile media containing various anti-neoplastic agents at therapeutic concentration was assessed to demonstrate the lack of contamination during our manufacturing process in the isolator.After 14 days of incubation in these media, the results show the absence of contamination of the manufactured batches.This leads us to conclude that using sterile drugs and sterile medical devices in a sterile isolator allows the manufacture of sterile therapeutic batches with excellent confidence.
Contamination of syringe plungers during the sampling of cyclophosphamide solutions
Authors: Favier Bertrand , Gilles Laurence , Latour Jean François , Desage Michel , Giammarile Francesco
Published: April 2005

The presence of cytotoxic agents in the urine of operators and in their environment has been demonstrated. The pharmacokinetics of the urinary elimination of cyclophosphamide suggests that these drugs are absorbed cutaneously during handling. In the framework of a more general study on the contamination of hospital environment, the present study addresses the possible presence of cytotoxic agents on the plungers of syringes. The report is based on results indicating that the bacterial contamination of a plunger may result in the contamination of the solution being sampled. The study was divided into two phases. The first phase consisted in measuring the contamination of the plungers of eight syringes used for handling cyclophosphamide. Cyclophosphamide was analysed by gas chromatography—mass spectrometry with a detection limit of 0.1ng/ml. The aim of the second phase was to localize the contamination on the plunger and thus determine the amount of drug that comes into contact with the gloves of the operators. The contamination was quantified by measuring the activity of metastable technetium. The results of the first phase showed that all the plungers were contaminated with cyclophosphamide amounts varying from 3.7 to 445.7 ng. The second phase showed that the infiltration of liquid onto the plunger depended on the solution being sampled. Almost no infiltration was seen with labelled water, but contamination appeared after the first sampling of a cyclophosphamide solution, then increased as a function of the number of times the plunger was pushed in and out. These results indicate that cyclophosphamide solutions infiltrate onto the plungers of syringes. They suggest that the general procedure for handling cytotoxic agents should be modified, and a regular replacement of syringes should be enforced. They also partly explain why the gloves of 50-90% operators are contaminated after a single preparation. The contamination seems to depend on the type of solution sampled and the number of samplings. Initial investigations by the manufacturer of the syringes had shown that the acid pH of cyclophosphamide solutions may affect the lubricant of the joint. Our study demonstrates that the contamination of plungers is one of the sources of environmental contamination for health workers handling antineoplastic agents, even in the absence of manipulation errors. More generally, these results demonstrate that the exposure of operators cannot be clearly described unless all existing sources of contamination in their environment are identified. The implementation of suitable procedures should thus take into account all possible sources of contamination, including technical facilities such as the use of a safety cabinet or an isolator.
Polyuria associated with high-dose methotrexate in two patients with acute lymphoblastic leukaemia
Authors: Lau Keith K , Weiss Aaron R , Jones Deborah P
Published: April 2005

Although methotrexate has an established safety profile in clinical practice, severe morbidity can still occur on rare occassions. We report two patients with leukemia treated with high dose methotrexate. Both patients developed profound polyuria that required aggressive fluid resuscitations during the treatments. Renal toxicity is a known complication of methotrexate, but polyuria associated with its use has not been reported before. Polyuria started shortly after the initiation of the medicine in both patients. The polyuria resolved as the drug level in blood became undetectable. The episodes of polyuria were transient and recurred every time when the patients received methotrexate. The clinical pictures were not compatible with classical drug induced nephrogenic diabetes insipidus. It is possible that the drug interferes with adenosine metabolism, which in turn alters the tubular ability of solute and fluid reabsorption.
Study of the effect of standardized chemotherapy order forms on prescribing errors and anti-emetic cost
Authors: Sano Harold S , Waddell J Aubrey , A Solimando Dominic , Doulaveris Paula , Myhand Rickey
Published: April 2005

Objectives. Many anti-neoplastic medication errors and excessive use of serotonin antagonist anti-emetic agents might be prevented by the use of a standardized chemotherapy order form (SCOF). Several studies showing a reduction in prescribing errors or control of inappropriate anti-emetic use through the use of SCOFs have been reported. No previously published study reported SCOFs were used to reduce both prescribing errors and anti-emetic cost. This study attempts to measure these outcomes in a haematology-oncology pharmacy service.

Methods. The study consisted of a four-month control period, followed by a four-month test period following dissemination of the standardized order forms. In each period, prescriber errors and anti-emetic use were monitored. During the control period, using clinical studies from the primary literature and anti-emetic guidelines, 64 SCOFs representing the most commonly used chemotherapy regimens in the medical oncology and gynaecology oncology services were developed by the haematology-oncology pharmacy. Differences in prescribing error rate and anti-emetic cost were compared between each period and with the institution’s historic prescribing error rate.

Results. During the control period, 1078 orders for oral and parenteral granisetron and ondansetron with combined total acquisition cost of $76 454.64 and a mean cost of $70.92 were dispensed. During the test period, the pharmacy dispensed 1121 orders with an acquisition cost of $73 331.61 and a mean cost of $65.42. A savings of $3123.03 resulted from a reduction of the amount prescribed in the test period. The difference in mean cost per order between the two periods was significant (P B0.037). Fifty-three prescribing errors out of 3592 medication orders were detected in the control period, while 12 errors out of 3585 medication orders were detected during the test period. A significant difference (P B0.0001) was detected between the two periods. There was a significant difference (P B0.0001) between the control period and the institution’s historic prescribing error rate and no difference between the test period and the institution’s historic prescribing error rate.

Conclusion. SCOFs significantly reduced serotonin antagonist anti-emetic cost and prescribing error rate over a four-month period.
The Role of Psychology in a Liberal Arts Education: An Interview with Diane F. Halpern
Authors: McCarthy Maureen A.
Published: April 2005

Diane F. Halpern is the Past-President of the American Psychological Association (APA) and Director of the Berger Institute for Work, Family, and Children and Professor of Psychology at Claremont McKenna College. Her earlier appointments include professor of psychology, Chair and Dean of Undergraduate Studies at California State University, San Bernardino. Her teaching, service to psychology, and research have been recognized with many awards, including the 2002 Outstanding Teaching Award from the Western Psychological Association, Wang Family Excellence Award, American Psychological Foundation Award for Distinguished Teaching, Distinguished Career Contributions to Education and Training, G. Stanley Hall Lecture, and the Outstanding Professor Award from California State University-Statewide.

Maureen McCarthy is the Director of Precollege and Undergraduate Programs for the APA. She provides leadership and management oversight of programs and initiatives to enhance the teaching and learning of psychology in high schools, community colleges, and undergraduate programs; coordinates programs with initiatives of national organizations, projects, and agencies that share the mission of enhancing teaching and faculty development; and initiates research pertaining to needs, achievements, and characteristics of undergraduate psychology. Effective spring 2005, she became a faculty member at Kennesaw State University, where she teaches experimental psychology and research methods.
Encouraging Active Learning Can Improve Students' Performance on Examinations
Authors: Yoder Janice D. , Hochevar Catherine M.
Published: April 2005

We tested the hypothesis that students in psychology of women classes would perform better on materials covered by multiple-choice exams when the first author presented these materials with active learning versus lecture, autonomous readings, and video presentations alone. Across 3 classes, we coded exam items according to how the instructor presented relevant materials and recorded classwide performance. Both between and within classes, students' performances were better on items testing materials covered with active learning techniques compared to other formats. These data provide empirical support for the efficacy of active learning techniques.
The Consequences of Including Seductive Details during Lecture
Authors: Harp Shannon F. , Maslich Amy A.
Published: April 2005

When text passages include seductive details (i.e., interesting, tangentially related adjuncts that are irrelevant to the lesson), students perform worse on recall (Garner, Gillingham, & White, 1989) and problem-solving tests (Harp & Mayer, 1997, 1998) than students reading the same material without seductive details. To determine whether including seductive details during lecture had a similar effect, students listened to a recorded lecture that either contained or did not contain seductive details and took a test on the material. As predicted, students who heard a lecture containing seductive details recalled significantly fewer main points and provided significantly fewer acceptable problem-solving solutions than students who heard the lecture without seductive details. These results suggest that lectures should not include seductive details.
Using Kitchen Appliance Analogies to Improve Students' Reasoning about Neurological Results
Authors: Vishton Peter M.
Published: April 2005

This article describes and evaluates a new technique for teaching students to interpret studies of patients with brain injuries. This technique asks students to consider how knives and blenders lose specific functionality when they are damaged. This approach better prepares students to make proper inferences from behavioral deficits observed after brain injury, specifically with reference to single and double dissociation. Significantly improved performance on multiple-choice and identification questions included in midterm examinations suggests that the impact of these thought experiments was substantive and long lasting.
Promoting Knowledge Transfer with Electronic Note Taking
Authors: Katayama Andrew D. , Shambaugh R. Neal , Doctor Tasneem
Published: April 2005

We investigated the differences between (a) copying and pasting text versus typed note-taking methods of constructing study notes simultaneously with (b) vertically scaffolded versus horizontally scaffold notes on knowledge transfer. Forty-seven undergraduate educational psychology students participated. Materials included 2 electronic chapter-length texts, 2 sets of computerized study notes, 2 tests (fact and application), and an attitudinal survey. Only the application test revealed a significant main effect for notes format. We conclude that keying in notes leads to higher retention of knowledge transfer than copying and pasting notes after a 1-week delay.
Increased keratinocyte proliferation by JUN-dependent expression of PTN and SDF-1 in fibroblasts
Authors: Florin Lore , Maas-Szabowski Nicole , Werner Sabine , Szabowski Axel , Angel Peter
Published: April 2005

In skin, fibroblasts of the connective tissue play a decisive role in epidermal homeostasis and repair by contributing to the regulation of keratinocyte proliferation and differentiation. The AP-1 transcription factor subunit JUN plays a crucial role in this mesenchymal-epithelial interplay by regulating the expression of two critical paracrine-acting cytokines, keratinocyte growth factor (KGF) and granulocyte-macrophage colony-stimulating factor (GM-CSF). We have performed gene expression profiling of wild-type and Jun–/– mouse embryonic fibroblasts to identify additional players involved in this complex network, and have found pleiotrophin (PTN) and the stromal cell-derived factor 1 (SDF-1) as novel JUN-regulated factors. Both cytokines are expressed by dermal fibroblasts in vivo, as shown by semi-quantitative RT-PCR and in situ hybridization on murine skin sections. Using a heterologous feeder layer co-culture system, we demonstrated that PTN and SDF-1 exert a mitogenic effect on primary human keratinocytes. Moreover, SDF-1-induced keratinocyte proliferation could be specifically inhibited by neutralizing antibodies against SDF-1 or its receptor, CXCR4. Consistent with its role in promoting keratinocyte growth, PTN was upregulated during cutaneous wound healing in vivo. Interestingly, co-cultivation with keratinocytes stimulated PTN expression but repressed SDF-1 production in fibroblasts, demonstrating the complexity of the paracrine regulatory cytokine networks that control skin homeostasis and regeneration.
Keratin-8-deficient mice develop chronic spontaneous Th2 colitis amenable to antibiotic treatment
Authors: Habtezion Aida , Toivola Diana M. , Butcher Eugene C. , Omary M. Bishr
Published: April 2005

Keratin 8 (K8) is the major intermediate filament protein present in intestinal epithelia. Depending on the mouse genetic background, absence of K8 causes embryonic lethality or colonic hyperplasia and colitis. We studied disease progression, the inflammatory responses, and role of luminal bacteria in K8-null mice in order to characterize the intestinal pathology of K8-associated colitis. Colon lymphocytes were isolated for analysis of their phenotype and cytokine production, and vascular and lymphocyte adhesion molecule expression in K8–/– mice of varying ages. K8–/– mice had a marked increase in TCRβ-positive/CD4-positive T cells infiltrating the colon lamina propria, in association with enhanced Th2 cytokine (IL-4, IL-5 and IL-13) production. K8–/– mice show early signs of inflammation even prior to weaning, that increases with age, and their epithelial cells overexpress MHC class II antigens. The chronic colitis is related to increased CD4-positive infiltrating T cells displaying memory and naive phenotypes, and an altered vascular endothelium with aberrant expression of peripheral node addressin. Analysis of normal gut-specific homing molecules, reveals an increased number of α4β7-positive cells and vascular mucosal addressin cell adhesion molecule-1 in K8-null colons. Antibiotic treatment markedly decreased colon inflammation and ion transporter AE1/2 mistargeting, indicating that luminal bacteria play an important role in the observed phenotype. Therefore, K8-null mice develop chronic spontaneous Th2-type colitis due to a primary epithelial rather than immune cell defect, which is amenable to antibiotic therapy. These mice provide a model to investigate epithelial-leukocyte and epithelial-microbial cross-talk.
Cytosolic tail sequences and subunit interactions are critical for synaptic localization of glutamate receptors
Authors: Chang Howard Chia-Hao , Rongo Christopher
Published: April 2005

AMPA-type glutamate receptors mediate excitatory synaptic transmission in the nervous system. The receptor subunit composition and subcellular localization play an important role in regulating synaptic strength. GLR-1 and GLR-2 are the Caenorhabditis elegans subunits most closely related to the mammalian AMPA-type receptors. These subunits are expressed in overlapping sets of interneurons, and contain type-I PDZ binding motifs in their carboxy-terminal cytosolic tail sequences. We report that GLR-1 and GLR-2 may form a heteromeric complex, the localization of which depends on either GLR-1 or GLR-2 tail sequences. Subunit interactions alone can mediate synaptic localization as endogenous GLR-1, or GLR-2 subunits can rescue the localization defects of subunits lacking tail sequences. Moreover, GLR-2 cytosolic tail sequences are sufficient to confer synaptic localization on a heterologous reporter containing a single-transmembrane domain. The localization of this GLR-2 reporter requires both a PDZ-binding motif in the GLR-2 tail sequence, and sequences outside of this motif. The PDZ protein LIN-10 regulates the localization of the reporter through the sequences outside of the PDZ-binding motif. Our results suggest that multiple synaptic localization signals reside in the cytosolic tail sequence of the receptor subunits, and that channel assembly can rescue the synaptic localization defects of individual mutant subunits as long as there are also wild-type subunits in the receptor complex.
Manipulation of redox signaling in mammalian cells enabled by controlled photogeneration of reactive oxygen species
Authors: Posen Yehudit , Kalchenko Vyacheslav , Seger Rony , Brandis Alexander , Scherz Avigdor , Salomon Yoram
Published: April 2005

Reactive oxygen species (ROS) comprise a group of noxious byproducts of oxidative processes which participate in the induction of many common diseases. However, understanding their role in the regulation of normal physiological redox signaling is currently evolving. Detailed study of the dynamic functions of ROS within the biological milieu is difficult because of their high chemical reactivity, short lifetime, minute concentrations and cytotoxicity at high concentrations. In this study, we show that increasing intracellular ROS levels, set off by controlled in situ photogeneration of a nontoxic bacteriochlorophyll-based sensitizer initiate responses in cultured melanoma cells. Using hydroethidine as detector, we determined light-dependent generation of superoxide and hydroxyl radicals in cell-free and cell culture models. Monitoring the ROS-induced responses revealed individual and differential behavior of protein kinases [p38, mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and Akt] as well as effects on the subcellular distribution of phosphorylated p38. Furthermore, alterations in morphology and motility and effects on cell viability as a function of time and photosensitizer doses were observed. Following mild ROS challenge, enzymatic and cellular changes were observed in the majority of the cells, without inducing extensive cell death. However, upon vigorous ROS challenge, a similar profile of the overall responses was observed, terminating in cell death. This study shows that precisely controlled photogeneration of ROS can provide simple, fine-tuned, noninvasive manipulation of ROS-sensitive cellular responses ranging from individual enzymes to gross behavior of target cells. The observations made with this tool enable a dynamic and causal correlation, presenting a new alternative for studying the role of ROS in cellular redox signaling.
Analysis of phosphoinositide binding domain properties within the myotubularin-related protein MTMR3
Authors: Lorenzo Óscar , Urbé Sylvie , Clague Michael J.
Published: April 2005

The myotubularins are a large family of phosphoinositide-specific phosphatases with substrate specificity for PtdIns3P and PtdIns(3,5)P2. In addition to an N-terminal PH-GRAM (PH-G) domain and a signature catalytic domain shared with other family members, MTMR3 contains a C-terminal FYVE domain. We show that the FYVE domain of MTMR3 is atypical in that it neither confers endosomal localisation nor binds to the lipid PtdIns3P. Furthermore the FYVE domain is not required for in vitro enzyme activity of MTMR3. In contrast, the PH-GRAM domain is able to bind to phosphoinositide lipids, of which the allosteric regulator PtdIns5P is the preferred partner. Consequently, generation of PtdIns5P at the plasma membrane by ectopic expression of the bacterial phosphatase IpgD leads to a translocation of MTMR3 that requires the PH-G domain. Deletion of the PH-G domain leads to loss of activity of MTMR3 in vitro, and surprisingly, when combined with an active site mutation, accumulates the protein on the Golgi complex.
Cyclic AMP mediates keratinocyte directional migration in an electric field
Authors: Pullar Christine E. , Isseroff R. Rivkah
Published: April 2005

Re-epithelialization of wounded skin is necessary for wound closure and restoration of barrier function and requires directional keratinocyte migration towards the center of the wound. The electric field (EF) generated immediately upon wounding could be the earliest signal keratinocytes receive to initiate directional migration and healing. Keratinocytes express many β2-adrenergic receptors (β2-ARs), but their role in the epidermis is unknown. We have previously shown that β-AR agonists decrease keratinocyte migration in a cyclic AMP (cAMP) independent mechanism involving the activation of protein phosphatase 2A (PP2A). Here, we ask whether β2-ARs play a role in keratinocyte galvanotaxis.

We report a bimodal response. When keratinocytes were exposed to higher concentrations of β-AR agonist (0.1 μM), their tracked migratory speed was inhibited, in both the presence (directional migration) and the absence (random migration) of a 100 mV mm–1 EF, as expected. At lower agonist concentrations (0.1 pM to 0.1 nM), there was no effect on migratory speed; however, all directionality was lost – essentially, cells were `blinded' to the directional cue. Preincubating the cells with β-antagonist restored directional migration, demonstrating that the `blindness' was β2-AR mediated. Incubation of keratinocytes with agents known to increase intracellular cAMP levels, such as sp-cAMP, pertussis toxin and forskolin, resulted in similar `blinding' to the EF, whereas random migration was unaffected. The inactive cAMP analog rp-cAMP had no effect on keratinocyte migration, whether directional or random. However, rp-cAMP pretreatment before β-agonist addition fully restored galvanotaxis, demonstrating the complete cAMP dependence of the attenuation of keratinocyte directional migration. This is the first report that cAMP is capable of mediating keratinocyte galvanotaxis. β-AR agonists and antagonists could be valuable tools for modulating re-epithelialization, an essential step in the wound-healing process. Thus, β-ARs regulate the two distinct components of keratinocyte directional migration differently: migration speed via a cAMP-independent mechanism and galvanotaxis by a cAMP-dependent one.
Alexander-disease mutation ofGFAPcauses filament disorganization and decreased solubility of GFAP
Authors: Hsiao Victoria C. , Tian Rujin , Long Heather , Der Perng Ming , Brenner Michael , Quinlan Roy A. , Goldman James E.
Published: April 2005

Alexander disease is a fatal neurological illness characterized by white-matter degeneration and the formation of astrocytic cytoplasmic inclusions called Rosenthal fibers, which contain the intermediate filament glial fibrillary acidic protein (GFAP), the small heat-shock proteins HSP27 and αB-crystallin, and ubiquitin. Many Alexander-disease patients are heterozygous for one of a set of point mutations in the GFAP gene, all of which result in amino acid substitutions. The biological effects of the most common alteration, R239C, were tested by expressing the mutated protein in cultured cells by transient transfection. In primary rat astrocytes and Cos-7 cells, the mutant GFAP was incorporated into filament networks along with the endogenous GFAP and vimentin, respectively. In SW13Vim– cells, which have no endogenous cytoplasmic intermediate filaments, wild-type human GFAP frequently formed filamentous bundles, whereas the R239C GFAP formed `diffuse' and irregular patterns. Filamentous bundles of R239C GFAP were sometimes formed in SW13Vim– cells when wild-type GFAP was co-transfected. Although the presence of a suitable coassembly partner (vimentin or GFAP) reduced the potential negative effects of the R239C mutation on GFAP network formation, the mutation affected the stability of GFAP in cells in a dominant fashion. Extraction of transfected SW13Vim– cells with Triton-X-100-containing buffers showed that the mutant GFAP was more resistant to solubilization at elevated KCl concentrations. Both wild-type and R239C GFAP assembled into 10 nm filaments with similar morphology in vitro. Thus, although the R239C mutation does not appear to affect filament formation per se, the mutation alters the normal solubility and organization of GFAP networks.
PECAM-1, α6 integrins and neutrophil elastase cooperate in mediating neutrophil transmigration
Authors: Wang Shijun , Dangerfield John P. , Young Rebecca E. , Nourshargh Sussan
Published: April 2005

The heterogeneous nature of the perivascular basement membrane (composed primarily of laminin and collagen type IV) suggests the existence of an elaborate array of adhesive interactions and possibly proteolytic events in leukocyte migration through this barrier. In this context, blockade of α6 integrins (laminin receptors), neutrophil elastase (NE) or both inhibited neutrophil migration through interleukin-1β (IL-1β)-stimulated mouse cremasteric venules, as observed by intravital microscopy. Furthermore, analysis of tissues by confocal microscopy indicated a synergistic role for α6 integrins and NE in mediating neutrophil migration through the perivascular basement membrane. Using a combined in vitro and in vivo experimental approach, the findings of this study also suggest that α6 integrins and NE are mobilized from intracellular stores to the cell surface of transmigrating mouse neutrophils, although these events occur via mechanisms dependent on and independent of platelet/endothelial-cell adhesion molecule 1 (PECAM-1, CD31), respectively. Despite different regulatory mechanisms, blockade of α6 integrins or NE inhibited migration of murine neutrophils through laminin-coated filters in vitro. Collectively, the findings suggest that, whereas regulation of the expression of α6 integrins and NE occur via different adhesive mechanisms, these molecules might act in a cooperative manner in mediating neutrophil migration through venular walls, in particular the perivascular basement membrane.
Blocking, descent and gravity waves: Observations and modelling of a MAP northerly föhn event
Authors: Jiang Qingfang , Doyle James D. , Smith Ronald B.
Published: April 2005

A northerly föhn event observed during the special observational period of the Mesoscale Alpine Programme is investigated based on observational analysis and numerical modelling. The focus of this study includes three dynamical processes associated with mountain perturbations and their interactions, namely, windward flow blocking, descent and warming on the lee side, and mountain waves. Observations indicate the presence of a deep weak‐flow layer underneath a stable layer, associated with Alpine‐scale blocking. Satellite imagery reveals a föhninduced cloud‐free area to the south of the Alps, which is consistent with flow descent diagnosed from radiosondes and constant‐volume balloons. Moderate‐amplitude stationary waves were observed by research aircraft over the major Alpine peaks. Satellite images and balloon data indicate the presence of stationary trapped‐wave patterns located to the north of the Alpine massif.

Satisfactory agreement is found between observations and a real‐data COAMPS simulation nested to 1 km resolution. COAMPS indicates the presence of trapped waves associated with a sharp decrease of Scorer parameter above a stable layer in the mid‐troposphere. Underneath the stable layer, moist low‐level flow is blocked to the north of the Alps. The warm air in the stable layer descends in the lee and recovers its altitude over a relatively short horizontal distance through a hydraulic jump.

Blocking reduces the effective mountain and hence significantly reduces mountain drag. A simple empirical formula for estimation of the effective mountain height, he, is derived based on numerical simulations. The formula states he/hc=√h/hc, where h is the real mountain height and hc is the critical mountain height to have flow stagnation. Copyright © 2005 Royal Meteorological Society.
Flavours of pseudo‐height
Authors: White A. A. , Beare R. J.
Published: April 2005

Various functions of pressure have been used as vertical coordinate in atmospheric dynamics. Some of these are called pseudo‐heights, or described as height‐like, although they could equally well be called pseudo‐thicknesses and described as thickness‐like. Three pseudo‐heights are reviewed here, and a unified treatment is presented in which two appear as specializations of the third. The common feature is a standard temperature profile which is a function only of pressure, and the two specializations arise when that function is chosen to represent either an isothermal atmosphere or an isentropic atmosphere. Neither of these choices—or any other reasonable function of pressure—itself introduces approximation. Under quasi‐geostrophic approximation, the need for a non‐zero mean‐state stratification means that a second reference‐temperature profile must be introduced if isentropic pseudo‐height is used as vertical coordinate. © Crown copyright 2005.
The structure of the lunar semi‐diurnal pressure tide L2
Authors: Goulter Stephen W.
Published: April 2005

The Hough structure of the lunar semi‐diurnal tide L2(p) in surface pressure is estimated using joint weightings by area and probable‐error structure, on annual and Lloyd seasonal scales, with new data.

Global representations, on seasonal and annual scales of the L2 wave, in both spherical harmonic and Hough function forms, are presented for the Haurwitz–Cowley dataset enlarged by the more recent tidal determinations of Hutchings and Palumbo. These are mainly for southern hemiphere island locations.

The asymmetric Hough eigenfunction terms H32 are considerably larger than previously estimated, both annually and seasonally, a possible artefact of the earlier data analysis technique. They are consistent and identified with the well‐known difference in phasing and amplitude of L2 by hemisphere.

The dependence of the Hough structure on the order of the fitting and on the more extreme residuals is examined. It is stable for the smallest models (spherical harmonic terms up to degree 6 and order 3), to removal of the largest residuals from a first fitting, and to the two largest datasets.

Seasonal changes in the Hough structure are discussed. The asymmetric results appear consistent with early frictional/thermal interpretations on the seasonal variation of L2. But the sensitivity of L2 to upper‐air temperature structure is not consistently shown in the analyses for the main mode H(2, 2). Copyright © 2005 Royal Meteorological Society.
Effects of stochastic parametrizations in the Lorenz '96 system
Authors: Wilks Daniel S.
Published: April 2005

Stochastic parametrization of the effects of unresolved variables is studied in the context of the Lorenz '96 system. These parametrizations are found to produce clear improvements in correspondence between the model and ‘true’ climatologies; they similarly provide clear improvements in all ensemble forecast verification measures investigated, including accuracy of ensemble means and ensemble probability estimation, and including measures operating on both scalar (each resolved forecast variable evaluated individually) and vector (all forecast variables evaluated simultaneously) predictands. Scalar accuracy measures for non‐ensemble (i.e. single integration) forecasts are, however, degraded. The results depend very strongly on both the amplitude (standard deviation) and time‐scale of the stochastic forcing, but only weakly on its spatial scale. In general there seems not to be a single clear optimum combination of time‐scale and amplitude, but rather there exists a range of combinations producing similar results. Copyright © 2005 Royal Meteorological Society.
An investigation of incremental 4D‐Var using non‐tangent linear models
Authors: Lawless A. S. , Gratton S. , Nichols N. K.
Published: April 2005

We investigate the convergence of incremental four‐dimensional variational data assimilation (4D‐Var) when an approximation to the tangent linear model is used within the inner loop. Using a semi‐implicit semi‐Lagrangian model of the one‐dimensional shallow water equations, we perform data assimilation experiments using an exact tangent linear model and using an inexact linear model (a perturbation forecast model). We find that the two assimilations converge at a similar rate and the analyses are also similar, with the difference between them dependent on the amount of noise in the observations. To understand the numerical results, we present the incremental 4D‐Var algorithm as a Gauss–Newton iteration for solving a least‐squares problem and consider its fixed points. Copyright © 2005 Royal Meteorological Society.