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Fibroblast growth factor 21 (FGF21) is a promising drug candidate for the treatment of type 2 diabetes. However, the use of wild type native FGF21 is challenging due to several limitations. Among these are its short half-life, its susceptibility to in vivo proteolytic degradation and its propensity to in vitro aggregation. We here describe a rationale-based protein engineering approach to generate a potent long-acting FGF21 analog with improved resistance to proteolysis and aggregation. A recombinant Fc-FGF21 fusion protein was constructed by fusing the Fc domain of human IgG1 to the N-terminus of human mature FGF21 via a linker peptide. The Fc positioned at the N-terminus was determined to be superior to the C-terminus as the N-terminal Fc fusion retained the βKlotho binding affinity and the in vitro and in vivo potency similar to native FGF21. Two specific point mutations were introduced into FGF21. The leucine to arginine substitution at position 98 (L98R) suppressed FGF21 aggregation at high concentrations and elevated temperatures. The proline to glycine replacement at position 171 (P171G) eliminated a site-specific proteolytic cleavage of FGF21 identified in mice and cynomolgus monkeys. The derived Fc-FGF21(RG) molecule demonstrated a significantly improved circulating half-life while maintaining the in vitro activity similar to that of wild type protein. The half-life of Fc-FGF21(RG) was 11 h in mice and 30 h in monkeys as compared to 1-2 h for native FGF21 or Fc-FGF21 wild type. A single administration of Fc-FGF21(RG) in diabetic mice resulted in a sustained reduction in blood glucose levels and body weight gains up to 5-7 days, whereas the efficacy of FGF21 or Fc-FGF21 lasted only for 1 day. In summary, we engineered a potent and efficacious long-acting FGF21 analog with a favorable pharmaceutical property for potential clinical development.

The French program Coriolis as part of the French oceanographic operational system produces the COriolis dataset for Re-Analysis (CORA) on a yearly basis which is based on temperature and salinity measurements on observed levels from different data types. The latest release of CORA covers the period 1990 to 2010. To qualify this dataset, several tests have been developed to improve in a homogeneous way the quality of the raw dataset and to fit the level required by the physical ocean re-analysis activities (assimilation and validation). These include some simple tests, climatological tests and a model background check based on a global ocean reanalysis. Visual quality control (QC) is performed on all suspicious temperature (T) and salinity (S) profiles identified by the tests and quality flags are modified in the dataset if necessary. In addition, improved diagnostic tools were developed – including global ocean indicators – which give information on the potential and quality of the CORA dataset for all applications. This Coriolis product is available on request through the MyOcean Service Desk (http://www.myocean.eu/).

The Optical Spectrograph and Infra-Red Imager System (OSIRIS) and the Atmospheric Chemistry Experiment (ACE) have been taking measurements from space since 2001 and 2003, respectively. This paper presents intercomparisons between ozone and NO2 measured by the ACE and OSIRIS satellite instruments and by ground-based instruments at the Polar Environment Atmospheric Research Laboratory (PEARL), which is located at Eureka, Canada (80° N, 86° W) and is operated by the Canadian Network for the Detection of Atmospheric Change (CANDAC). The ground-based instruments included in this study are four zenith-sky differential optical absorption spectroscopy (DOAS) instruments, one Bruker Fourier transform infrared spectrometer (FTIR) and four Brewer spectrophotometers. Ozone total columns measured by the DOAS instruments were retrieved using new Network for the Detection of Atmospheric Composition Change (NDACC) guidelines and agree to within 3.2%. The DOAS ozone columns agree with the Brewer spectrophotometers with mean relative differences that are smaller than 1.5%. This suggests that for these instruments the new NDACC data guidelines were successful in producing a homogenous and accurate ozone dataset at 80° N. Satellite 14–52 km ozone and 17–40 km NO2 partial columns within 500 km of PEARL were calculated for ACE-FTS Version 2.2 (v2.2) plus updates, ACE-FTS v3.0, ACE-MAESTRO (Measurements of Aerosol Extinction in the Stratosphere and Troposphere Retrieved by Occultation) v1.2 and OSIRIS SaskMART v5.0x ozone and Optimal Estimation v3.0 NO2 data products. The new ACE-FTS v3.0 and the validated ACE-FTS v2.2 partial columns are nearly identical, with mean relative differences of 0.0 ± 0.2% for ozone and −0.2 ± 0.1% for v2.2 minus v3.3 NO2. Ozone columns were constructed from 14–52 km satellite and 0–14 km ozonesonde partial columns and compared with the ground-based total column measurements. The satellite-plus-sonde measurements agree with the ground-based ozone total columns with mean relative differences of 0.1–7.3%. For NO2, partial columns from 17 km upward were scaled to noon using a photochemical model. Mean relative differences between OSIRIS, ACE-FTS and ground-based NO2 measurements do not exceed 20%. ACE-MAESTRO measures more NO2 than the other instruments, with mean relative differences of 25–52%. Seasonal variation in the differences between partial columns is observed, suggesting that there are systematic errors in the measurements, the photochemical model corrections, and/or in the coincidence criteria. For ozone spring-time measurements, additional coincidence criteria based on stratospheric temperature and the location of the polar vortex were found to improve agreement between some of the instruments. For ACE-FTS v2.2 minus Bruker FTIR, the 2007–2009 spring-time mean relative difference improved from −5.0 ± 0.4% to −3.1 ± 0.8% with the dynamical selection criteria. This was the largest improvement, likely because both instruments measure direct sunlight and therefore have well-characterized lines-of-sight compared with scattered sunlight measurements. For NO2, the addition of a ±1° latitude coincidence criterion improved spring-time intercomparison results, likely due to the sharp latitudinal gradient of NO2 during polar sunrise. The differences between satellite and ground-based measurements do not show any obvious trends over the missions, indicating that both the ACE and OSIRIS instruments continue to perform well.

The updated 2009 edition of the spectroscopic database GEISA (Gestion et Etude des Informations Spectroscopiques Atmosphériques; Management and Study of Atmospheric Spectroscopic Information) is described in this paper. GEISA is a computer-accessible system comprising three independent sub-databases devoted, respectively, to: line parameters, infrared and ultraviolet/visible absorption cross-sections, microphysical and optical properties of atmospheric aerosols. In this edition, 50 molecules are involved in the line parameters sub-database, including 111 isotopologues, for a total of 3,807,997 entries, in the spectral range from 10-6 to 35,877.031cm-1.The successful performances of the new generation of hyperspectral sounders depend ultimately on the accuracy to which the spectroscopic parameters of the optically active atmospheric gases are known, since they constitute an essential input to the forward radiative transfer models that are used to interpret their observations. Currently, GEISA is involved in activities related to the assessment of the capabilities of IASI (Infrared Atmospheric Sounding Interferometer; http://smsc.cnes.fr/IASI/index.htm) on board the METOP European satellite through the GEISA/IASI database derived from GEISA. Since the Metop-A (http://www.eumetsat.int) launch (19 October 2006), GEISA is the reference spectroscopic database for the validation of the level-1 IASI data. Also, GEISA is involved in planetary research, i.e. modeling of Titan's atmosphere, in the comparison with observations performed by Voyager, or by ground-based telescopes, and by the instruments on board the Cassini-Huygens mission.GEISA, continuously developed and maintained at LMD (Laboratoire de Météorologie Dynamique, France) since 1976, is implemented on the IPSL/CNRS (France) "Ether" Products and Services Centre WEB site (http://ether.ipsl.jussieu.fr), where all archived spectroscopic data can be handled through general and user friendly associated management software facilities. More than 350 researchers are registered for on line use of GEISA. © 2011 Elsevier Ltd., SCOPUS: ar.j, info:eu-repo/semantics/published

Background Few validated instruments exist to measure pediatric code team skills. The goal of this study was to develop an instrument for the assessment of resuscitation competency and self-appraisal using multirater and gap analysis methodologies. Methods Multirater assessment with gap analysis is a robust methodology that enables the measurement of self-appraisal as well as competency, offering faculty the ability to provide enhanced feedback. The Team Performance during Simulated Crises Instrument (TPDSCI) was grounded in the Accreditation Council for Graduate Medical Education competencies. The instrument contains 5 competencies, each assessed by a series of descriptive rubrics. It was piloted during a series of simulation-based interdisciplinary pediatric crisis resource management education sessions. Course faculty assessed participants, who also did self-assessments. Internal consistency and interrater reliability were analyzed using Cronbach α and intraclass correlation (ICC) statistics. Gap analysis results were examined descriptively. Results Cronbach α for the instrument was between 0.72 and 0.69. The overall ICC was 0.82. ICC values for the medical knowledge, clinical skills, communication skills, and systems-based practice were between 0.87 and 0.72. The ICC for the professionalism domain was 0.22. Further examination of the professionalism competency revealed a positive skew, 43 simulated sessions (98%) had significant gaps for at least one of the competencies, 38 sessions (86%) had gaps indicating self-overappraisal, and 15 sessions (34%) had gaps indicating self-underappraisal. Conclusions The TPDSCI possesses good measures of internal consistency and interrater reliability with respect to medical knowledge, clinical skills, communication skills, systems-based practice, and overall competence in the context of simulated interdisciplinary pediatric medical crises. Professionalism remains difficult to assess. These results provide an encouraging first step toward instrument validation. Gap analysis reveals disparities between faculty and self-assessments that indicate inadequate participant self-reflection. Identifying self-overappraisal can facilitate focused interventions.

The protein composition of inclusion bodies (IBs) formed in recombinant Escherichia coli producing high levels of porcine somatotropin (pST) was analyzed by one- and two-dimensional protein gel electrophoresis. Recombinant pST is exclusively recovered from the insoluble cell fraction. Results indicate that, in addition to the main species of pST, subspecies with different isoelectric points and degradative fragments are contained within IBs. The presence of outer membrane proteins in IB fractions results from coprecipitation of cell debris during IB preparation and not from specific in vivo or in vitro interaction of these proteins with IBs. Cells producing pST contain up to three IBs located in the cytoplasm. The implication of high level gene expression on the uniformity of the desired product is discussed.

Major limitations of our present knowledge of the global distribution of CO2 in the atmosphere are the uncertainty in atmospheric transport and the sparseness of in situ concentration measurements. Limb viewing spaceborne sounders such as the Atmospheric Chemistry Experiment Fourier transform spectrometer (ACE-FTS) offer a vertical resolution of a few kilometres for profiles, which is much better than currently flying or planned nadir sounding instruments can achieve. After having demonstrated the feasibility of obtaining CO2 vertical profiles in the 5–25 km altitude range with an accuracy of about 2 ppm in a previous study, we present here the results of five years of ACE-FTS observations in terms of monthly mean profiles of CO2 averaged over 10° latitude bands for northern mid-latitudes. These results are compared with in-situ aircraft measurements and with simulations from two different air transport models. Key features of the measured altitude distribution of CO2 are shown to be accurately reproduced by the ACE-FTS retrievals: variation in altitude of the seasonal cycle amplitude and extrema, seasonal change of the vertical gradient, and mean growth rate. We show that small but significant differences from model simulations could result from an over estimation of the model circulation strength during the northern hemisphere spring. Coupled with column measurements from a nadir viewing instrument, it is expected that occultation measurements will bring useful constraints to the surface carbon flux determination.

RNA interference (RNAi) depends on the production of small RNA to regulate gene expression in eukaryotes. Two RNAi systems exist to control repetitive selfish elements in Neurospora crassa. Quelling targets transgenes during vegetative growth, whereas meiotic silencing by unpaired DNA (MSUD) silences unpaired genes during meiosis. The two mechanisms require common RNAi proteins, such as RNA-directed RNA polymerases, Dicers, and Argonaute slicers. We have previously demonstrated that, while Quelling depends on the redundant dicer activity of DCL-1 and DCL-2, only DCL-1 is required for MSUD. Here, we show that QDE-2-interacting protein (QIP), an exonuclease that is important for the production of single-stranded siRNA during Quelling, is also required for MSUD. QIP is crucial for sexual development and is shown to colocalize with other MSUD proteins in the perinuclear region.

Routine quantitation of recombinant human granulocyte colony-stimulating factor charge isoforms in the purified protein product requires development of a reliable analytical method. In this report, isoelectric focusing gel electrophoresis, peptide mapping, and cation-exchange high-performance liquid chromatography are compared and evaluated in the analysis of charge isomers that may be present in the recombinant factor. Due to a lack of sensitivity and reliability, isoelectric focusing gel electrophoresis and peptide mapping are not recommended. However, peptide mapping can distinguish aberrant peptides with differences in charges and provide separation for subsequent structural characterization. By this approach, an N-terminally blocked formylmethionyl species was identified to be the minor charge isoform in the purified preparations of recombinant human granulocyte colony-stimulating factor. In contrast to electrophoresis and peptide mapping, a strong cationic-exchange chromatographic procedure was found to be the most selective, sensitive, and reproducible analytical method. The sensitivity and reliability of the method were evaluated and validated using the formylmethionyl isoform and several deamidated analogs (Gln → Glu) made by site-directed mutagenesis. Recombinant human granulocyte colony-stimulating factor preparations contain a very low to undetectable level of the formylmethionine isoform and have no detectable deamidated isoforms.

The folding and oxidation of recombinant human granulocyte colony-stimulating factor solubilized from Escherichia coli inclusion bodies was investigated. During the folding process, two intermediates, I1 and I2, were detected by kinetic studies using high performance liquid chromatography. I1 exists transiently and disappears quickly with the concomitant formation of I2. In contrast, I2 requires a longer time to fold into the final oxidized form, N. CuSO4 catalysis increases the folding rate of I2 from I1, while CuSO4 and elevated temperature (37 degrees C) have a dramatic effect on the folding rate of N from I2. These observations suggest the following sequential oxidative folding pathway. [sequence: see text] Peptide map analysis of the iodoacetate-labeled intermediates revealed that I1 represents the fully reduced granulocyte colony-stimulating factor containing 5 free cysteines; I2 is the partially oxidized species containing a single Cys36-Cys42 disulfide bond; and N, the final folded form, has two disulfide bonds. The physicochemical properties and biological activities of I1, I2, N, and several Cys—-Ser analogs made by site-directed mutagenesis were further investigated. In guanidine hydrochloride-induced denaturation studies, the disulfide-reduced intermediates and the analogs missing either of the disulfide bonds are conformationally less stable than those of the wild type molecule or the analog with the free Cys at position 17 changed to Ser. Recombinant human granulocyte colony stimulating factor lacking either disulfide bond or both has overall secondary and tertiary structures different from those of the wild type molecule and exhibits lower biological activity. These studies show that disulfide bond formation is crucial for maintaining the molecule in a properly folded and biologically active form.

Stem cell factor (SCF) is a novel, early-acting hematopoietic factor. It was isolated from the medium of a rat cell line in a soluble, processed form (Zsebo et al., 1990, Cell 63, 195). The cloned human and rat genes encode the soluble form plus additional C-terminal amino acids including a hydrophobic transmembrane domain (Martin et al., 1990, Cell 63, 203). We have recombinantly expressed forms of human and rat SCF corresponding to the soluble, processed form in Escherichia coli and in Chinese hamster ovary (CHO) cells. After expression in E. coli, folding and oxidation of the SCF polypeptides are required. The SCFs expressed in CHO cells are secreted into the medium in active state and, like the natural SCF, are glycosylated. Purification of the recombinant SCFs is described. Biological and biochemical characterization includes activity toward responsive human and mouse cell lines, N-terminal amino acid sequences, disulfide bond linkages, and sites of glycosylation.

Ten dogs were given mitoxantrone at a dose of 5 mg/m2 body surface area intravenously. Recombinant canine granulocyte colony-stimulating factor (rcG-CSF) was administered subcutaneously daily for 20 days after an infusion of mitoxantrone in five of these dogs to determine the effect of the hematopoietic growth factor on the duration and severity of myelosuppression. The median neutrophil counts dropped below normal (

Immunomodulatory effects of recombinant bovine granulocyte colony-stimulating factor were evaluated in periparturient dairy cows. Eleven of 21 cows were experimentally infected with Staphylococcus aureus in one mammary quarter prior to the study. Cows were assigned to four groups in a randomized complete block design to evaluate the effects of recombinant bovine granulocyte colony-stimulating factor (5μg/kg of body weight or placebo injected subcutaneously once daily beginning 14 d prepartum through 10 d postpartum) on infected and uninfected cows during the periparturient period. Blood lymphocytes were isolated and evaluated from 5 wk before expected parturition through 7 wk postpartum. Lymphocyte function was evaluated using a blastogenesis assay, a mitochondrial methylthiazoltetrazolium cleavage activity assay, and an in vitro assay of IgM production. Serum concentrations of IgM, IgG 1 , conglutinin, and hemolytic complement were also determined. Injections of cows with recombinant bovine granulocyte colony-stimulating factor resulted in enhanced lymphocyte blastogenesis and mitochondrial methylthiazoltetrazolium cleavage activity in unstimulated cultures, higher serum IgM, and increased in vitro IgM production by B lymphocytes. These data provide support for the use of recombinant bovine granulocyte colony-stimulating factor to alleviate immunosuppression in periparturient cows.

Transforming growth factor beta (TGF-beta) is a potent and selective growth inhibitor of early hematopoietic progenitors and leukemic cells. The cellular mechanism(s) underlying this antiproliferative effect is, however, currently unknown. In the present study, we demonstrate that TGF-beta inhibits the expression of granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin 3 (IL-3), and granulocyte-CSF (G-CSF) receptors on murine factor-dependent and independent hematopoietic progenitor cell lines without a significant change in receptor affinity. A maximum reduction in GM-CSF receptor numbers of 65% to 77% was observed by 96-hour incubation with TGF-beta. The TGF- beta induced trans-down-modulation of GM-CSF receptors was prolonged, noncytotoxic but reversible, and not due to endogenous production of GM- CSF. The TGF-beta induced reduction in CSF receptor numbers preceded TGF-beta's growth inhibitory action. In addition, the ED50 (1 to 10 pmol/L) for TGF-beta's CSF receptor modulatory and antiproliferative effect was similar. The effect of TGF-beta on cell surface CSF receptor expression was specific, because the expression of other cell surface proteins (Ly 5 and Ly 17) was not affected by TGF-beta treatment, and because other growth inhibitors (tumor necrosis factor and interferon) did not affect CSF receptor expression. These data suggest that the downregulation of the growth of hematopoietic progenitor cells by TGF- beta involves reducing the cell surface expression on growth factor receptors.

The Atmospheric Chemistry Experiment (ACE), also known as SCISAT, was launched on 12 August 2003, carrying two instruments that measure vertical profiles of atmospheric constituents using the solar occultation technique. One of these instruments, the ACE Fourier Transform Spectrometer (ACE-FTS), is measuring volume mixing ratio (VMR) profiles of nitrous oxide (N2O) from the upper troposphere to the lower mesosphere at a vertical resolution of about 3–4 km. In this study, the quality of the ACE-FTS version 2.2 N2O data is assessed through comparisons with coincident measurements made by other satellite, balloon-borne, aircraft, and ground-based instruments. These consist of vertical profile comparisons with the SMR, MLS, and MIPAS satellite instruments, multiple aircraft flights of ASUR, and single balloon flights of SPIRALE and FIRS-2, and partial column comparisons with a network of ground-based Fourier Transform InfraRed spectrometers (FTIRs). Overall, the quality of the ACE-FTS version 2.2 N2O VMR profiles is good over the entire altitude range from 5 to 60 km. Between 6 and 30 km, the mean absolute differences for the satellite comparisons lie between −42 ppbv and +17 ppbv, with most within ±20 ppbv. This corresponds to relative deviations from the mean that are within ±15%, except for comparisons with MIPAS near 30 km, for which they are as large as 22.5%. Between 18 and 30 km, the mean absolute differences are generally within ±10 ppbv, again excluding the aircraft and balloon comparisons. From 30 to 60 km, the mean absolute differences are within ±4 ppbv, and are mostly between −2 and +1 ppbv. Given the small N2O VMR in this region, the relative deviations from the mean are therefore large at these altitudes, with most suggesting a negative bias in the ACE-FTS data between 30 and 50 km. In the comparisons with the FTIRs, the mean relative differences between the ACE-FTS and FTIR partial columns are within ±6.6% for eleven of the twelve contributing stations. This mean relative difference is negative at ten stations, suggesting a small negative bias in the ACE-FTS partial columns over the altitude regions compared. Excellent correlation (R=0.964) is observed between the ACE-FTS and FTIR partial columns, with a slope of 1.01 and an intercept of −0.20 on the line fitted to the data.

A number of proteinases are induced and secreted into the culture medium of Tritirachium album Limber when the nitrogen source is limited to exogenous proteins. We have constructed a cDNA library using the polyadenylated RNA isolated during the nutritional induction with bovine serum albumin. A full-length clone of a gene for a new proteinase (named proteinase R) was identified from this library. This clone contained sequences coding for the 108-amino-acid prepro-leader as well as for the 279-amino-acid mature proteinase. Proteinase R apparently belongs to the subtilisin group of serine proteases that contains disulphide bonds. Homology between proteinase R and proteinase K was found to be about 87% at the nucleotide as well as at the amino acid level. The Brookhaven Protein Data Base co-ordinate file of proteinase K was used as a template to study the proteinase R substitutions in three-dimensional space. The majority of the substitutions of proteinase R with respect to proteinase K were found to be on the exterior of the protein model.

The Transfusion Safety Study (TSS) is a multicenter, cooperative investigation of factors that may determine the occurrence and modify the expression of transusion-transmitted infections. A flow cytometry laboratory was established in each of the six participating centers in order to avoid alterations in cell phenotypes which may be caused by shipping delays, temperature changes, and handling. As a consequence, in order to assure compatibility of results, stringent standardization, quality control, and proficiency testing procedures were developed. This paper documents (i) the effect of time from phlebotomy to specimen staining and then to analysis for the antibodies used in the study; (ii) the effects of variations in light scatter cursor location for certain antibodies; (iii) a quality control program and data management and analysis system, each specifically designed for the study; and (iv) presents extensive data on age- and sex-related reference (normal) ranges for the several individual and paired monoclonal antibodies used in the study. Problems encountered, including obtaining reliable absolute lymphocyte counts, interference by nucleated erythrocytes, and sources of variability in results, are discussed. This study is meant to serve as a reference for future TSS publications.

The purification and characterization of a metalloproteinase inhibitor (MI) from bovine aortic endothelial cells, and the demonstration that it is related to, but distinct from, tissue inhibitor of metalloproteinase (TIMP), have previously been reported [De Clerck, Y. A., Yean, T.-D., Ratzkin, B. J., Lu, H.S. & Langley, K. E. (1989) J. Biol. Chem. 264, 17445-17453]. The cDNA cloning of the bovine MI and its human homolog is now reported. The bovine cDNA cloning used probes designed on the basis of NH2-terminal amino acid sequence of bovine MI. The human cDNA cloning in turn used probes representing parts of the bovine cDNA nucleotide sequence. Both cDNAs encode leader sequences of 26 amino acids and mature protein sequences of 194 amino acids. The amino acid sequences of the mature proteins are 94% identical. The human MI cDNA was expressed in Escherichia coli, and a preparation containing anticollagenase activity was recovered. The amino acid sequence of mature human MI is 38% identical to the sequence for human TIMP, and the 12 cysteines in MI and TIMP are aligned almost identically. Thus MI and TIMP comprise an inhibitor family.

Recombinant TNF was capable of inducing differentiation of human leukemic ML-1 cells in the monocytic pathway. Recombinant interleukin 6 did not have the activity but it could significantly increase the activity of recombinant TNF. Both of these molecules were found to play a similar role in PWM-induced conditioned medium from human lymphocytes (LCM). The differentiation inducing effect of LCM could be partially neutralized by antibody to interleukin 6. Fractionation of LCM also identified interleukin 6 as the factor that synergizes with TNF.

Interleukin-4 (IL-4) is a multifunctional lymphokine which promotes the growth and/or maturation of multiple cell types. We have examined the ability of IL-4 to promote the phenotypic maturation of subsets of human thymocytes. When cultured in serum-free medium supplemented with recombinant IL-4, a subset of immature CD3-CD45RA- human thymocytes ceased to express the CD1 common thymocyte antigen and acquired phenotypic features characteristic of relatively mature thymocytes, such as high-density expression of the CD3 antigen and de novo expression of the CD45RA isoform of the common leukocyte antigen family. These changes, which were not seen in cells cultured in medium alone, occurred over an 8-9 day period and were accompanied by a significant increase in cell size. The CD45RA+ cells that derived from these immature CD3-CD45RA- precursors were mainly CD4-CD8- or CD8+ cells, and a significant proportion of these cells expressed the T cell receptor delta chain. IL-4 also increased expression of the CD45RA antigen on the more mature CD3+ thymocyte population. However, the CD45RA+ cells derived from IL-4 stimulated CD3+ thymocyte precursors expressed either the CD4 or the CD8 antigen, and virtually all expressed alpha/beta TCR chains. Studies of cell viability and cell growth indicated that these findings were due to direct changes in the phenotype of responsive cells rather than the growth or selective survival of a small number of mature thymocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

The first three Canadian Arctic ACE validation campaigns were held during polar sunrise at Eureka, Nunavut, Canada (80° N, 86° W) from 2004 to 2006 in support of validation of the ACE (Atmospheric Chemistry Experiment) satellite mission. Three or four zenith-sky viewing UV-visible spectrometers have taken part in each of the three campaigns. The differential slant column densities and vertical column densities from these instruments have been compared following the methods of the UV-visible Working Group of the NDACC (Network for Detection of Atmospheric Composition Change). The instruments are found to partially agree within the required accuracies for both species, although both the vertical and slant column densities are more scattered than required. This might be expected given the spatial and temporal variability of the Arctic stratosphere in spring. The vertical column densities are also compared to integrated total columns from ozonesondes and integrated partial columns from the ACE-FTS (ACE-Fourier Transform Spectrometer) and ACE-MAESTRO (ACE-Measurements of Aerosol Extinction in the Stratosphere and Troposphere Retrieved by Occultation) instruments on board ACE. For both species, the columns from the ground-based instruments and the ozonesondes are found to generally agree within their combined error bars. The ACE-FTS ozone partial columns and the ground-based total columns agree within 4.5%, averaged over the three campaigns. The ACE-MAESTRO ozone partial columns are generally smaller than those of the ground-based instruments, by an average of 9.9%, and are smaller than the ACE-FTS columns by an average of 14.4%. The ACE-FTS NO2 partial columns are an average of 13.4% smaller than the total columns from the ground-based instruments, as expected. The ACE-MAESTRO NO2 partial columns are larger than the total columns of the ground-based instruments by an average of 2.5% and larger than the partial columns of the ACE-FTS by an average of 15.5%.

Peroxiredoxin 5 (PRDX5) is a mammalian thioredoxin peroxidase ubiquitously expressed in tissues. Its role as antioxidant enzyme has been previously supported in different pathological situations. In this study, we determined the complete human PRDX5 genomic organization and isolated the 5'-flanking region of the gene. Human PRDX5 gene is composed of six exons and five introns similarly to other chordate PRDX5 genes. Several single nucleotide polymorphisms were identified. Six out of them have amino acid substitutions in protein-coding region. Analysis of the 5'-flanking region of human PRDX5 revealed the presence of a TATA-less promoter containing a canonical CpG island and several putative response elements for transcription factors. To analyze the regulatory mechanisms controlling human PRDX5 expression, we characterized the 5'-flanking region by cloning various segments of this region in front of a luciferase reporter sequence. Transfection in HepG2 cells indicate that the 5'-flanking region contains regulatory elements for constitutive expression of human PRDX5. Multiple transcription start sites were also identified by 5'-RACE-PCR in human liver. Moreover, although no corresponding proteins were reported, we present new alternative splicing variants encoded specifically by human PRDX5 gene. The characterization of human PRDX5 gene revealed the complexity of its regulation and a high variability of sequences that might be associated with pathological situations.

The neural network methodology is applied to the sea surface salinity retrieval from SMOS brightness temperatures. The direct model for simulating the brightness temperatures is the Small Slope Approximation model (SSA). Different cases are compared to analyze the retrieval quality. The effect of a bias on the brightness temperatures and of the instrumental accuracy expected on the SMOS measurements are evaluated. The inversion algorithm is improved when adding ancillary parameters (sea surface temperature, wind speed and a priori salinity).

In order to make a qualitative assessment on the error of retrieved sea surface salinity (SSS), the simulating tool developed by P. Waldteufel tuned to SMOS characteristics is used in combination with numerical models outputs, remotely sensed data and in-situ data. A space-time average is carried out in order to reduce the SSS error. Its impact is presented for some specific regions.

We describe regulatory effects that a novel neurotrophin-1/B cell-stimulating factor-3 (NNT-1/BSF-3; also reported as cardiotrophin-like cytokine) has on B cell function. NNT-1/BSF-3 stimulates B cell proliferation and Ig production in vitro. NNT-1/BSF-3-transgenic mice, engineered to express NNT-1/BSF-3 in the liver under control of the apolipoprotein E promoter, show B cell hyperplasia with particular expansion of the mature follicular B cell subset in the spleen and the prominent presence of plasma cells. NNT-1/BSF-3-transgenic mice show high serum levels of IgM, IgE, IgG2b, IgG3, anti-dsDNA Abs, and serum amyloid A. NNT-1/BSF-3-transgenic mice also show non-amyloid mesangial deposits that contain IgM, IgG, and C3 and are characterized by a distinctive ultrastructure similar to that of immunotactoid glomerulopathy. NNT-1/BSF-3-transgenic mice produce high amounts of Ag-specific IgM, IgA, and IgE and low amounts of IgG2a and IgG3. Normal mice treated with NNT-1/BSF-3 also produce high amounts of Ag-specific IgE. NNT-1/BSF-3 regulates immunity by stimulating B cell function and Ab production, with preference for Th2 over Th1 Ig types.

G-CSF is an extremely well-known and well-characterized molecule. Both the natural glycosylated form and the E. coli-produced nonglycosylated form are biologically active. A thorough understanding of the primary, secondary, and tertiary structures has enabled a rational approach to purification, folding, and formulation. Although the product can be considered mature, and testing and development of the second-generation formulation are complete, chemical and physical analysis of the product continue. It is this continued effort to understand the chemistry and stability of the product that ensure a safe and efficacious molecule.

Publisher Summary This chapter discusses the near real-time processing and validation of T/P and ERS-2 altimeter data, developing the use of altimeter data for climate studies (DUACS). The goal of the DUACS EC project was to develop and validate a Near Real Time (NRT) altimeter data processing system for climate and seasonal prediction studies. Altimeter data from TOPEX/POSEIDON Interim Geophysical Data Record (IGDR) and ERS-2 Fast Delivery Products (FDP) are fully processed, combined, and validated every week. They are delivered, at the earliest, 2 days after the last gathered measurement. Altimeter products consist of a long-track sea level anomaly (SLA) data and the maps of SLA. After a roughly 2 month delay imposed by the processing and distribution of the best available delayed time homogenous historical (HH) data from TOPEX/POSEIDON and ERS-2, the NRT SLA data are compared to HH SLA data. The NRT SLA data has a very good accuracy with respect to HH data. NRT data can, thus, be used to monitor large scale climatic signals, such as El Nino, with accuracy on the order of 2 cm root mean square (RMS). It can also provide an accurate fine-scale description of different current systems and of the mesoscale variability.

Signal transduction pathways with shared components must be insulated from each other to avoid the inappropriate activation of multiple pathways by a single stimulus. Scaffold proteins are thought to contribute to this specificity by binding select substrates.We have studied the ability of scaffold proteins to influence signaling by the yeast kinase Ste11, a MAPKKK molecule that participates in three distinct MAP kinase pathways: mating, filamentation, and HOG. We used protein fusions to force Ste11 to associate preferentially with a subset of its possible binding partners in vivo, including Ste5, Ste7, and Pbs2. Signaling became confined to a particular pathway when Ste11 was covalently attached to these scaffolds or substrates. This pathway bias was conferred upon both stimulus-activated and constitutively active forms of Ste11. We also used membrane-targeted derivatives of the mating pathway scaffold, Ste5, to show that stimulus-independent signaling initiated by this scaffold remained pathway specific. Finally, we demonstrate that loss of pathway insulation has a negative physiological consequence, as nonspecific activation of both the HOG and mating pathways interfered with proper execution of the mating pathway.The signaling properties of these kinase fusions support a model in which scaffold proteins dictate substrate choice and promote pathway specificity by presenting preferred substrates in high local concentration. Furthermore, insulation is inherent to scaffold-mediated signaling and does not require that signaling be initiated by pathway-specific stimuli or activator proteins. Our results give insight into the mechanisms and physiological importance of pathway insulation and provide a foundation for the design of customized signaling proteins.

During the last decades, livestock breeders attempted to develop strategies for decreasing fat content in meat. One of these strategies consists in immunization against adipocytes. Polyclonal antisera have been raised against fat cells or adipocyte plasma membranes of different species, and in vivo reduction of fat depots has been observed in some cases. Facing the problems of specificity and reproducibility inherent to polyclonal antisera, monoclonal antibodies could offer a promising alternative, which has been developed only in the porcine species.

The use of nuclear morphometry as an intermediate endpoint biomarker is described in a Phase I, dose-seeking trial of chemoprevention of cervical cancer, using the agent alpha-difluoromethylornithine (DFMO). Thirty patients with grade III cervical intraepithelial neoplasia (CIN III) were enrolled, and these received daily doses of DFMO at 0.06-1.0 mg/m(2) for a period of 1 month. Fifteen patients were observed to have a complete or partial regressive response to the agent, as assessed by histopathology. No significant differences in cell feature measurements were found between responders and nonresponders in specimens obtained before treatment, indicating that it may be difficult to predict response on the basis of these measurements. In specimens collected after treatment, large differences in morphometric features were observed between responders and nonresponders, indicating a differential effect of DFMO. Significantly modulated features were considered in terms of their correlations with CIN grade, which was determined from an independent set of measurements from archival tissue. Differences between features were consistent with a deletion of cells with high grade nuclei in the responders, and with the persistence of a more heterogeneous population of high grade cells in the nonresponders. Based on an independent set of measurements from archival material, a morphometric index of progression was derived, yielding a quantitative measure of the degree of nuclear atypia in these lesions. When applied to this trial, the morphometric index was seen to be specifically and consistently decreased in responsive lesions, and unchanged in nonresponders. The study indicates that morphometric features fulfill the requirements for an intermediate endpoint biomarker of cervical cancer chemoprevention.

Osteoprotegerin (OPG) is a secreted protein that inhibits osteoclast formation and activity and appears to be a critical regulator of bone mass and metabolism. In the current study, mice were challenged with various cytokines and hormones (interleukin-1beta, tumor necrosis factor-alpha, parathyroid hormone, parathyroid hormone-related protein, and 1alpha,25-dihydroxyvitamin D3) that are known to increase bone resorption and cause hypercalcemia and treated concurrently with either a recombinant chimeric Fc fusion form of human OPG, with enhanced biological activity (cOPG) (2.5 mg/kg/day) or vehicle. Mice receiving these bone-resorbing factors became hypercalcemic by day 3 after commencing treatment and had increased bone resorption as evidenced by elevated osteoclast numbers on day 5. Concurrent cOPG treatment prevented hypercalcemia (p < 0.05) and maintained osteoclast numbers in the normal range (p < 0.001). The demonstration that cOPG can inhibit bone resorption suggests that this molecule may be useful in the treatment of diseases including hyperparathyroidism, humoral hypercalcemia of malignancy, osteoporosis, and inflammatory bone disease, which are characterized, in part, by increases in osteoclastic bone resorption.

The cyclic adenosine-monophosphate (cAMP) pathway is generally recognized as one of the essential pathways for the adipose conversion of rodent preadipocytes in vitro. However, divergent effects of cAMP on adipocyte differentiation have also been reported. Since there is very little data on non-rodent preadipose cells, the aim of the present work was to analyze the effects of classic activators of the cAMP pathway on the proliferation and differentiation of porcine preadipocytes grown either in serum-free or in serum-containing medium. In both media, the addition of 10 microM forskolin from day 1 after cell plating to day 3 or 7 did not affect cell proliferation. Such stimulations also failed to enhance preadipocyte differentiation, as assessed by the measurement of lipoprotein lipase (LPL) and glycerol 3-phosphate dehydrogenase (GPDH) activities, two markers of adipose conversion. Similar results were obtained when various concentrations of forskolin (0.1 nM-100 microM) were added for 2 days either during the growth phase (days 1-3) or after confluence (days 5-7). Addition of methylisobutylxanthine (MIX) or 8-bromo-cAMP was also found inefficient to stimulate porcine preadipocytes differentiation clearly. By contrast, post-confluence treatment of the murine 3T3-L1 cell line with either forskolin or MIX markedly enhanced lipid accumulation and led to a dramatic increase in GPDH activity (up to 120 times). This indicates that similar culture conditions are adipogenic for the murine 3T3-L1 preadipocytes but not for porcine preadipose cells. In summary, this work clearly highlights the finding that porcine preadipocytes do not respond to classic activators of the cAMP pathway like rodent cells do. This calls in question again the general model proposed for the action of this pathway in adipose conversion and suggests that the mechanisms regulating adipocyte differentiation may differ among species.

Several studies have shown an association between high nuclear grade or necrosis of ductal carcinoma in situ (DCIS) lesions and the risk of local disease recurrence in patients with DCIS treated surgically with less than mastectomy. Although criteria for separating low from high nuclear grade lesions have been published, no information exists regarding interobserver reproducibility (IR). To assess IR in the classification of DCIS, six surgical pathologists from four institutions used the Lagios grading system to grade 125 DCIS lesions. Before meeting to evaluate the cases, a training set of 12 glass slides, including cases chosen to present conflicting cues for classification, was mailed to the participants with a written criteria summary. This was followed by a working session in which criteria were reviewed and agreed on. The pathologists then graded the lesions independently. The area of interest was marked on each slide before grading. After initial grading, the pathologists met again to resolve discrepant lesion classifications. A complete agreement among raters was achieved in 43 (35%) cases, with five of six raters agreeing in another 45 (36%) cases. In no case did two raters differ by more than one grade. The pairwise kappa agreement values ranged from fair to substantial (0.30 to 0.61). Generalized kappa value indicated moderate agreement (0.46, standard error = 0.02). Kappa statistics for the distinction between grades 1 and 2 and 2 and 3 were 0.29 and 0.48, respectively, (standard error = 0.02). Only one of the six raters differed significantly in scoring. With adherence to specific criteria, IR in the classification of DCIS cases can be obtained in most cases. Although these pathologists made a few grading system modifications, further refinements are needed, especially if grading will influence future therapy.

Chemical modification and mutagenesis of methionines in recombinant human granulocyte colony-stimulating factor (G-CSF) were investigated. Selective oxidation of G-CSF by H2O2 and t-butyl hydroperoxide leads to generation of different oxidized forms. Four modified forms were isolated and shown to contain 1 to 4 oxidized methionyl residues. All methionines in G-CSF are reactive, with reaction kinetics following the order of Met1>Met138>Met127>>>Met122. H2O2 oxidation of Met122 is relatively slow and is biphasic with a faster second reaction phase being affected by the oxidation of Met127. All oxidized forms retain gross G-CSF conformation similar to that of the native molecule and are able to bind the soluble G-CSF receptor. However, G-CSF form oxidized at both Met127 and Met122 is unstable and exhibits decreased ability to dimerize the receptor after exposure to acid or elevated temperature. All modified forms, except Met1-oxidized G-CSF, also show significantly lower biological activity. Our data suggest that Met138 is solvent accessible and its surrounding microenvironment may be critical for G-CSF function, whereas Met127 is less accessible to solvent and Met122 is near the hydrophobic core. Oxidation at both Met127 and Met122 results in alterations of G-CSF structure that affect the apparent molecular size, polarity, and stability and lead to the loss of G-CSF biological function. G-CSF variants with Leu replacement at Met127 or at Met138 are not completely resistant to oxidation-induced inactivation, while the variant with Leu replacement at both sites is more stable and can retain in vitro biological activity following oxidation.

The effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) were analyzed on the proliferation and differentiation of cultured porcine preadipocytes. In both chemically-defined and serum-containing media, short term treatment (2 days), either during the growth phase or from confluence, significantly enhanced cell proliferation, as assessed by tritiated thymidine incorporation and protein content assay. Addition of TPA during the growth phase in serum-free medium had no effect on lipoprotein lipase (LPL), glycerol 3-phosphate dehydrogenase (GPDH) and malic enzyme (ME) activities after 14 days of culture. By contrast, similar treatments in serum-containing medium significantly increased all these enzyme activities. Addition of TPA from confluence in both media stimulated LPL activity on day 14 as well as ME activity on day 17. These results were confirmed by morphologic studies. In conclusion, this study demonstrates that a short stimulation of the protein kinase C pathway can enhance the differentiation of porcine preadipocytes. Such a positive effect is however dependent on the time of TPA addition as well as the culture conditions, indicating a complex regulation of the protein kinase C pathway in this cell type.

In the 1980s, human and murine forms of many hematopoietic colony stimulating factors were cloned. One factor that was purified, cloned, and produced in commercial quantities was granulocyte colony-stimulating factor (G-CSF, Filgrastim), a protein that acts on the neutrophil lineage. Neutrophils are the body’s major defence against infections. The initial licensing indication for Filgrastim was the amelioration of chemotherapy-induced neutropenia. It has been approved in more than 75 countries for a variety of uses, including aplastic anaemia, severe chronic neutropenia, and the mobilisation of peripheral blood progenitor cells for transplantation. Filgrastim has an excellent safety profile with the only common side effect of administration reported being mild to moderate bone pain. Current issues being investigated include evaluation of the cost benefit of Filgrastim in various clinical settings and impact on survival in dose-intensive chemotherapy with Filgrastim given as an adjunct treatment.

Background: Scepticism has been expressed regarding the accuracy of computed tomography for evaluating patients with blunt abdominal trauma in low-volume institutions. Diagnostic peritoneal lavage has been suggested as a more reliable method, and recently ultrasound has been proposed as a quick alternative. We sought to determine the accuracy of computed tomography at our low-volume center, which lacks 24-hour, in-house computed tomography dedicated radiologists. Methods: A prospective case series of patients who had computed tomographic evaluation requested for suspected blunt abdominal trauma were enrolled over a 1-year study period. Results: Fifty-five patients were enrolled. Injuries were identified in 12 patients, six of whom required intervention. The diagnostic accuracies for the detection of injury were 86% and 90.5% for radiology residents and attending radiologists, respectively. The likelihood ratios for the detection of an injury requiring intervention were identical for both groups of interpreters and were 0.17 for negative result and 35.6 for positive result. Conclusion: The accuracy of computed tomography for evaluating blunt abdominal trauma at a low-volume institution is comparable to that reported from higher-volume centers and may be the procedure of choice, averting the expected higher incidence of nontherapeutic laparotomy attendant with more widespread application of diagnostic peritoneal lavage and the missed injuries that may be expected from the occasional ultrasonographer.

Soluble Escherichia coli-derived recombinant human stem cell factor (rhSCF) forms a non-covalently associated dimer. We have determined a dimer association constant (Ka) of 2-4 x 10(8) M-1, using sedimentation equilibrium and size exclusion chromatography. SCF has been shown previously to be present at concentrations of approximately 3.3 ng/ml in human serum. Based on the dimerization Ka, greater than 90% of the circulating SCF would be in the monomeric form. When 125I-rhSCF was added to human serum and the serum analyzed by size exclusion chromatography, 72-49% of rhSCF was monomer when the total SCF concentration was in the range of 10-100 ng/ml, consistent with the Ka determination. Three SCF variants, SCF(F63C), SCF (V49L,F63L), and SCF(A165C), were recombinantly expressed in Escherichia coli, purified, and characterized. The dimer Ka values, biophysical properties, and biological activities of these variants were studied. Dimerization-defective variants SCF(F63C)S-CH2CONH2 and SCF(V49L,F63L) showed substantially reduced mitogenic activity, while the activity of the Cys165-Cys165 disulfide-linked SCF(A165C) dimer was 10-fold higher than that of wild type rhSCF. The results suggest a correlation between dimerization affinity and biological activity, consistent with a model in which SCF dimerization mediates dimerization of its receptor, Kit, and subsequent signal transduction.

A mouse monoclonal antibody of the IgG2b subclass was raised against porcine adipocyte plasma membranes. This antibody did not cross-react in immunocytofluorescence with any tested cell-type or tissue other than porcine adipocytes. Complement-mediated cytotoxicity was demonstrated in primary cultures of porcine stromal-vascular cells. When the antibody and complement were added to already differentiated cultures, the treatment resulted in elimination of lipid-filled preadipocytes, whereas an early treatment of cultures prevented the appearance of these cells. In vivo, injection of newborn pigs with 1 mg/kg of monoclonal antibody on d 2 and 5 of life produced a more than 20% reduction of subcutaneous and leaf fat lipids at 35 d of age, whereas the lipid content of the longissimus muscle remained unaffected. These results demonstrate that early systemic treatment of pigs with a specific anti-adipocyte antibody reduces the fat mass. In addition to their potential in vivo use, monoclonal antibodies directed against adipose determinants may be useful tools for studying adipocyte lineage.

As part of the design of the Silicone Study, a new classification of proliferative vitreoretinopathy (PVR) was developed that distinguishes the different types of contraction found in PVR. In contrast to the original Retina Society system that emphasized the post-equatorial retinal pathology (posterior PVR), the Silicone Study classification system included the characteristic types of contraction found in both the equatorial region and the pre-equatorial retina and vitreous base (anterior PVR).The authors contrast (1) preoperative and intraoperative findings and (2) vision and anatomic outcomes in the cohort of anterior PVR eyes with the cohort of posterior-only PVR eyes. For the cohort of eyes randomized to perfluoropropane gas (C3F8) or silicone oil, the authors carry out univariate and multivariate analyses to assess the predictive value of baseline and intraoperative parameters on vision and anatomic outcome.Anterior PVR was present in 321 eyes (79%) and was more prevalent in eyes that had undergone an unsuccessful vitrectomy before study entry than in eyes that underwent a primary vitrectomy for PVR (88% versus 73%; P0.001). Compared with eyes that had posterior PVR at the preoperative examination, eyes that had anterior PVR tended to (1) be graded (Retina Society classification system) as D-1 or worse (86% versus 49%; P0.0001), (2) have worse (2/200) visual acuity (93% versus 86%; P = 0.003), (3) have more hypotony (24% versus 11%; P = 0.03), more edema (8% versus 2%; P = 0.04), more aqueous flare (P = 0.02), more macular pucker (69% versus 52%; P = 0.005), and more intravitreal contraction (21% versus 6%; P = 0.002). When compared with eyes that had anterior PVR, eyes with posterior PVR had a better outcome at the 6-month postoperative examination: complete attachment of the retina (76% versus 62%; P = 0.04), visual acuity of 5/200 or better (64% versus 45%; P = 0.006), and normal intraocular pressure (86% versus 71%; P = 0.04). For eyes with anterior PVR, significant predictors of poor (5/200) visual acuity were a preoperative PVR grade D-1 or worse and the use of C3F8 gas as the intraocular tamponade.The Silicone Study classification of anterior PVR permits greater specificity in characterizing PVR and is prognostic of anatomic and vision outcome. Eyes with anterior PVR and clinically significant posterior PVR changes had a better visual prognosis if silicone oil was used. With the current understanding of the pathoanatomy of anterior PVR and the recent development of new surgical techniques, the incidence of anterior PVR in eyes that previously underwent vitrectomy may decline, and the prognosis in eyes with anterior PVR may improve.

We have isolated the cDNA and the genomic clones encoding a novel serine proteinase, named proteinase T, from the fungus Tritirachium album Limber. The coding region of the gene is interrupted by two introns. The amino acid sequence of proteinase T as deduced from the nucleotide sequence is about 56% identical to that of proteinase K. Four cysteines are present in the mature proteinase, probably in the form of disulfide bonds. We have also purified the native proteinase from Tritirachium album Limber grown in the presence of 2% skim milk. Proteinase T is extremely stable at 50 degrees C. The thermal stability is not affected in the presence of 1% SDS either at pH 8.0 or 10.0. We have expressed the cDNA of proteinase T in Escherichia coli. The authenticity of the proteinase has been characterized by Western blotting and amino terminal analysis of the recombinant product. High level expression of proteinase T in E. coli as well as the refolding process to generate active proteinase will be discussed in detail.