Meeting report: international workshop on endocrine disruptors: exposure and potential impact on consumers health.

The French Agency for Food, Environmental and Occupational Health and Safety (Anses) hosted a two-day workshop on Endocrine Disruptors: Exposure and Potential Impact on Consumers Health, bringing together participants from international organizations, academia, research institutes and from German, Swedish, Danish and French governmental agencies. The main objective of the workshop was to share knowledge and experiences on endocrine disruptors (ED) exposure and potential impact on consumers' health, to identify current risk assessment practices and knowledge gaps and issue recommendations on research needs and future collaboration. The following topics were reviewed: (1) Definition of ED, (2) endpoints to be considered for Risk assessment (RA) of ED, (3) non-monotonic dose response curves, (4) studies to be considered for RA (regulatory versus academic studies), (5) point of departure and uncertainty factors, (6) exposure assessment, (7) regulatory issues related to ED. The opinions expressed during this workshop reflect day-to-day experiences from scientists, regulators, researchers, and others from many different countries in the fields of risk assessment, and were regarded by the attendees as an important basis for further discussions. Accordingly, the participants underlined the need for more exchange in the future to share experiences and improve the methodology related to risk assessment for endocrine disrupters.

Metabolic effects of interventions to increase exercise in adults with type 2 diabetes.

AIMS/HYPOTHESIS: The aim of this meta-analysis was to integrate the results of primary research testing the effect of diabetes self-management interventions that included recommendations to increase exercise on metabolic outcomes among adults with type 2 diabetes. MATERIALS AND METHODS: Extensive literature searching strategies were used to identify published and unpublished intervention studies that measured glycated haemoglobin outcomes. Primary study results were coded. Fixed- and random-effects meta-analytic procedures included moderator analyses. RESULTS: Data were synthesised across 10,455 subjects from 103 research reports. The overall mean weighted effect size for two-group comparisons was 0.29 (higher mean for treatment than control). This effect size is consistent with a difference in HbA1c means of 0.45% (e.g. 7.38% for treatment subjects vs 7.83% for control subjects). For single-group studies, the overall mean weighted effect size was 0.32-0.34. Control group subjects experienced no improvement in metabolic control during participation in the studies. Interventions that targeted multiple health behaviours resulted in smaller effect size estimates (0.22) than interventions that focused only on exercise behaviours (0.45). Funded studies reported greater improvements in metabolic controls. Studies with a greater proportion of female subjects reported lower effect sizes. Baseline HbA1c and BMI were unrelated to metabolic outcomes. CONCLUSIONS/INTERPRETATION: These findings suggest that self-management interventions that include exercise recommendations improve metabolic control, despite considerable heterogeneity in the magnitude of the intervention effect. Interventions that emphasise exercise may be especially effective in improving metabolic control. Primary research testing interventions in randomised trials to confirm causal relationships would be constructive.

Testing gene function early in the B cell lineage in mb1-cre mice.

The mb1 gene encodes the Ig-alpha signaling subunit of the B cell antigen receptor and is expressed exclusively in B cells beginning at the very early pro-B cell stage in the bone marrow. We examine here the efficacy of the mb1 gene as a host locus for cre recombinase expression in B cells. We show that by integrating a humanized cre recombinase into the mb1 locus we obtain extraordinarily efficient recombination of loxP sites in the B cell lineage. The results from a variety of reporter genes including the splicing factor SRp20 and the DNA methylase Dnmt1 suggest that mb1-cre is probably the best model so far described for pan-B cell-specific cre expression. The availability of a mouse line with efficient cre-mediated recombination at an early developmental stage in the B lineage provides an opportunity to study the role of various genes specifically in B cell development and function.

A point mutation in the IL-12R beta 2 gene underlies the IL-12 unresponsiveness of Lps-defective C57BL/10ScCr mice.

Lps-defective C57BL/10ScCr (Cr) mice are homozygous for a deletion encompassing Toll-like receptor 4 that makes them refractory to the biological activity of LPS. In addition, these mice exhibit an inherited IL-12 unresponsiveness resulting in impaired IFN-gamma responses to different microorganisms. By positional cloning methods, we show here that this second defect of Cr mice is due to a mutation in a single gene located on mouse chromosome 6, in close proximity to the Igkappa locus. The gene is IL-12Rbeta2. Cr mice carry a point mutation creating a stop codon that is predicted to cause premature termination of the translated IL-12Rbeta2 after a lysine residue at position 777. The truncated beta2 chain can still form a heterodimeric IL-12R that allows phosphorylation of Janus kinase 2, but, unlike the wild-type IL-12R, can no longer mediate phosphorylation of STAT4. Because the phosphorylation of STAT4 is a prerequisite for the IL-12-mediated induction of IFN-gamma, its absence in Cr mice is responsible for their defective IFN-gamma response to microorganisms.

The absence of SLP65 and Btk blocks B cell development at the preB cell receptor-positive stage.

Mice deficient for the adapter protein SLP65 (BLNK) show a partial block in early B cell development, reduced numbers of mature B cells in the periphery, an absence of B1 cells and a reduction of IgM and IgG3 serum immunoglobulin levels. A strikingly similar phenotype is observed in Btk-deficient mice. To investigate the consequences of mutations in both SLP65 and Btk, we generated SLP65/ Btk double-mutant mice by crossing the single-mutant mice. Analysis of the double-mutant mice reveals a much more severe defect in B cell development. B cells in the SLP65/Btk double-mutant mice are arrested at the preB cell stage and, surprisingly, express the preB cell receptor. Normally, preB cell receptor expression in wild-type mice is restricted to a very small fraction of B cells making it difficult to identify these cells in the bone marrow. Together, the data demonstrate the synergistic role of SLP65 and Btk in B cell development and describe a situation where large numbers of preB cell receptor-positive cells accumulate in the bone marrow and spleen.

Transcripts of Fliz1, a nuclear zinc finger protein, are expressed in discrete foci of the murine fetal liver.

The origin and expansion of hematopoietic progenitor and stem cells during fetal development and their differentiation into mature effector cells are thought to be driven by the activation of developmental stage- and cell-lineage-specific genes. To gain further insight into the molecular mechanisms regulating the expansion and differentiation of fetal hematopoietic progenitor and stem cells, we performed differential display RT-PCR analysis on fractionated murine E12 fetal liver cells. We identified a novel transcript predicted to encode a protein of 305 amino acids with a calculated molecular mass of 35 kDa, containing a charged domain and three putative C(3)H-type zinc fingers. The fetal liver zinc-finger protein 1 (Fliz1) transcript is approximately 1.8 kb and is variably expressed both during embryogenesis and in adult tissues. Fliz1 expression was detected in discrete cell foci in the fetal liver and in LIN(-)/ckit(+) cells. Nuclear localization studies revealed that Fliz1 is targeted to the nucleus. Thus, Fliz1 is a newly identified nuclear protein expressed in hematopoietic progenitor cells of the developing fetal liver.

The B lymphocyte-specific coactivator BOB.1/OBF.1 is required at multiple stages of B-cell development.

The transcriptional coactivator BOB.1/OBF.1 confers B-cell specificity on the transcription factors Oct1 and Oct2 at octamer site-containing promoters. A hallmark of the BOB.1/OBF.1 mutation in the mouse is the absence of germinal center development in secondary lymphoid organs, demonstrating the requirement for BOB.1/OBF.1 in antigen-dependent stages of B-cell differentiation. Here we analyzed earlier stages of B lymphopoiesis in BOB.1/OBF.1-deficient mice. Examination of B-cell development in the bone marrow revealed that the numbers of transitional immature (B220(+) IgM(hi)) B cells were reduced and that B-cell apoptosis was increased. When in competition with wild-type cells, BOB.1/OBF.1(-/-) bone marrow cells exhibited defects in repopulating the bone marrow B-cell compartment and were unable to establish a presence in the periphery of host mice. The defective bone marrow populations in BOB.1/OBF.1(-/-) mice were rescued by conditional expression of a BOB.1/OBF.1 transgene controlled by the tetracycline gene expression system. However, the restored populations did not restore the numbers of IgD(hi) B cells in the periphery, where the BOB.1/OBF.1 transgene was not expressed. These results show that BOB.1/OBF.1(-/-) B cells exhibit multistage defects in B-cell development, including impaired production of transitional B cells and defective maturation of recirculating B cells.

Inherited IL-12 unresponsiveness contributes to the high LPS resistance of the Lps(d) C57BL/10ScCr mouse.

LPS(d) mouse strains are characterized by the presence of a defective LPS/tlr4 gene that make them refractory to the biological activity of LPS. One of the mouse strains commonly used to study LPS defects is the C57BL/10ScCr (Cr) strain. However, unlike other LPS(d) strains, the Cr strain also has a heavily impaired IFN-gamma response to micro-organisms. As a consequence, unlike other LPS(d) mouse strains, they do not acquire a partial LPS susceptibility when treated with sensitizing bacteria. Because IL-12 is important for the microbial induction of IFN-gamma, we investigated whether the production or function of IL-12 might be defective in Cr mice. IL-12 mRNA (p35 and p40) was present in the spleen of untreated Cr mice, IL-12p40 mRNA was inducible in mice injected with live or killed Salmonella typhimurium, and IL-12 (p70) was inducible in macrophages by bacteria. Thus, Cr mice exhibit normal IL-12 responses. In functional tests, splenocytes of untreated or of S. typhimurium-infected mice failed to produce IFN-gamma when stimulated with murine rIL-12 or with a combination of IL-12 and murine rIL-18 or Con A. Furthermore, Cr mice were identical with IL-12p35/p40 and IL-12 receptor beta(1) knockout mice in their impaired in vivo and in vitro IFN-gamma responses to bacteria. Thus, Cr mice carry a second genetic defect unrelated to the Lps/tlr4 mutation that underlies the IL-12 unresponsiveness and contributes to the LPS resistance and impaired innate immune response in this strain.

Regulation of SRp20 exon 4 splicing.

SR proteins are essential splicing factors involved in the use of both constitutive and alternative exons. We previously showed that the SR proteins SRp20 and ASF/SF2 have antagonistic activities on SRp20 pre-mRNA splicing. SRp20 activates exon 4 recognition in its pre-mRNA, whereas ASF/SF2 inhibits this recognition. In experiments aimed at testing the specificity of SRp20 and ASF/SF2 for exon 4 splicing regulation, we show here that this specificity lies in the RNA binding domains of SRp20 and ASF/SF2 and not in the RS domains. Surprisingly, a deletion of 14 amino acids at the end of ASF/SF2-RBD2 converts ASF/SF2 from an inhibitor to an activator of exon 4 splicing. We found that ASF3 also inhibits exon 4 recognition, thus acting similarly to ASF/SF2, while SC35 activates a cryptic 5' splice site downstream of exon 3 and, in doing so, represses exon 4 use. In contrast, Tra2 and the SR proteins 9G8 and SRp40 do not appear to affect exon 4 splicing.

The BOB.1 / OBF.1 co-activator is essential for octamer-dependent transcription in B cells.

The BOB.1 / OBF.1 / OCA-B protein (henceforth designated as BOB.1 / OBF.1) is a B cell-specific co-activator of the Oct1 and Oct2 transcription factors. It is involved in mediating the transcriptional activity of these proteins. Surprisingly, animals deficient for BOB.1 / OBF.1 showed normal expression of genes that contain an octamer motif in their regulatory regions. Here we have addressed the role of BOB.1 / OBF.1 for octamer-dependent transcription. We show that promoters exclusively dependent on functional octamer motifs are completely inactive in BOB.1 / OBF. 1-deficient B cells. The lack of activity is a direct consequence of lack of the co-activator. To show this, a hormone-regulated conditional allele of BOB.1 / OBF.1 was introduced into the BOB.1 / OBF.1-deficient B cells. This resulted in the hormone-dependent transcriptional activity of octamer-dependent reporters in these cells. The BOB.1 / OBF.1 requirement for octamer promoter function was also observed when an authentic immunoglobulin kappa-promoter was assayed. BOB.1 / OBF.1 dependence could not be overcome by including the strong enhancer element from the immunoglobulin heavy chain gene. Induction of pre-B cells with lipopolysaccharide led to increased Oct2 levels but did not significantly increase octamer-dependent transcription in BOB.1 / OBF.1-deficient B cells. Thus, these results demonstrate that BOB.1 / OBF.1 itself is a non-redundant protein in B cells and absolutely required for octamer-dependent transcriptional activity.

Bacterial induction of beta interferon in mice is a function of the lipopolysaccharide component.

We investigated the reason for the inability of lipopolysaccharide (LPS)-resistant (Lps-defective [Lps(d)]) C57BL/10ScCr mice to produce beta interferon (IFN-beta) when stimulated with bacteria. For this purpose, the IFN-beta and other macrophage cytokine responses induced by LPS and several killed gram-negative and gram-positive bacteria in LPS-sensitive (Lps-normal [Lps(n)]; C57BL/10ScSn and BALB/c) and Lps(d) (C57BL/10ScCr and BALB/c/l) mice in vitro and in vivo were investigated on the mRNA and protein levels. In addition, double-stranded RNA (dsRNA) was used as a nonbacterial stimulus. LPS and all gram-negative bacteria employed induced IFN-beta in the Lps(n) mice but not in the Lps(d) mice. All gram-positive bacteria tested failed to induce significant amounts of IFN-beta in all four of the mouse strains used. As expected, all other cytokines tested (tumor necrosis factor alpha, interleukin 1alpha [IL-1alpha], IL-6, and IL-10) were differentially induced by gram-negative and gram-positive bacteria. Stimulation with dsRNA induced IFN-beta and all other cytokines mentioned above in all mouse strains, regardless of their LPS sensitivities. The results suggest strongly that LPS is the only bacterial component capable of inducing IFN-beta in significant amounts that are readily detectable under the conditions used in this study. Consequently, in mice, IFN-beta is inducible only by gram-negative bacteria, but not in C57BL/10ScCr or other LPS-resistant mice.

Abnormal development and function of B lymphocytes in mice deficient for the signaling adaptor protein SLP-65.

During signal transduction through the B cell antigen receptor (BCR), several signaling elements are brought together by the adaptor protein SLP-65. We have investigated the role of SLP-65 in B cell maturation and function in mice deficient for SLP-65. While the mice are viable, B cell development is affected at several stages. SLP-65-deficient mice show increased proportions of pre-B cells in the bone marrow and immature B cells in peripheral lymphoid organs. B1 B cells are lacking. The mice show lower IgM and IgG3 serum titers and poor IgM but normal IgG immune responses. Mutant B cells show reduced Ca2+ mobilization and reduced proliferative responses to B cell mitogens. We conclude that while playing an important role, SLP-65 is not always required for signaling from the BCR.

Blastocyst formation is blocked in mouse embryos lacking the splicing factor SRp20.

SRp20 is a splicing factor belonging to the highly conserved family of SR proteins [1] [2] [3] [4], which have multiple roles in the regulation of constitutive and alternative splicing in vivo. It has been suggested that SR proteins are involved in bringing together the splice sites during spliceosome assembly [5]. SR proteins show partial redundancy, as each single SR protein can restore splicing activity to a splicing-deficient cytoplasmic extract (termed S-100 extract). Nevertheless, several studies demonstrate that individual SR proteins have different effects on the selection of specific alternative splice sites, and they recognize distinct RNA sequences [6] [7] [8] [9] [10] [11] [12]. Also, inactivation of two SR proteins, B52/SRp55 in Drosophila [13] or ASF/SF2 in the chicken cell line DT40 [14], is lethal, indicating the existence of nonredundant functions. Here, using Cre-loxP-mediated recombination in mice to inactivate the SRp20 gene, we found that it is essential for mouse development. Mutant preimplantation embryos failed to form blastocysts and died at the morula stage. Immunofluorescent staining showed that SRp20 is present in oocytes and early stages of embryonic development. This is the first report of mice deficient for a member of the SR protein family. Our experiments confirm that, although similar in structure, the SR proteins are not functionally redundant.

SH3P7 is a cytoskeleton adapter protein and is coupled to signal transduction from lymphocyte antigen receptors.

Lymphocytes respond to antigen receptor engagement with tyrosine phosphorylation of many cellular proteins, some of which have been identified and functionally characterized. Here we describe SH3P7, a novel substrate protein for Src and Syk family kinases. SH3P7 migrates in sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a 55-kDa protein that is preferentially expressed in brain, thymus, and spleen. It contains multiple amino acid sequence motifs, including two consensus tyrosine phosphorylation sites of the YXXP type and one SH3 domain. A region of sequence similarity, which we named SCAD, was found in SH3P7 and three actin-binding proteins. The SCAD region may represent a new type of protein-protein interaction domain that mediates binding to actin. Consistent with this possibility, SH3P7 colocalizes with actin filaments of the cytoskeleton. Altogether, our data implicate SH3P7 as an adapter protein which links antigen receptor signaling to components of the cytoskeleton.

The life, legacy, and premature death of Felix Mendelssohn.

Felix Mendelssohn is one of the great classical composers of all time. During his short lifetime in the first half of the nineteenth century, he reached enormous heights as a composer, conductor, and leader in the world of music. Nearly one hundred years after his death, the Nazi regime attempted, unsuccessfully, to erase his music and his memory from history. Since the end of World War II, there has been a resurgence in interest in the life and music of Felix Mendelssohn and that of his sister, Fanny. Felix Mendelssohn died in 1947 at the age of 38. Both of his sisters died suddenly at the ages of 42 and 45. There is insufficient laboratory or post-mortem data to make a medical diagnosis with certainty. However, based on the information available to us, we speculate that Mendelssohn suffered a subarachnoid or intracerebral hemorrhage. The differential diagnosis of familial stroke syndrome is discussed.

SLP-65: a new signaling component in B lymphocytes which requires expression of the antigen receptor for phosphorylation.

The B cell antigen receptor (BCR) consists of the membrane-bound immunoglobulin (Ig) molecule as antigen-binding subunit and the Ig-alpha/Ig-beta heterodimer as signaling subunit. BCR signal transduction involves activation of protein tyrosine kinases (PTKs) and phosphorylation of several proteins, only some of which have been identified. The phosphorylation of these proteins can be induced by exposure of B cells either to antigen or to the tyrosine phosphatase inhibitor pervanadate/H2O2. One of the earliest substrates in B cells is a 65-kD protein, which we identify here as a B cell adaptor protein. This protein, named SLP-65, is part of a signaling complex involving Grb-2 and Vav and shows homology to SLP-76, a signaling element of the T cell receptor. In pervanadate/H2O2-stimulated cells, SLP-65 becomes phosphorylated only upon expression of the BCR. These data suggest that SLP-65 is part of a BCR transducer complex.

Evaluation of local anesthesia techniques for small incision cataract surgery.

PURPOSE: To evaluate the surgical experiences and patient preference with 3 local anesthesia techniques for small incision cataract surgery. SETTING: Department of Ophthalmology, Hjørring Hospital, Denmark. METHODS: This prospective, randomized study included 66 patients having simultaneous bilateral cataract surgery. There were 3 test groups, each containing 2 of the following local anesthesia techniques: retro/peribulbar (RBA), sub-Tenon's (STA), or topical (TA). Each patient served as his or her own control. No medical sedation was used. Patient response to each anesthesia technique was evaluated by the surgeon based on surgical difficulties, a nurse using hand-holding tension and verbal interaction, and a visual analog pain score. Patients were also asked which of the 2 techniques they preferred and their reasons. RESULTS: No local anesthesia techniques interfered with surgery. The order of a positive pain/discomfort response during surgery was TA > STA > RBA. Significantly more pain occurred with application of RBA than with STA or TA. No postoperative pain was recorded with any method. Fifty-six percent of patients said they preferred 1 technique over the other; 16% of patients having STA would not do so again, 19% would not have TA again, and 40% would not have RBA again. The main reasons for preferring STA and TA were fear of or pain from a retrobulbar injection. The main reasons for preferring RBA were less awareness, anxiety, and surgical pain. Immediate visual recovery seemed to be of minor importance in patients' choice of an anesthesia technique. CONCLUSION: Although less discomfort/pain occurred during surgery with RBA, patients preferred STA and TA primarily because of the inconvenience or pain of the retrobulbar injection. Although medical sedation was not used in this study, the pain/discomfort ratio from surgery was not greater than in studies using intravenous sedation, indicating that the use of medical sedation should be re-evaluated.

Homotypic interaction of the heat-stable antigen is not responsible for its co-stimulatory activity for T cell clonal expansion.

The heat-stable antigen (HSA) is an important co-stimulatory molecule on antigen-presenting cells (APC). However, the receptor on T cells that receives the co-stimulatory signal from HSA has not been identified. Because the HSA is transiently expressed on T cells after the T cell receptor/CD3 complex is engaged, and because it can bind to itself in a homotypic fashion, it has been proposed that homotypic interaction of HSA is responsible for its co-stimulatory activity. Here we test this hypothesis using mice that have a targeted mutation of the HSA gene, as well as novel transgenic mice that constitutively express HSA on T cells. We show that HSA-deficient T cells remain responsive to co-stimulation by HSA. Furthermore, constitutive expression of HSA does not enhance T cell response to co-stimulatory by HSA. Taken together, our results demonstrate that homotypic interaction of HSA is not responsible for co-stimulation mediated by HSA expressed on APC.

The splicing factor SRp20 modifies splicing of its own mRNA and ASF/SF2 antagonizes this regulation.

SRp20 is a member of the highly conserved SR family of splicing regulators. Using a variety of reporter gene constructs, we show that SRp20 regulates alternative splicing of its own mRNA. Overexpression of SRp20 results in a reduction in the level of exon 4-skipped SRp20 transcripts and activates the production of transcripts containing exon 4. These exon 4-included transcripts encode a truncated protein lacking the C-terminal RS domain. We provide evidence that SRp20 probably enhances the recognition of the otherwise unused, weak splice acceptor of exon 4. The recognition of exons with weak splice acceptor sites may be a general activity of SRp20. Unexpectedly, ASF/SF2, another member of the SR family, antagonizes the effect of SRp20 on SRp20 pre-mRNA splicing and suppresses the production of the exon 4-included form. Our results indicate that ASF/SF2 suppresses the use of the alternative exon 4, most likely by inhibiting the recognition of the splice donor of exon 4. These results demonstrate, for the first time, an auto-regulatory activity of an SR protein which is antagonized by a second SR protein.

Regulated expression and RNA processing of transcripts from the Srp20 splicing factor gene during the cell cycle.

Eukaryotic splicing factors belonging to the SR family are essential splicing factors consisting of an N-terminal RNA-binding region and a C-terminal RS domain. They are believed to be involved in alternative splicing of numerous transcripts because their expression levels can influence splice site selection. We have characterized the structure and transcriptional regulation of the gene for the smallest member of the SR family, SRp20 (previously called X16). The mouse gene encoding SRp20, termed Srp20, consists of one alternative exon and six constitutive exons and was mapped to a 2-centimorgan interval on chromosome 17. When cells are transfected with SRp20 genomic DNA, both standard and alternatively spliced transcripts and corresponding proteins are produced. Interestingly, in starved (G0) cells, the amount of SRp20 mRNA containing the alternative exon is large, whereas the amount of the standard SRp20 mRNA without the alternative exon is small. When starved cells are stimulated with serum, the alternative form is lost and the standard form is induced. These results suggest that splicing could be regulated during the cell cycle and that this could be, at least in part, due to regulated expression of SR proteins. Consistent with this, experiments with synchronized cells showed an induction of SRp20 transcripts in late G1 or early S. We have also characterized the promoter of SRp20. It lies within a GC-rich CpG island and contains two consensus binding sites for E2F, a transcription factor thought to be involved in regulating the cell cycle. These motifs may be functional since reporter constructs with the SRp20 promoter can be stimulated by cotransfection with E2F expression plasmids.