Expression of the adaptor protein hematopoietic Src homology 2 is up-regulated in response to stimuli that promote survival and differentiation of B cells

Analysis of hematopoietic Src homology 2 (HSH2) protein expression in mouse immune cells demonstrated that it is expressed at low levels in resting B cells but not T cells or macrophages. However, HSH2 expression is up-regulated within 6-12 h in response to multiple stimuli that promote activation, differentiation, and survival of splenic B cells. HSH2 expression is increased in response to anti-CD40 mAb, the TLR ligands LPS and CpG DNA, and B lymphocyte stimulator (BLyS), a key regulator of peripheral B cell survival and homeostasis. Stimulation of B cells with anti-CD40 mAb, LPS, CpG DNA, or BLyS has previously been shown to induce activation of NF-kappaB. In agreement with this finding, up-regulation of HSH2 expression in response to these stimuli is blocked by inhibitors of NF-kappaB activation and is potentiated by stimulation with PMA, suggesting that HSH2 expression is dependent on NF-kappaB activation. In contrast to CD40, BAFF receptor, TLR4, and TLR9 mediated signaling, stimulation of splenic B cells via the BCR was not observed to induce expression of HSH2 unless the cells had been stimulated previously through CD40. Finally, HSH2 expression is down-regulated in splenic B cells in response to stimulation with IL-21, which has been shown to induce apoptosis, even in the presence of anti-CD40 mAb, LPS, or CpG DNA. IL-21 stimulation also results in down-regulation of antiapoptotic proteins such as Bcl-x(L) and up-regulation of proapoptotic proteins like Bim. Therefore, HSH2 expression is coordinately up-regulated with known antiapoptotic molecules and directly correlates with B cell survival.     

Ambulatory sleep recording in a healthcare network: a feasibility study

Sleep disorders have a high prevalence: around 20% of insomniacs, 10% hypersomnolent including 2 to 4% of sleep disordered breathing in the general adult population. The low availability of sleep centres implies the research of alternative recording techniques in the natural setting of the patient. The objective was to evaluate an ambulatory recorder and its integration in a managed healthcare network. Fifteen patients had a full set-up at home and ten patients were hooked-up in the hospital but recorded at home. Technical failures occurred in 2/15 with full polysomnographic recordings. Integration within an experimental sleep network is in progress. This managed care network will include training of general practitioners, teletransmissions between GP and sleep specialists for a graded use of available resources including ambulatory monitoring.     

The adaptor protein HSH2 attenuates apoptosis in response to ligation of the B cell antigen receptor complex on the B lymphoma cell line, WEHI-231

Signals transduced by the B cell antigen receptor (BCR) play a central role in regulating the functional response of the cell to antigen. Depending on the nature of the antigenic signal and the developmental or differentiation state of the B cell, antigen receptor signaling can promote either apoptosis or survival and activation. Understanding the molecular mechanisms underlying BCR-mediated apoptosis constitutes an important area of research because aberrations in programmed cell death can result in the development of autoimmunity or cancer. Expression of the adaptor protein hematopoietic Src homology 2 (HSH2) was found to significantly decrease BCR-mediated apoptosis in the murine WEHI-231 cell line. Analysis of signal transduction pathways activated in response to BCR ligation revealed that HSH2 does not significantly alter total protein tyrosine phosphorylation or Ca2+ mobilization. HSH2 does not potentiate the activation-dependent phosphorylation of AKT either. With respect to MAPK activation, HSH2 was not observed to alter the activation of ERK or p38 in response to BCR ligation, but it does significantly potentiate JNK activation. Analysis of processes directly associated with apoptosis revealed that HSH2 inhibits mitochondrial depolarization to a significant degree, whereas it has only a slight effect on caspase activation and poly ADP-ribose polymerase cleavage. BCR-induced apoptosis of WEHI-231 cells is associated with the loss of endogenous HSH2 expression within 12 h, whereas inhibition of apoptosis in response to CD40-mediated signaling leads to stabilization of HSH2 expression. Thus, endogenous HSH2 expression correlates directly with survival of WEHI-231 cells, which supports the hypothesis that HSH2 modulates the apoptotic response through its ability to directly or indirectly promote mitochondrial stability.     

Hypomagensemia with secondary hypocalcemia in a female with balanced X;9 translocation: mapping of the Xp22 chromosome breakpoint

Magnesium-dependent hypocalcaemia (HSH), a rare inherited disease, is caused by selective disorders of magnesium absorption. Both X-linked and autosomal recessive modes of inheritance have been reported for HSH; this suggests a genetically heterogeneous condition. A balanced de novo t(X;9)(p22;q12) translocation has been reported in a female manifesting hypomagnesemia with secondary hypocalcemia. In a lymphoblastoid cell line, derived from this patient, the normal X chromosome is preferentially inactivated, suggesting that the patient's phenotype is caused by disruption of an HSH gene in Xp22. In an attempt to define more precisely the position of the X breakpoint, we have constructed a hybrid cell line retaining the der(X)(Xqter-Xp22.2::9q12-9qter) in the absence of the der(9) and the normal X chromosome. Southern blot analysis of this hybrid and in situ hybridization on metaphase chromosomes have localized the breakpoint between DXS16 and the cluster (DXS207, DXS43), in Xp22.2. Thus, if a gene involved in HSH resides at or near the translocation breakpoint, our findings should greatly facilitate its isolation.     

Differential expression of the adaptor protein HSH2 controls the quantitative and qualitative nature of the humoral response

Endogenous expression of the adaptor protein hematopoietic Src homology 2-containing adaptor protein (HSH2) is regulated in a dynamic manner during B cell maturation and differentiation. Developing B cells lack detectable HSH2, whereas transitional 1 and 2 B cells in the periphery exhibit increasing levels of expression. Mature follicular B cells exhibit decreased expression of HSH2 compared with transitional 2 B cells, and expression is further downregulated in germinal center B cells. In contrast, marginal zone B cells and B1a/b B cells exhibit high-level HSH2 expression. Regulation of HSH2 expression plays a critical role in determining the outcome of the humoral immune response as demonstrated using HSH2 transgenic (Tg) mice. Constitutive expression of HSH2 in the B lineage at levels comparable to B1a/b B cells results in decreased serum Ig titers for all subclasses with the exception of IgA. HSH2 Tg mice immunized with T-dependent or T-independent Ags exhibit a moderate decrease in the production of Ag-specific IgM, whereas class-switched isotypes are decreased by ～80-90% compared with control mice. Analysis of HSH2 Tg B cell activation in vitro demonstrated that HSH2 selectively regulates the B cell response to TNF family receptors (i.e., CD40 and BAFF-R), but not BCR- or TLR-dependent signals. These data demonstrate that changes in HSH2 expression have profound effects on the humoral immune response.     

Conservation of structure and subunit interactions in yeast homologues of splicing factor 3b (SF3b) subunits.

Human SAP 49, a subunit of the multimeric splicing factor 3b (SF3b), contains two RNA recognition motifs (RRMs) and binds another SF3b subunit called SAP 145, whose yeast homologue is CUS1. Here we show that the predicted yeast open reading frame YOR319w (HSH49) encodes an essential yeast splicing factor. Using bacterially expressed proteins, we find that yeast HSH49 binds CUS1. Mutations that alter putative RNA-binding residues of either HSH49 RRM are lethal in vivo, but do not prevent binding to CUS1 in vitro, suggesting that the predicted RNA-binding surfaces of HSH49 are not required for interaction with CUS1. In vivo interaction tests show that HSH49 and CUS1 associate primarily through the N-terminal RRM of HSH49. Recombinant HSH49 protein has a general RNA-binding activity that does not require CUS1. The parallels in structure and interaction between two SF3b subunits from yeast implies that the mechanism of SF3b action is highly conserved.     

人细胞核dUTPase的克隆表达及其酶学活性

以阿尔茨海默病 (Alzheimer’sdisease ,AD)患者脑cDNA文库质粒为模板 ,用PCR方法扩增得到人细胞核dUTP焦磷酸酶 (dUTPase)的cDNA ,将其克隆到谷胱甘肽 S 转移酶 (GST)融合表达载体pGEX 4T 1中 ,并在大肠杆菌BL2 1中获得高效表达 .表达的融合蛋白GST dUTPase经过谷胱甘肽 Sepharose 4B亲和层析 ,凝血酶酶切和SephacrylS 10 0纯化 ,得到高纯度dUTPase蛋白 .通过SDS PAGE ,氨基酸组成分析 ,N端氨基酸序列测定以及HPLC测Mr 结果与期望值一致 .通过检测该酶水解dUTP释放的焦磷酸 (PPi)来测定表达产物dUTPase蛋白及GST dUTPase融合蛋白的酶活性 ,发现两蛋白都具有正常的酶水解dUTP活性 ,但融合蛋白的活性比dUTPase蛋白低 7～ 8倍 .同时研究了Mg2 +和EDTA对酶活性的影响     

Synthése,conformation et méthanolyse des chlorures de 2,3,4-tri-O-chlorosulfonyl-β- et α-L-fucopyranosyle

Treatment of α-L-fucose with sulfuryl chloride at low temperature gave mainly 2,3,4-tri-O-chlorosulfonyl-β-L-fucopyranosyl chloride (1) and a small proportion of the α-anomer (2). Both compounds adopt a ¹C₄ chair conformation. Methanolysis of 1 in the presence of silver carbonate and anhydrous calcium sulfate gave methyl 2,3,4-tri-O-chlorosulfonyl-α-L-fucopyranoside (the β-anomer being only present in small proportion), further converted into methyl α-L-fucopyranoside by treatment with a basic resin and a catalytic amount of sodium iodide. Methanolysis of 1 in the presence of sodium iodide gave directly methyl α-L-fucopyranoside, in a more rapid but less stereoselective way. Methanolysis of 2 in the presence of silver carbonate is very slow and gave, after removal of the chlorosulfonyl groups, methyl β-L-fucopyranoside with a rather poor stereoselectivity.     

Regulation of hydrogenase activity in vegetative cells of Anabaena variabilis.

Heterocyst-free (NH4+-grown) cultures of the cyanobacterium Anabaena variabilis produce a hydrogenase which is reversibly inhibited by light and O2. White or red light at an intensity of 5,000 lx inhibited greater than 95% of the activity. Oxygen at concentrations as low as 0.5% inhibited more than 85% of the hydrogenase in the vegetative cells of CO2-NH4+-grown cultures. The vegatative cell hydrogenase is also sensitive to strong oxidants like ferricyanide. In the presence of strong reductants like S2O4(2-), hydrogenase activity was not inhibited by light. However, hydrogenase activity in the heterocysts was insensitive to both light (greater than 5,000 lx) and O2 (10%). Heterocysts and light-insensitive hydrogenase activity appear simultaneously during differentiation of the vegetative cells into heterocysts (an NH4+-grown culture transferred to NH4+-free, N2-containing medium). This light-insensitive hydrogenase activity was detected several hours before the induction of nitrogenase activity. These results suggest a mode of regulation of hydrogenase in the vegetative cells of A. variabilis that is similar to "redox control" of hydrogenase and other "anaerobic" proteins in enteric bacteria like Escherichia coli.     

Wood utilization is dependent on catalase activities in the filamentous fungus Podospora anserina

Catalases are enzymes that play critical roles in protecting cells against the toxic effects of hydrogen peroxide. They are implicated in various physiological and pathological conditions but some of their functions remain unclear. In order to decipher the role(s) of catalases during the life cycle of Podospora anserina, we analyzed the role of the four monofunctional catalases and one bifunctional catalase-peroxidase genes present in its genome. The five genes were deleted and the phenotypes of each single and all multiple mutants were investigated. Intriguingly, although the genes are differently expressed during the life cycle, catalase activity is dispensable during both vegetative growth and sexual reproduction in laboratory conditions. Catalases are also not essential for cellulose or fatty acid assimilation. In contrast, they are strictly required for efficient utilization of more complex biomass like wood shavings by allowing growth in the presence of lignin. The secreted CATB and cytosolic CAT2 are the major catalases implicated in peroxide resistance, while CAT2 is the major player during complex biomass assimilation. Our results suggest that P. anserina produces external H(2)O(2) to assimilate complex biomass and that catalases are necessary to protect the cells during this process. In addition, the phenotypes of strains lacking only one catalase gene suggest that a decrease of catalase activity improves the capacity of the fungus to degrade complex biomass.     

Primary and secondary structural homologies between the HIS4 gene product of Saccharomyces cerevisiae and the hisIE and hisD gene products of Escherichia coli and Salmonella typhimurium

Summary A detailed comparative analysis of the Escherichia coli and Salmonella typhimurium hisIE and hisD gene products and the functionally equivalent, single, HIS4 gene product of Saccharomyces cerevisiae permitted several insights concerning the relationship between these genes. Our analysis supports the idea that HIS4 results from the fusion of hisIE and hisD. The comparison permitted a more precise definition of the functional domains of hisI/HIS4A and hisE/HIS4B as well as the two functional domains of hisD/HIS4C. The homologies between the bacterial and yeast sequences suggest a region of the hisD/HIS4C protein that may constitute one of the active centres. A large fragment at the amino terminal region of the yeast protein is missing from the bacterial hisIE gene product and is probably not needed for catalytic activity. Another region of non-homology in the yeast protein is probably a peptide bridge connecting the HIS4AB domain to HIS4C. Although the overall homology at the level of amino acid sequence is modest (about 38%) there is a striking similarity when the hydropathic patterns and predicted secondary structural configurations of these proteins are compared.     

Decomposition and nitrogen dynamics of wheat residues and impact on wheat growth stages

N mineralisation and immobilisation were quantified in field conditions in the presence or in the absence of wheat residues. The incubation study was conducted in cylinders placed in microplots (no plants were grown in cylinders), and the rest of each microplot was sowed with the wheat crop (Triticum durum var. Massa). N mineralisation and immobilisation depend on the presence or the absence of wheat residues. In absence of residues, a linear model of regression was developed to follow the clear nitrogen mineralisation at different soil levels. Nitrogen mineralisation (mg kg-1), during the five months of wheat development, showed the following decreasing order: 0-15 cm (132.6) > 15-30 cm (120.6) > 30-45 cm (91.3). The mineralisation rate was 24.1, 22.9 and 18.9 mg kg-1 d-1 for 0-15, 15-30 and 30-45 cm levels, respectively. The supply of wheat residues resulted in a five months N immobilisation process. At level 0-15 cm the immobilisation (mg kg-1) showed the following decreasing order: (61.6) > (46.4) > (30.0) for the supply of wheat residues at seeding time, and 15 and 30 d before seeding respectively. At the other levels, the same decreasing order was recorded. The supply of 8 t ha-1 of wheat residues at seeding time, and 15 or 30 d before seeding, decreased the dry matter yield and N accumulation in wheat crop. In consequence, there was no synchronism between the nitrogen liberated by wheat residues decomposition and the wheat growth.     

A differential geometry approach for biomedical image processing

We show in this paper how simple considerations about bio-arrays images lead to a peak segmentation allowing the genes activity analysis. Bio-arrays images have a particular structure and the aim of the paper is to present a mathematical method allowing their automatic processing. The differential geometry approach used here can be also employed for other types of images presenting grey level peaks corresponding to a functional activity or to a chemical concentration. The mathematical method is based on elementary techniques of differential geometry and dynamical systems theory and provides a simple efficient algorithm when the peaks to segment are isolated.     

The aged: medical and social aspects

Physical health is affected by physiological aging that impacts on all tissues and organs, notably sensorial systems (hearing, sight), the locomotory and the immunological systems (lowering of resistance to infections). There is an increase with age in the incidence of many cancers (particularly breast, prostate, and colon cancers) and cardiovascular diseases. Regular check-ups are useful in order to take appropriate measures in time. It is important that people maintain regular physical activity and a balanced diet even up to an advanced age and the elderly must learn to adapt themselves to the ever-changing abilities of their organism. It is possible to slow down the aging process through good hygiene and often to maintain autonomy until the end of life. Mental health is threatened by impairment of mental functions, depressive tendencies, and the risk of senile dementia that cannot be foreseen or avoided. It appears that keeping intellectually active and having a good level of education impact favorably on mental aging. Social health depends, for a large part, on the way society accepts and treats the elderly. They must be kept integrated into society and allowed to live at home for as long as possible. Any measures of rejection, discrimination, and exclusion should be opposed. The dignity of the elderly must be respected and activities giving them a feeling of usefulness should be encouraged. It is important to help families who care for their parents at home, to develop and evaluate healthcare networks, and encourage medical professionals and social services to work together. The change in the demographic structure of France is a considerable phenomenon requiring a long-term strategy and not only superficial and cosmetic measures.     

Characterization of histones and chromatin of the hepatopancreas in Palaemon serratus (Crustacea Natantia)]

The chromatin of shrimp hepatopancreas has been extracted from isolated nuclei and characterized. Nuclei were prepared in the presence of Cu++ and phenyl methyl sulfonyl fluoride in order to inhibit the nuclease and protease activities throughout the different purification steps. The purified nuclei are heterogenous in size and show a density of 1,367 g/ml determined on saccharose - glucose gradients. After washing in 0,14 M NaCl and then in 10(-2) M Tris-HCL, pH = 7,6, the nuclei were disrupted in water. The solubilized chromatin was precipitated in 0,15 M.NaCl. This chromatin is characterized by a high level of RNA (RNA/DNA = 0,38) and of non histone proteins (NHP/DNA = 0,6). The denaturation curve showed only one Tm at 69 degrees in 2.10(-4) M.EDTA. When the chromatin was extracted in the presence of staphylococcal nuclease, the Tm reached 80 degrees C. The kinetics of the digestion by the staphylococcal nuclease have been studied and show that 10 per cent of hydrolysis occurs within the first minute. The repeat length of DNA as determined with the polymers of higher order is 189 +/- 5 base pairs. The existence of nucleosomes was confirmed by electron microscopy. The superstructure of chromatin was not completely destroyed after solubilisation with a Potter. The histones were studied by gel electrophoresis after differential staining. The most important feature consists in the presence of two H1, two H2A and two H4. The acetylation levels of the histones were followed after injection of 14C-acetate in vivo. The subfraction H1, 0 was acetylated. Only one H3 was present and the two H2A fractions showed the same level of acetylation. H2B migrated faster than the H2A fractions like in Echinoderms. The two H4 fractions corresponded to two differently acetylated forms. Shrimp hepatopancreas histones were fractionated by molecular sieving on Biogel P 100 and characterized according to their electrophoretic properties as well as their amino-acid content. The amino-acid compositions of the different histone fractions were nearer to Echinoderm and Sipunculid histones, than Calf thymus homologue histones. All the fractions show a weaker basicity. The H3 fraction was the only one showing a lesser variability when compared to Calf thymus H3. The non histone proteins were extracted in 10(-2) M Tris-HCL, pH = 8 and 0.1 per cent SDS. A series of 50 proteins was detected. 80 per cent of the total amount of protein was localized in a molecular weight range comprised between 40 000 and 80 000 daltons. These proteins were compared to the histones and total proteins of sonicated chromatin solubilized by SDS in order to detect proteasic effects.