Temporal,cell-specific,and tissue-preferential expression of myrosinase genes during embryo and seedling development in Sinapis alba

The expression of the genes for two types of myrosinase (EC 3.2.3.1), designated MA and MB, during embryo and seedling development was investigated in Sinapis alba L. by in-situ and RNA slot-blot analyses. The expression of MA and MB genes followed similar temporal profiles during embryogenesis, but MB mRNA was present in considerably higher amounts than MA mRNA. In the embryo, both MA and MB genes are activated in cotyledons and axis. The MB genes are preferentially expressed in the cotyledons whereas MA genes are preferentially expressed in the axis. In the developing seedling, MA mRNA was not present in the organs investigated. By contrast, MB mRNA was found in appreciable amounts in hypocotyls, cotyledons and developing leaves. The MB genes seem to be activated preferentially in tissues undergoing rapid cell division and — or cell expansion.Abbreviations DAP days after pollination - MA, MB A type, B type myrosinases in Sinapis alba Anna-Stina Höglund (Uppsala Genetic Center) is gratefully acknowledged for valuable discussion, Anders Gobl (Department of Immunology, Uppsala University) for kindly advice with the labeling of probes and Qingzhu Zhai (Department of Pharmaceutical Biosciences, Uppsala University) for help with seed harvest. This work was supported by grants from the Swedish Research Council for Forestry and Agriculture.     

Effect of cocaine and morphine on neutral endopeptidase activity of human peripheral blood mononuclear cells cultured with lectins

We have tested the effect of alkaloids (cocaine, morphine) and enkephalins on neutral endopeptidase of peripheral blood mononuclear cells activated by lectins. When treated with concanavalin A and cocaine, peripheral blood mononuclear cells showed an enhanced activity (+110 per cent) of the membrane neutral endopeptidase, which was not related to the expression of the common acute lymphoblastic leukemia antigen at the cell surface, although both molecules have the identical amino acid sequence. Phytohemagglutinin-P, morphine and synthetic enkephalins did not induce the activity of neutral endopeptidase nor the expression of common acute lymphoblastic leukemia antigen. Our findings suggested that the drugs of abuse, cocaine and morphine, affected specific membrane constituents without altering proliferation, subcellular localization of membrane enzymes or the surface immune phenotype of peripheral blood mononuclear cells.     

Human Cytidine Triphosphate Synthetase 1 Interacting Proteins

We investigated the interacting proteins and intracellular localization of CTP synthetase 1 (CTPS1) in mammalian cells. CTPS1 interacted with a GST- peptidyl prolyl isomerase, Pin1 fusion (GST-Pin1) in a Ser 575 (S575) phosphorylation-dependent manner. Immunoprecipitation experiments demonstrated that CTPS1 also bound tubulin, and thirteen additional coimmunoprecipitating proteins were identified by mass spectrometry. Immunolocalization experiments showed that tubulin and CTPS1 colocalized subcellularly. Taxol treatment enhanced this but cotreatment of cells with the CTPS inhibitor, cyclopentenyl cytosine (CPEC), and taxol failed to disrupt the colocalization. Thus, these studies provide novel information on the potential interacting proteins that may regulate CTPS1 function or intracellular localization.     

An oxidative and salinity stress induced peroxisomal ascorbate peroxidase from Avicennia marina: Molecular and functional characterization

APX (EC, 1.11.1.11) has a key role in scavenging ROS and in protecting cells against their toxic effects in algae and higher plants. A cDNA encoding a peroxisomal ascorbate peroxidase, Am-pAPX1, was isolated from salt stressed leaves of Avicennia marina (Forsk.) Vierh. by EST library screening and its expression in the context of various environmental stresses was investigated. Am-pAPX1 contains an ORF of 286 amino acids coding for a 31.4kDa protein. The C-terminal region of the Am-pAPX1 ORF has a putative transmembrane domain and a peroxisomal targeting signal (RKKMK), suggesting peroxisomal localization. The peroxisomal localization of Am-pAPX1 was confirmed by stable transformation of the GFP-(Ala)(10)-Am-pAPX1 fusion in tobacco. RNA blot analysis revealed that Am-pAPX1 is expressed in response to salinity (NaCl) and oxidative stress (high intensity light, hydrogen peroxide application and excess iron). The isolated genomic clone of Am-pAPX1 was found to contain nine exons. A fragment of 1616bp corresponding to the 5' upstream region of Am-pAPX1 was isolated by TAIL-PCR. In silico analysis of this sequence reveals the presence of putative light and abiotic stress regulatory elements.     

Indoleamine 2,3-Dioxygenase Tissue Distribution and Cellular Localization in Mice: Implications for Its Biological Functions

Earlier studies have suggested that indoleamine 2,3-dioxygenase (IDO) has a wide tissue distribution in mammals. However, detailed information on its cellular localization and also the levels of expression in various tissues is still scarce. In the present study, we sought to determine the cellular localization of IDO and also to quantify the level of its expression in various mouse tissues by using the branched DNA signal amplification assay, Western blotting, and immunohistochemical staining. The highest levels of constitutive IDO expression were found to be selectively present in the caput of epididymis, except for its initial segment. IDO expression was also detected inside the luminal compartment and even in the stereocilia within this region. In the prostate, high levels of IDO were selectively expressed in the capsular cells. In addition, high levels of IDO expression were also selectively detected in certain types of cells in the placenta, spleen, thymus, lung, and digestive tract. Notably, the morphological features of most of the positively stained cells in these organs closely resembled those of antigen-presenting cells. Based on the tissue distribution and cellular localization characteristics of IDO, it is hypothesized that its expression may serve two main functions: one is to deplete tryptophan in an enclosed microenvironment (such as in the epididymal duct lumen) to prevent bacterial or viral infection, and the other is to produce bioactive tryptophan catabolites that would serve to suppress T-cell–mediated immune responses against self-antigens, fetal antigens, or allogeneic antigens, in different situations. (J Histochem Cytochem 58:17–28, 2010)     

Immunological detection of a cysteine protease in the skin and other tissues

Summary Monospecific antibody directed to cysteine protease of 2-day-old rat epidermis recently characterized as being different from the proteases previously reported was produced in rabbits. By immunofluorescence microscopy and immunoperoxidase staining with an avidin-biotin-peroxidase method the protease was found to be present in the epidermis of rodents of different ages as well as that of humans, but not in the dermis. The staining in germinative cells was more intense than in cells in the superficial layers. It appeared as irregular patches in the nuclei and stained more diffusely in the cytoplasm where small granular components, strongly stained, were identified. The staining patterns in granular cells showed accumulation of the antigen in a granular form. The morphology and distribution of granules resembled those of keratohyalin-like granules in the nucleus and dense homogenous deposits in the cytoplasm. In cornified cells the reaction product was localized by the plasma membrane where concentration of the dense homogenous deposits occurred, suggesting that the cysteine protease is one component of the unique and characteristic structure of differentiated keratinocytes. In addition, the cysteine protease antigen having the same molecular weight as the epidermal enzyme was detected in liver, kidney and lung indicating a wider tissue distribution of the protease. The significance of the protease in regulation of cellular functions remains to be investigated.     

Calmodulin,a calmodulin acceptor protein,and calcimedins: Unique antibody localizations in hamster sperm

A calmodulin acceptor protein has been identified in isolated hamster caudal sperm by immunofluoresence and Western transfer techniques. The protein shows a localization in sperm heads identical to calmodulin. Fluorescence of both calmodulin and the acceptor protein are lost by treatment with MgCl₂, conditions which release the acrosome. These results are consistent with the proposed function of calmodulin in a sperm function.