Modulation of immunity in young-adult and aged squirrel, Funambulus pennanti by melatonin and p-chlorophenylalanine

Background  

Our interest was to find out whether pineal gland and their by melatonin act as modulator of immunosenescence. Parachlorophenylalanine (PCPA) – a β adrenergic blocker, is known to perform chemical pinealectomy (Px) by suppressing indirectly the substrate 5-hydroxytryptamine (5-HT) for melatonin synthesis and thereby melatonin itself. The role of PCPA, alone and in combination with melatonin was recorded in immunomodulation and free radical load in spleen of young adult and aged seasonal breeder Indian palm squirrel Funambulus pennanti.     

cDNA microarray analysis of bovine embryo gene expression profiles during the pre-implantation period

Background

After fertilization, embryo development involves differentiation, as well as development of the fetal body and extra-embryonic tissues until the moment of implantation. During this period various cellular and molecular changes take place with a genetic origin, e.g. the elongation of embryonic tissues, cell-cell contact between the mother and the embryo and placentation. To identify genetic profiles and search for new candidate molecules involved during this period, embryonic gene expression was analyzed with a custom designed utero-placental complementary DNA (cDNA) microarray.

Methods

Bovine embryos on days 7, 14 and 21, extra-embryonic membranes on day 28 and fetuses on days 28 were collected to represent early embryo, elongating embryo, pre-implantation embryo, post-implantation extra-embryonic membrane and fetus, respectively. Gene expression at these different time points was analyzed using our cDNA microarray. Two clustering algorithms such as k-means and hierarchical clustering methods identified the expression patterns of differentially expressed genes across pre-implantation period. Novel candidate genes were confirmed by real-time RT-PCR.

Results

In total, 1,773 individual genes were analyzed by complete k-means clustering. Comparison of day 7 and day 14 revealed most genes increased during this period, and a small number of genes exhibiting altered expression decreased as gestation progressed. Clustering analysis demonstrated that trophoblast-cell-specific molecules such as placental lactogens (PLs), prolactin-related proteins (PRPs), interferon-tau, and adhesion molecules apparently all play pivotal roles in the preparation needed for implantation, since their expression was remarkably enhanced during the pre-implantation period. The hierarchical clustering analysis and RT-PCR data revealed new functional roles for certain known genes (dickkopf-1, NPM, etc) as well as novel candidate genes (AW464053, AW465434, AW462349, AW485575) related to already established trophoblast-specific genes such as PLs and PRPs.

Conclusions

A large number of genes in extra-embryonic membrane increased up to implantation and these profiles provide information fundamental to an understanding of extra-embryonic membrane differentiation and development. Genes in significant expression suggest novel molecules in trophoblast differentiation.     

Topological characterization of an inner membrane (1-->3)-beta-D-glucan (curdlan) synthase from Agrobacterium sp. strain ATCC31749

The crdS gene of Agrobacterium sp. strain ATCC31749 encodes the curdlan synthase (CrdS) protein based on the homology of the derived CrdS protein sequence with those of beta-glycosyl transferases with repetitive action patterns (Stasinopoulos et al. [1999] Glycobiology, 9, 31-41). Here we show that chemical (NTG) mutagenesis of crdS abolishes curdlan production and the induced mutations can be complemented by a cloned crdS amplicon, thus providing genetic confirmation that crdS is essential for curdlan production. When expressed in the native Agrobacterium or in Escherichia coli, the largely hydrophobic CrdS protein exhibited an Mr of approximately 60 kDa (compared with the predicted mass of 73,121 Da) and was located in the inner membrane of both bacteria. By analyzing reciprocal fusions between crdS and the reporter genes, lacZ and phoA, and assessing the sensitivity of CrdS in spheroplasts to proteinase K, CrdS was shown to be an integral membrane protein with seven transmembrane helices and an Nout-Cin disposition. A central large and relatively hydrophilic cytoplasmic region carries the substrate-binding and catalytic D,D,D35QxxRW motif. The amino acid sequence of this domain of CrdS was threaded onto the 3D structure of the comparable domain of the SpsA protein, a member of the family GT-2 glycosyl transferases, and enabled the identification of corresponding amino acids involved in binding UDP in CrdS. Analysis of Agrobacterium membrane preparations using blue native-PAGE provided preliminary evidence that CrdS occurs in multimeric protein complexes of approximately 420 kDa and approximately 500 kDa.     

Enzymic,phylogenetic, and structural characterization of the unusual papain-like protease domain of Plasmodium falciparum SERA5

Serine repeat antigen 5 (SERA5) is an abundant antigen of the human malaria parasite Plasmodium falciparum and is the most strongly expressed member of the nine-gene SERA family. It appears to be essential for the maintenance of the erythrocytic cycle, unlike a number of other members of this family, and has been implicated in parasite egress and/or erythrocyte invasion. All SERA proteins possess a central domain that has homology to papain except in the case of SERA5 (and some other SERAs), where the active site cysteine has been replaced with a serine. To investigate if this domain retains catalytic activity, we expressed, purified, and refolded a recombinant form of the SERA5 enzyme domain. This protein possessed chymotrypsin-like proteolytic activity as it processed substrates downstream of aromatic residues, and its activity was reversed by the serine protease inhibitor 3,4-diisocoumarin. Although all Plasmodium SERA enzyme domain sequences share considerable homology, phylogenetic studies revealed two distinct clusters across the genus, separated according to whether they possess an active site serine or cysteine. All Plasmodia appear to have at least one member of each group. Consistent with separate biological roles for members of these two clusters, molecular modeling studies revealed that SERA5 and SERA6 enzyme domains have dramatically different surface properties, although both have a characteristic papain-like fold, catalytic cleft, and an appropriately positioned catalytic triad. This study provides impetus for the examination of SERA5 as a target for antimalarial drug design.     

Morphometric analysis of photoreceptive,neuronal and endocrinal cell differentiation of avian pineal cells: an in vitro immunohistochemical study on the developmental transition from neuronal to photo-endocrinal property

Little is known about the developmental origin, determination and differentiation of different pineal immunoreactive cells in the avian group, and an experimental establishment is then required to explain the differentiation of cell types (i.e. photosensory, neural and secretory types). The present in vitro study suggests that the avian pineal organ is made up of multiple types of cells with different immunoreactivity at the ontogenic state (from embryonic day 9 to day 14), before it acquires the final photoendocrinal nature of the mature state. The morphometric analysis suggests that the developmental changes in the morphology of the quail pinealocytes appear to represent a condensed expression of the phylogenic development in the ontogeny. Several types of immunoreactive cells from a neuronal line were suppressed with maturation of developing pineal glands, while other cell types such as photoreceptive and endocrinal lines became more prominent. The melatonin level in the culture medium presented a high value up to 72 hr of culture, followed by a decrease as well as dampening of the level at the end of the culture possibly because the cultures were maintained in dark. The results of the present study, a combined analysis of morphometry and RIA, open a new line for research into the pineal development and cell differentiation.     

Hemodynamic Effects of the Non-Peptidic Angiotensin-(1-7) Agonist AVE0991 in Liver Cirrhosis

Background & Aims

Although in cirrhosis with portal hypertension levels of the vasoconstrictor angiotensin II are increased, this is accompanied by increased production of angiotensin (Ang)-(1–7), the endogenous ligand of the Mas receptor (MasR), which blunts hepatic fibrosis and decreases hepatic vascular resistance. Therefore, we investigated the effects of the non-peptidic Ang-(1–7) agonist, AVE0991, in experimental cirrhosis.

Methods

Cirrhosis was induced by bile duct ligation (BDL) or carbon tetrachloride (CCl₄) intoxication. The coloured microsphere technique assessed portal and systemic hemodynamic effects of AVE0991 in vivo. Hepatic expression of eNOS, p-eNOS, iNOS, JAK2, ROCK and p-Moesin were analyzed by western blots. Activities of ACE and ACE2 were investigated fluorometrically. Moreover, fibrosis was assessed in BDL rats receiving AVE0991.

Results

In vivo, AVE0991 decreased portal pressure (PP) in both rat models of cirrhosis. Importantly, systemic effects were not observed. The hepatic effects of AVE0991 were based on upregulation of vasodilating pathways involving p-eNOS and iNOS, as well as by downregulation of the vasoconstrictive pathways (ROCK, p-Moesin). Short-term treatment with AVE0991 decreased the activity of ACE2, long-term treatment did not affect hepatic fibrosis in BDL rats.

Conclusions

The non-peptidic agonist of Ang-(1–7), AVE0991, decreases portal pressure without influencing systemic pressure. Thus, although it does not inhibit fibrosis, AVE0991 may represent a promising new therapeutic strategy for lowering portal pressure.     

Novel Regulation of CD80/CD86-induced Phosphatidylinositol 3-Kinase Signaling by NOTCH1 Protein in Interleukin-6 and Indoleamine 2,3-Dioxygenase Production by Dendritic Cells

Dendritic cells (DC) play a critical role in modulating antigen-specific immune responses elicited by T cells via engagement of the prototypic T cell costimulatory receptor CD28 by the cognate ligands CD80/CD86, expressed on DC. Although CD28 signaling in T cell activation has been well characterized, it has only recently been shown that CD80/CD86, which have no demonstrated binding domains for signaling proteins in their cytoplasmic tails, nonetheless also transduce signals to the DC. Functionally, CD80/CD86 engagement results in DC production of the pro-inflammatory cytokine IL-6, which is necessary for full T cell activation. However, ligation of CD80/CD86 by CTLA4 also induces DC production of the immunosuppressive enzyme indoleamine 2,3-dioxygenase (IDO), which depletes local pools of the essential amino acid tryptophan, resulting in blockade of T cell activation. Despite the significant role of CD80/CD86 in immunological processes and the seemingly opposing roles they play by producing IL-6 and IDO upon their activation, how CD80/CD86 signal remains poorly understood. We have now found that cross-linking CD80/CD86 in human DC activates the PI3K/AKT pathway. This results in phosphorylation/inactivation of its downstream target, FOXO3A, and alleviates FOXO3A-mediated suppression of IL-6 expression. A second event downstream of AKT phosphorylation is activation of the canonical NF-κB pathway, which induces IL-6 expression. In addition to these downstream pathways, we unexpectedly found that CD80/CD86-induced PI3K signaling is regulated by previously unrecognized cross-talk with NOTCH1 signaling. This cross-talk is facilitated by NOTCH-mediated up-regulation of the expression of prolyl isomerase PIN1, which in turn increases enzyme activity of casein kinase II. Subsequently, phosphatase and tensin homolog (which suppresses PI3K activity) is inactivated via phosphorylation by casein kinase II. This results in full activation of PI3K signaling upon cross-linking CD80/CD86. Similar to IL-6, we have found that CD80/CD86-induced IDO production by DC at late time points is also dependent upon the PI3K → AKT → NF-κB pathway and requires cross-talk with NOTCH signaling. These data further suggest that the same signaling pathways downstream of DC CD80/CD86 cross-linking induce early IL-6 production to enhance T cell activation, followed by later IDO production to self-limit this activation. In addition to characterizing the pathways downstream of CD80/CD86 in IL-6 and IDO production, identification of a novel cross-talk between NOTCH1 and PI3K signaling may provide new insights in other biological processes where PI3K signaling plays a major role.     

Involvement of the Reck tumor suppressor protein in maternal and embryonic vascular remodeling in mice

Background

The accessibility of the developing zebrafish pharyngeal dentition makes it an advantageous system in which to study many aspects of tooth development from early initiation to late morphogenesis. In mammals, hedgehog signaling is known to be essential for multiple stages of odontogenesis; however, potential roles for the pathway during initiation of tooth development or in later morphogenesis are incompletely understood.

Results

We have identified mRNA expression of the hedgehog ligands shha and the receptors ptc1 and ptc2 during zebrafish pharyngeal tooth development. We looked for, but did not detect, tooth germ expression of the other known zebrafish hedgehog ligands shhb, dhh, ihha, or ihhb, suggesting that as in mammals, only Shh participates in zebrafish tooth development. Supporting this idea, we found that morphological and gene expression evidence of tooth initiation is eliminated in shha mutant embryos, and that morpholino antisense oligonucleotide knockdown of shha, but not shhb, function prevents mature tooth formation. Hedgehog pathway inhibition with the antagonist compound cyclopamine affected tooth formation at each stage in which we applied it: arresting development at early stages and disrupting mature tooth morphology when applied later. These results suggest that hedgehog signaling is required continuously during odontogenesis. In contrast, over-expression of shha had no effect on the developing dentition, possibly because shha is normally extensively expressed in the zebrafish pharyngeal region.

Conclusion

We have identified previously unknown requirements for hedgehog signaling for early tooth initiation and later morphogenesis. The similarity of our results with data from mouse and other vertebrates suggests that despite gene duplication and changes in the location of where teeth form, the roles of hedgehog signaling in tooth development have been largely conserved during evolution.