Strongylocins, novel antimicrobial peptides from the green sea urchin, Strongylocentrotus droebachiensis

Sea urchins possess an innate immune system and are regarded as a potential source for the discovery of new antimicrobial peptides (AMPs). Here we report the purification and characterization of two novel antibacterial peptides (5.6 and 5.8kDa) from coelomocyte extracts of the green sea urchin, Strongylocentrotus droebachiensis. These are the first reported AMPs isolated from sea urchins. The cDNA encoding the peptides and genomic sequences was isolated and sequenced. The two peptides (named strongylocins 1 and 2) have putative isoforms (1b and 2b), similar to two putative proteins from the purple sea urchin S. purpuratus. The native strongylocins are cationic, defensin-like peptides (cysteine-rich), but show no similarity to other known AMPs concerning the cysteine distribution pattern. Strongylocin 1 consists of 83 amino acids that include a preprosequence of 35 amino acids, whereas strongylocins 2a and 2b are composed of 89 and 90 amino acids, respectively, where 38 amino acids represent a preprosequence. No introns were found in the cloned gene of strongylocin 1b, whereas three introns and four exons were found in strongylocins 1a and 2a/b. The latter gene organization was also found in genes coding for putative strongylocins in S. purpuratus. The molecular mass difference between the native peptide and the deduced strongylocin 2 suggests that the first amino acid is bromotryptophan. The native peptides display potent activities against Gram-negative and Gram-positive bacteria.     

The important significance of filopodial elongation of phagocytic granular cells of the silkworm, Bombyx mori, in recognition of foreignness

The significance of filopodial elongation of granular phagocytes of Bombyx mori in recognition of foreignness was examined using a larval epidermis obtained from the same individual of B.mori as a self material and intra- or interspecific epidermis and glass as non-self ones. Incubation of cells on self or intraspecific epidermis markedly decreased the filopodial elongation of attached cells, as compared with the incubation on glass. Interspecific epidermis from other Lepidopterans (Papilio xuthus and Pieris melete) have not received filopodial elongation of B.mori granular cells to a lesser degree than B.mori epidermis. Moreover, granular cells directly fixed with glutaraldehyde soon after bleeding were also observed to fold their filopodia. These results seemed to provide experimental evidence that filopodial elongation was probably stimulated by non-self recognition by granular phagocytes.     

Progressively restricted expression of a homeo box gene within the aboral ectoderm of developing sea urchin embryos

A homeo box-containing gene, Hbox1 is expressed in an unusual and highly conserved spatial pattern in embryos of two different species of sea urchin, Tripneustes gratilla and Strongylocentrotus purpuratus. Hybridization in situ shows that this mRNA accumulates initially throughout the aboral ectoderm; however, between blastula and pluteus stages, the region containing Hbox1 mRNA retracts gradually until only a small area around the vertex is labeled in pluteus larvae. Aboral ectoderm appears cytologically uniform and also accumulates uniform levels of other tissue-specific mRNAs. Therefore, the Hbox1 pattern reveals a previously unsuspected heterogeneity of aboral ectoderm cells and a polarity within this tissue. In S. purpuratus, the Hbox1 gene product probably is not involved in initial specification of cell fate, as this message does not achieve a significant fraction of its peak abundance until almost hatching blastula stage, well after the time aboral ectoderm cells have initiated a tissue-specific program of gene expression. RNA blot and RNase protection analyses revealed low levels of Hbox1 mRNA in all adult tissues examined. However, this message was not detectable in mature eggs, suggesting that the Hbox1 gene does not have a maternal function. In addition to highly conserved spatial and temporal patterns of expression, the homeo box genes of these two urchin species also are conserved highly in sequences outside the homeo domain, despite the divergence of these two species (30-45 my). Two notable features of the protein shared with several vertebrate homeo proteins are a short conserved sequence encoded by an exon upstream of that encoding the homeo domain and a large region of high serine and proline content.     

The spherule cells of Holothuria polii Delle Chiaie, 1823 (Aspidochirota, Holothuroidea) during brown body formation: an ultrastructural study

Spherule cells are specific types of coelomocytes found in both the coelomic fluids and the connective tissues of many echinoderm groups and are characterised by large membrane-bound inclusions which completely fill their cytoplasm. In holothurians they are present in massive number in the coelomic fluids and are employed in brown body formation. Brown bodies are products of encapsulation and mainly consist of phagocytic amoebocytes and spherule cells: they surround foreign particles too large to be ingested by circulating phagocytes. During brown body formation, phagocytic amoebocytes flatten out over the surface of foreign particles to form unpigmented nodules which eventually aggregate into a single brown body in which many spherule cells are entrapped. Morphological modifications of spherule cells were studied in Holothuria polii following the induction of brown body formation by intracoelomic injection of sheep erythrocytes. Our ultrastructural observations provide evidence that the granules undergo typical exocytosis after previous disorganisation of their content and suggest a specific secretory activity for the spherule cells. The possible functional role of the secreted vacuolar material in brown body formation is discussed.     

Subcellular localization and complements of GABA(A) and GABA(C) receptors on bullfrog retinal bipolar cells

gamma-Aminobutyric acid (GABA) receptors on retinal bipolar cells (BCs) are highly relevant to spatial and temporal integration of visual signals in the outer and inner retina. In the present work, subcellular localization and complements of GABA(A) and GABA(C) receptors on BCs were investigated by whole cell recordings and local drug application via multi-barreled puff pipettes in the bullfrog retinal slice preparation. Four types of the BCs (types 1-4) were identified morphologically by injection of Lucifer yellow. According to the ramification levels of the axon terminals and the responses of these cells to glutamate (or kainate) applied at their dendrites, types 1 and 2 of BCs were supposed to be OFF type, whereas types 3 and 4 of BCs might be ON type. Bicuculline (BIC), a GABA(A) receptor antagonist, and imidazole-4-acetic acid (I4AA), a GABA(C) receptor antagonist, were used to distinguish GABA receptor-mediated responses. In all BCs tested, not only the axon terminals but also the dendrites showed high GABA sensitivity mediated by both GABA(A) and GABA(C) receptors. Subcellular localization and complements of GABA(A) and GABA(C) receptors at the dendrites and axon terminals were highly related to the dichotomy of OFF and ON BCs. In the case of OFF BCs, GABA(A) receptors were rather evenly distributed at the dendrites and axon terminals, but GABA(C) receptors were predominantly expressed at the axon terminals. Moreover, the relative contribution of GABA(C) receptors to the axon terminals was prevalent over that of GABA(A) receptors, while the situation was reversed at the dendrites. In the case of ON BCs, GABA(A) and GABA(C) receptors both preferred to be expressed at the axon terminals; relative contributions of these two GABA receptor subtypes to both the sites were comparable, while GABA(C) receptors were much less expressed than GABA(A) receptors. GABA(A), but not GABA(C) receptors, were expressed clusteringly at axons of a population of BCs. In a minority of BCs, I4AA suppressed the GABA(C) responses at the dendrites, but not at the axon terminal, implying that the GABA(C) receptors at these two sites may be heterogeneous. Taken together, these results suggest that GABA(A) and GABA(C) receptors may play different roles in the outer and inner retina and the differential complements of the two receptors on OFF and ON BCs may be closely related to physiological functions of these cells.     

Immunolocalization of heparinbinding growth factors (HBGF) types 1 and 2 in rat liver. Selective hyperexpression of HBGF-2 in carbon tetrachloride-induced fibrosis

Ito cells play a major role in liver fibrosis but the mechanisms controlling their activation in vivo are poorly understood. Heparin-binding growth factors (H BGF) types 1 and 2 are mitogenic for cultured Ito cells. They have been found in liver extracts but their cellular localization is unknown. We have studied by immunohistochemistry KBGF-1 and -2 expression in normal rat liver and in carbon tetrachloride (CCl₄)-induced fibrosis. In normal liver, HBGF-1 was present only in sinusoidal cells whereas HBGF-2 was also detected in endothelial cells lining major vessels. At the acute stage of CC1₄ intoxication, HBGF-2 was expressed in centrilobular clusters of mononuclear phagocytes that were surrounded by many HBGF-2-negative Ito cells. In the later stages, HBGF-2 was expressed by Ito cells within the fibrous bands. No modulation of HBGF-1 expression was noted at any stage. These results suggest that (1) at the acute stage of CC1₄ intoxication, HBGF-2 produced by mononuclear phagocytes could participate in the recruitment of Ito cells; and (2) during the CCl₄-induced fibrotic process, HBGF-2 could contribute to Ito cell proliferation and the synthesis of fibrosis components. In this in vivo model of hepatic fibrosis, the hyperexpression of HBGF-2 is a relatively specific event since the expression of a structurally related molecule, HBGF-1 was not modulated.     

Conserved microRNA miR-8 in fat body regulates innate immune homeostasis in Drosophila

Antimicrobial peptides (AMPs) constitute a major arm of the innate immune system across diverse organisms. In Drosophila, septic injury by microbial pathogens rapidly induces the production of the AMPs in fat body via well elucidated pathways such as Toll and IMD. However, several epithelial tissues were reported to locally express AMPs without septic injury via poorly characterized ways. Here, we report that microRNA miR-8 regulates the levels of AMPs basally expressed in Drosophila. The levels of AMPs such as Drosomycin and Diptericin are significantly increased in miR-8 null animals in non-pathogen stimulated conditions. Analysis of various larval tissues revealed that the increase of Drosomycin is fat body specific. Supporting this observation, re-introduction of miR-8 only in the fat body restored the altered AMP expression in miR-8 null flies. Although loss of miR-8 impedes PI3K in the fat body, inhibition of PI3K does not phenocopy the AMP expression of miR-8 null flies, indicating that miR-8 regulates AMP independently of PI3K. Together, our findings suggest a role of miR-8 in systemic immune homeostasis in generally non-pathogenic conditions in flies.     

NLRC4-driven production of IL-1β discriminates between pathogenic and commensal bacteria and promotes host intestinal defense

Intestinal phagocytes transport oral antigens and promote immune tolerance, but their role in innate immune responses remains unclear. Here we found that intestinal phagocytes were anergic to ligands for Toll-like receptors (TLRs) or commensals but constitutively expressed the precursor to interleukin 1β (pro-IL-1β). After infection with pathogenic Salmonella or Pseudomonas, intestinal phagocytes produced mature IL-1β through the NLRC4 inflammasome but did not produce tumor necrosis factor (TNF) or IL-6. BALB/c mice deficient in NLRC4 or the IL-1 receptor were highly susceptible to orogastric but not intraperitoneal infection with Salmonella. That enhanced lethality was preceded by impaired expression of endothelial adhesion molecules, lower neutrophil recruitment and poor intestinal pathogen clearance. Thus, NLRC4-dependent production of IL-1β by intestinal phagocytes represents a specific response that discriminates pathogenic bacteria from commensal bacteria and contributes to host defense in the intestine.     

Expression and Distribution of Leptospiral Outer Membrane Components during Renal Infection of Hamsters

The outer membrane of pathogenic Leptospira species grown in culture media contains lipopolysaccharide (LPS), a porin (OmpL1), and several lipoproteins, including LipL36 and LipL41. The purpose of this study was to characterize the expression and distribution of these outer membrane antigens during renal infection. Hamsters were challenged with host-derived Leptospira kirschneri to generate sera which contained antibodies to antigens expressed in vivo. Immunoblotting performed with sera from animals challenged with these host-derived organisms demonstrated reactivity with OmpL1, LipL41, and several other proteins but not with LipL36. Although LipL36 is a prominent outer membrane antigen of cultivated L. kirschneri, its expression also could not be detected in infected hamster kidney tissue by immunohistochemistry, indicating that expression of this protein is down-regulated in vivo. In contrast, LPS, OmpL1, and LipL41 were demonstrated on organisms colonizing the lumen of proximal convoluted renal tubules at both 10 and 28 days postinfection. Tubular epithelial cells around the luminal colonies had fine granular cytoplasmic LPS. When the cellular inflammatory response was present in the renal interstitium at 28 days postinfection, LPS and OmpL1 were also detectable within interstitial phagocytes. These data establish that outer membrane components expressed during infection have roles in the induction and persistence of leptospiral interstitial nephritis.     

Androgen receptor is frequently expressed in HER2-positive, ER/PR-negative breast cancers

The estrogen receptor (ER)/progesterone receptor (PR)-negative breast carcinomas (BCs) encompass three molecular subtypes: one with human epidermal growth factor receptor 2 (HER) overexpression, one normal like, and the triple negative. The androgen receptor (AR) is expressed in 70–90% of invasive BCs. The aim of our study is to detect the expression of AR in a series of ER/PR-negative BCs to ascertain if there is clinical significance in relation to BC molecular subtypes. A immunohistochemical study for all receptors and cytokeratin expression was performed in 232 cases of ER/PR-negative BCs. According to cytokeratin expression, BCs were classified into two groups: luminal-type BCs (44.2%) and basal-like-type BCs (55.8%). According to the expression of HER2, 59.3% were triple-negative BCs (when ER, PR, and HER2 were negative) and 40.7% were HER2-positive BCs. AR expression was observed in 128 tumors (56.6%). One hundred and ten cases (48.8%) had >10% and 18 (7.8%) had <10% of positively stained cells. AR immunoreactivity was found in 31.2% basal-like BCs, while in the luminal group 71.1% of cases were positive, showing highly significant correlation (p < 10⁻⁸). Regarding HER2 status, 76.7% of HER2-positive BC cases were AR positive compared with only 30.4% of triple-negative BC types, showing a strong statistically significant correlation. In conclusion, we show that AR is frequently expressed in ER/PR-negative BCs and that expression of HER2 and AR is highly correlated (p < 0.005). Our results point out the role of AR and HER2 in the pathogenesis of BCs and suggest the potential role of AR in clinical management of ER/PR-negative BCs.