The β-1,3-glucan-binding protein from the crayfish Pacifastacus leniusculus,when reacted with a β-1,3-glucan,induces spreading and degranulation of crayfish granular cells

Summary A -1,3-glucan-binding protein (GBP) was purified from crayfish plasma, and incubated with laminarin (L), a -1,3-glucan. The GBP reacted with laminarin (GBP-L) induced strong spreading and partial degranulation of isolated and separated crayfish granular haemocytes. However, neither the GBP nor laminarin alone induced any changes in the crayfish granular cells. When monolayers of granular haemocytes were incubated with 20 g of GBP-L, more than 82% of the haemocytes were affected. The activity of GBP-L on granular cells was dose-dependent and a plateau was reached at 10 g of GBP-L. The degranulation of crayfish haemocytes induced by GBP-L seemed to occur by a regulated exocytosis, since it was strongly inhibited by specific blockers of this process such as SITS or calmidazolium. Monospecific anti-GBP antibodies also totally blocked the effect of GBP-L on crayfish granular cells. Indirect immunofluoresence staining demonstrated that the GBP-L could bind to the surface of granular cells, whereas GBP did not bind or bound very weakly to the haemocyte surface.     

Excision of pIJ408 from the chromosome of Streptomyces glaucescens and its transfer into Streptomyces lividans

Summary Streptomyces glaucescens GLA000 contains the integrated 15 kb DNA element pIJ408 which, during mating of the parent strain with S. lividans, can be transferred into recipient cells. In S. lividans cells, pIJ408 was found in an autonomously replicating form and in a chromosomally integrated state. In the majority of the S. lividans transconjugants studied, a deletion derivative pIJ408. 1 (12.4 kb) occurred. The deletion form was found in some strains only as a free plasmid, in others it was also chromosomally integrated. The integration region of pIJ408 was subcloned and precisely mapped by hybridization, restriction and sequencing analyses. The DNA junction fragments of the integrated plasmid in S. glaucescens, as well as the DNA fragment containing the attachment site of the S. lividans chromosome, were also cloned, submitted to detailed restriction analysis and sequenced. The attachment site of pIJ408 (attP) and the junctions of its integrated form with the chromosomal DNA in S. glaucescens (attL and attR) contain an identical 43 bp sequence. The chromosomal attachment site in S. lividans (attB) differs from the S. glaucescens att sequence by a single base substitution. The similarities between attachment sites of SLP1, pMEA100, pSAM2 and pIJ408 are discussed.     

Epithelial V-like Antigen (EVA), a Novel Member of the Immunoglobulin Superfamily, Expressed in Embryonic Epithelia with a Potential Role as Homotypic Adhesion Molecule in Thymus Histogenesis

Thymus development depends on a complex series of interactions between thymocytes and the stromal component of the organ. To identify regulated genes during this codependent developmental relationship, we have applied an RNA fingerprinting technique to the analysis of thymus expansion and maturation induced in recombinase-deficient mice injected with anti-CD3 antibodies. This approach led us to the identification of a gene encoding a new member of the immunoglobulin superfamily, named epithelial V-like antigen (EVA), which is expressed in thymus epithelium and strongly downregulated by thymocyte developmental progression. This gene is expressed in the thymus and in several epithelial structures early in embryogenesis. EVA is highly homologous to the myelin protein zero and, in thymus-derived epithelial cell lines, is poorly soluble in nonionic detergents, strongly suggesting an association to the cytoskeleton. Its capacity to mediate cell adhesion through a homophilic interaction and its selective regulation by T cell maturation might imply the participation of EVA in the earliest phases of thymus organogenesis.     

Exclusion of the Sonic Hedgehog gene as responsible for Currarino syndrome and anorectal malformations with sacral hypodevelopment

Anorectal malformations (ARMs) are common congenital anomalies that account for 1:4 digestive malformations. ARM patients show different degrees of sacral hypodevelopment while the hemisacrum is characteristic of the Currarino syndrome (CS). Cases of CS present an association of ARM, hemisacrum and presacral mass. A gene responsible for CS has recently been mapped in 7q36. Among the genes localized in this critical region, sonic hedgehog (SHH) was thought to represent a candidate gene for CS as well as for ARM with different levels of sacral hypodevelopment according to its role in the differentiation of midline mesoderm. By linkage analysis we confirmed the critical region in one large family with recurrence of CS. In addition, the screening of SHH in 7 CS and in 15 sporadic ARM patients with sacral hypodevelopment allowed us to exclude its role in the pathogenesis of these disorders. Received: 10 August 1998 / Accepted: 12 November 1998     

XBP1 promotes NRASG12D pre-B acute lymphoblastic leukaemia through IL-7 receptor signalling and provides a therapeutic vulnerability for oncogenic RAS

Activating point mutations of the RAS gene act as driver mutations for a subset of precursor-B cell acute lymphoblastic leukaemias (pre-B ALL) and represent an ambitious target for therapeutic approaches. The X box-binding protein 1 (XBP1), a key regulator of the unfolded protein response (UPR), is critical for pre-B ALL cell survival, and high expression of XBP1 confers poor prognosis in ALL patients. However, the mechanism of XBP1 activation has not yet been elucidated in RAS mutated pre-B ALL. Here, we demonstrate that XBP1 acts as a downstream linchpin of the IL-7 receptor signalling pathway and that pharmacological inhibition or genetic ablation of XBP1 selectively abrogates IL-7 receptor signalling via inhibition of its downstream effectors, JAK1 and STAT5. We show that XBP1 supports malignant cell growth of pre-B NRAS^G12D ALL cells and that genetic loss of XBP1 consequently leads to cell cycle arrest and apoptosis. Our findings reveal that active XBP1 prevents the cytotoxic effects of a dual PI3K/mTOR pathway inhibitor (BEZ235) in pre-B NRAS^G12D ALL cells. This implies targeting XBP1 in combination with BEZ235 as a promising new targeted strategy against the oncogenic RAS in NRAS^G12D-mutated pre-B ALL.     

Preliminary studies on noncovalent hyperbranched polymers based on PNA and DNA building blocks

In this work, we report thermodynamic, kinetic, and microrheological studies relative to the formation of PNA‐ and PNA/DNA‐based noncovalent polymeric systems, useful tools for biotechnological and bioengineering applications. We realized two kinds of systems: a PNA‐based system formed by a self‐assembling PNA tridendron, and a PNA/DNA hybrid system formed by a PNA tridendron and a DNA linker. The formation of a three‐dimensional polymeric network, by means of specific Watson–Crick base pairing, was investigated by a detailed UV and CD spectroscopic study. Preliminary microrheology experiments were performed on both systems to evaluate their viscoelastic properties which resulted in agreement with the formation of soluble hyperbranched polymers that could be useful for drug/gene delivery, as well as for encapsulating organic pollutants of different shapes and sizes in environmental applications. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Histochemical and ultrastructural analysis of the epidermal gland cells of Branchiomma luctuosum (Polychaeta, Sabellidae)

Abstract. This histochemical and ultrastructural study describes the epidermal gland cells of a tubicolous polychaete, Branchiomma luctuosum . The histochemistry was carried out using standard techniques and FITC-labelled lectins. Four types of secretory cells were identified in two categories: orthochromatic cells (Type 1) and metachromatic cells (Types 2, 3, and 4). The secretory product of the Type-1 orthochromatic cells contains neutral glycoproteins with Galβ1,3GalNAc residues. Metachromatic cells produce acidic, mainly sulfated, glycoconjugates with Galβ1,3GalNAc residues (Type 2) or glucosidic and/or mannosidic residues (Types 3 and 4). In sialylated chains, terminal sialic acid is bound to the penultimate GalNAc and Galβ1,3GalNAc residues. The complex composition of the mucus produced by epidermal gland cells of B. luctuosum may be correlated with its different functions. Ultrastructural studies of the epidermal gland cells showed differing morphology, and the presence in the gland cells of Types 3 and 4 of a funnel-shaped structure for the extrusion of the secretory material.     

Trace elements and melanoma.

Melanoma incidence has been steadily increasing in recent years in most western countries, thus suggesting a role of environmental risk factors. Among these determinants, it has been hypothesized that some trace elements of nutritional and toxicological interest may be implicated in the etiology of the disease. We examined patients with newly diagnosed melanoma of the skin and population controls from the Modena province northern Italy. Clinical and dietary data were collected through questionnaires, and toenails were sampled for trace element determination. Levels of cadmium, chromium, lead, selenium, zinc, copper and iron in toenails were measured by inductively coupled plasma optical emission spectrometry and by neutron activation analysis. Data obtained from 58 cases and 58 controls indicated higher levels of copper and lower concentrations of iron in melanoma patients, whilst no other differences were seen for the remaining elements. Patterns of correlations of zinc and copper with the estimated intake of some dietary factors were different between cases and controls. Results of the present study suggest that abnormal intake or metabolism of copper and of iron might be implicated in the etiology of melanoma, whilst they do not indicate an involvement of exposure to cadmium, chromium, lead, selenium and zinc in this disease.     

Intracellular interaction of interleukin-15 with its receptor alpha during production leads to mutual stabilization and increased bioactivity

We show that co-expression of interleukin 15 (IL-15) and IL-15 receptor alpha (IL-15Ralpha) in the same cell allows for the intracellular interaction of the two proteins early after translation, resulting in increased stability and secretion of both molecules as a complex. In the absence of co-expressed IL-15Ralpha, a large portion of the produced IL-15 is rapidly degraded immediately after synthesis. Co-injection into mice of IL-15 and IL-15Ralpha expression plasmids led to significantly increased levels of the cytokine in serum as well as increased biological activity of IL-15. Examination of natural killer cells and T lymphocytes in mouse organs showed a great expansion of both cell types in the lung, liver, and spleen. The presence of IL-15Ralpha also increased the number of CD44(high) memory cells with effector phenotype (CD44(high)CD62L-). Thus, mutual stabilization of IL-15 and IL-15Ralpha leads to remarkable increases in production, stability, and tissue availability of bioactive IL-15 in vivo. The in vivo data show that the most potent form of IL-15 is as part of a complex with its receptor alpha either on the surface of the producing cells or as a soluble extracellular complex. These results explain the reason for coordinate expression of IL-15 and IL-15Ralpha in the same cell and suggest that the IL-15Ralpha is part of the active IL-15 cytokine rather than part of the receptor.