Structural characterization of glycosphingolipids from the eggs of Schistosoma mansoni and Schistosoma japonicum

The granulomatous pathology in human intestinal schistosomiasisis induced primarily by the egg antigens of schistosome, a parasitictrematode. Glycolipids and glycoproteins were extracted fromthe eggs of the two major species which infect human, Schistosomamansoni and Schistosoma japonicum, for structural characterizationbased on highly sensitive mass spectrometric analysis coupledwith chemical derivatization. Here, we demonstrate that a seriesof uniquely multifucosylated glycosphingolipids constitute themajor egg glycolipids of S.mansoni but not of S.japonicum. TheS.mansoni glycosphingolipids were found to be extended by varyingnumbers of an unusual repeating unit,     

Metabolite profiles of Stachybotrys isolates from water-damaged buildings and their induction of inflammatory mediators and cytotoxicity in macrophages

The metabolite profiles of 20 Stachybotrys spp.isolates from Finnish water-damaged buildings were compared with their biological activities. Effects of purified compounds on cytotoxicity and production of inflammatory mediators such as nitric oxide, IL-6 and TNFα in murine RAW264.7 macrophage cells were studied. The 11isolates belonging to the satratoxin-producing chemotype were highly cytotoxic to the macrophages. The isolates inducing inflammatory mediators all belonged to the atranone-producing chemotype, but pure atranones B, and D did not elicit a response in the bioassay. Altogether, cytotoxicity ofStachybotrys sp. isolates appear to be related to satratoxin production whereas the specific component inducing inflammatory responses in atranone-producing isolates remains obscure. This revised version was published online in June 2006 with corrections to the Cover Date.     

Impaired Th2 development and increased mortality during Schistosoma mansoni infection in the absence of CD40/CD154 interaction

The role of CD40/CD154 interaction during infection has primarily focused on pathogens that drive inflammatory Th1 responses. In this study, we show that CD40/CD154 interaction is a fundamental requirement for Th2 response development to the parasitic helminth Schistosoma mansoni. Compared with infected wild-type mice, greatly reduced levels of Th2-associated cytokines were measured both in vitro and in vivo, and no IgE or IgG1 was detected in infected CD154(-/-) mice. In the absence of an overt Th2 response, no exaggerated Th1 response was mounted by CD154(-/-) mice. Infected CD154(-/-) mice suffered severe morbidity and mortality, even though parasitemias in wild-type and CD154(-/-) mice did not differ significantly. These data indicate that CD40/CD154 interaction is required to allow development of a Th2-dominated immune response to S. mansoni and support the view that failure to develop such a response can have fatal consequences.     

Improved tumor selectivity of radiolabeled peptides by receptor and antigen dual targeting in the neurotensin receptor model

Radiolabeled peptides are emerging tools for diagnosis and therapy of tumors overexpressing receptors. However, binding to receptors expressed by nontumor tissues may cause toxicity. The objective of this study was to specifically enhance the binding affinity of labeled peptides to tumor cells, as opposed to receptor-positive nontumor cells, to ensure targeting selectivity. This was achieved by the simultaneous binding of hapten-bearing peptides to their receptor and to a tumor-associated antigen, mediated by a bispecific antibody directed to the tumor antigen and to the hapten. Binding of labeled neurotensin analogues bearing the DTPA(indium) hapten (NT-DTPA(111In)) to human colorectal carcinoma cells (HT29), which express the neurotensin receptor (NTR1) and carcinoembryonic antigen (CEA), was studied in the presence of a bispecific antibody (BsmAb) directed to CEA and to DTPA(indium). In vitro dual binding of NT-DTPA in the presence of BsmAb was about 6.5-fold higher than monovalent binding to NTR1 and 3.5-fold higher than the sum of the monovalent bindings to NTR1 or to CEA, suggesting cooperativity. Increased binding under bivalent conditions translated into increased internalization. In vivo pretargeting with BsmAb enhanced tumor uptake and tumor retention. Hapten bearing peptides binding simultaneously an overexpressed cell-surface receptor and a tumor antigen show increased selectivity to target tumor cells as compared to cells only expressing the cell surface receptor. Better resistance to enzymatic degradation and optimized administration protocols should further enhance in vivo targeting selectivity and may allow the development of radiopharmaceuticals labeled with isotopes suitable for radiotherapy such as 131I or 90Y.     

Extracellular hemoglobin combined with an O2-generating material overcomes O2 limitation in the bioartificial pancreas

The bioartificial pancreas encapsulating pancreatic islets in immunoprotective hydrogel is a promising therapy for Type 1 diabetes. As pancreatic islets are highly metabolically active and exquisitely sensitive to hypoxia, maintaining O₂ supply after transplantation remains a major challenge. In this study, we address the O₂ limitation by combining silicone-encapsulated CaO₂ (silicone-CaO₂) to generate O₂ with an extracellular hemoglobin O₂-carrier coencapsulated with islets. We showed that the hemoglobin improved by 37% the O₂-diffusivity through an alginate hydrogel and displayed antioxidant properties neutralizing deleterious reactive O₂ species produced by silicone-CaO₂. While the hemoglobin alone failed to maintain alginate macroencapsulated neonate pig islets under hypoxia, silicone-CaO₂ alone or combined to the hemoglobin restored islet viability and insulin secretion and prevented proinflammatory metabolism (PTGS2 expression). Interestingly, the combination took the advantages of the two individual strategies, improved neonate pig islet viability and insulin secretion in normoxia, and VEGF secretion and PDK1 normalization in hypoxia. Moreover, we confirmed the specific benefits of the combination compared to silicone-CaO₂ alone on murine pseudo-islet viability in normoxia and hypoxia. For the first time, our results show the interest of combining an O₂ provider with hemoglobin as an effective strategy to overcome O₂ limitations in tissue engineering.     

CABYR, a novel calcium-binding tyrosine phosphorylation-regulated fibrous sheath protein involved in capacitation

To reach fertilization competence, sperm undergo an incompletely understood series of morphological and molecular maturational processes, termed capacitation, involving, among other processes, protein tyrosine phosphorylation and increased intracellular calcium. Hyperactivated motility and an ability to undergo the acrosome reaction serve as physiological end points to assess successful capacitation. We report here that acidic (pI 4.0) 86-kDa isoforms of a novel, polymorphic, testis-specific protein, designated calcium-binding tyrosine phosphorylation-regulated protein (CABYR), were tyrosine phosphorylated during in vitro capacitation and bound (45)Ca on 2D gels. Acidic 86-kDa calcium-binding forms of CABYR increased during in vitro capacitation, and calcium binding to these acidic forms was abolished by dephosphorylation with alkaline phosphatase. Six variants of CABYR containing two coding regions (CR-A and CR-B) were cloned from human testis cDNA libraries, including five variants with alternative splice deletions. A motif homologous to the RII dimerization domain of PK-A was present in the N-terminus of CR-A in four CABYR variants. A single putative EF handlike motif was noted in CR-A at aas 197-209, while seven potential tyrosine phosphorylation-like sites were noted in CR-A and four in CR-B. Pro-X-X-Pro (PXXP) modules were identified in the N- and C-termini of CR-A and CR-B. CABYR localizes to the principal piece of the human sperm flagellum in association with the fibrous sheath and is the first demonstration of a sperm protein that gains calcium-binding capacity when phosphorylated during capacitation.     

N(4)C-alkyl-N(4)C cross-linked DNA: bending deformations in duplexes that contain a -CNG- interstrand cross-link

Short DNA duplexes containing a 1,3-N(4)C-alkyl-N(4)C interstrand cross-link that joins the two C residues of a -CNG- sequence were prepared using either a phosphoramidite or convertible nucleoside approach. The alkyl cross-link consists of 2, 4, or 7 methylene groups. The duplexes, which contain a seven-base-pair core and A(3)/T(3) complementary 3'-overhanging ends, were characterized by enzymatic digestion and MALDI-TOF mass spectrometry. Ultraviolet thermal denaturation studies showed that the duplexes denature in a cooperative manner and that the length of the cross-link affects the thermal stability. Thus, the transition temperature of the ethyl cross-linked duplex, 42 degrees C, is 16 degrees C higher than the melting temperature of the corresponding non-cross-linked control, whereas the transition temperatures of the butyl and heptyl cross-linked duplexes, 73 and 72 degrees C, respectively, are 46-47 degrees C higher. The reduced molecularity of denaturation of the cross-linked duplexes versus melting of the non-cross-linked duplex most likely accounts for these differences. Examination of molecular models suggests that the ethyl cross-link is too short to span the distance between the two C residues at the site of the cross-link in B-form DNA without causing distortion of the helix, whereas less and no distortion would be expected for the butyl and heptyl cross-links, respectively. The circular dichroism spectra, which show greatest deviation in the ethyl cross-linked duplex from B-form DNA, are consistent with this expectation. Anomalous mobilities on native polyacrylamide gels of multimers produced by self-ligation of each of the cross-linked duplexes suggest that the ethyl and butyl cross-linked duplexes undergo bending deformations, whereas multimers derived from the heptyl cross-linked duplex migrated normally. The bending angle was estimated to be 20 degrees, 13 degrees, and 0 degrees for the ethyl, butyl, and heptyl cross-linked duplexes, respectively. Thus, it appears that the degree of bending in these N(4)C-alkyl-N(4)C cross-linked duplexes is controlled by the length of the cross-link.