Purification and characterization of placental heparanase and its expression by cultured cytotrophoblasts

The role of different extracellular matrix (ECM)-degrading enzymesin the normal functioning of the placenta is well documented.Heparan sulphate proteoglycan (HSPG) is an integral constituentof the placental and decidual ECM. Because this proteoglycanspecifically interacts with various macromolecules in the ECM,its degradation may disassemble the matrix. Hence, in the caseof the placenta, this may facilitate normal placentation andtrophoblast invasion. Crude placental specimens were collectedfrom first and third trimester placentas. Heparanase (endo-P-glucuronidase)was isolated and purified by ammonium sulphate precipitationfollowed by sequential chromatographies on carboxymethyl-, heparin-and ConA-Sepharose columns. The placental enzyme was furthercharacterized for its molecular weight and specific inhibitionby heparin, and was shown to resemble heparanase expressed byhighly metastatic tumour cells and activated cells of the immunesystem. In order to locate the source of heparanase activityin the placenta, primary cytotrophoblast cultures were established.Intact cells, as well as conditioned medium and cell lysates,were analysed for heparanase activity using metabolically sulphate-labelledECM as a natural substrate. Heparanase was highly active inlysates of cytotrophoblasts. This activity was also expressedby intact cytotrophoblasts seeded on ECM, but no activity couldbe detected in the culture medium. Incubation of the cytotrophoblastsin contact with ECM resulted in release of ECM-bound basic fibroblastgrowth factor (bFGF). We propose that the cytotrophoblasticheparanase facilitates placentation, through cytotrophoblastextravasation and localized neovascularization. cytotrophoblast/extracellular matrix/heparanase/heparan sulphate proteoglycan/placenta     

A protein kinase A-dependent molecular switch in synapsins regulates neurite outgrowth

Cyclic AMP (cAMP) promotes neurite outgrowth in a variety of neuronal cell lines through the activation of protein kinase A (PKA). We show here, using both Xenopus laevis embryonic neuronal culture and intact X. laevis embryos, that the nerve growth-promoting action of cAMP/PKA is mediated in part by the phosphorylation of synapsins at a single amino acid residue. Expression of a mutated form of synapsin that prevents phosphorylation at this site, or introduction of phospho-specific antibodies directed against this site, decreased basal and dibutyryl cAMP-stimulated neurite outgrowth. Expression of a mutation mimicking constitutive phosphorylation at this site increased neurite outgrowth, both under basal conditions and in the presence of a PKA inhibitor. These results provide a potential molecular approach for stimulating neuron regeneration, after injury and in neurodegenerative diseases.     

Loss of heterozygosity and mutational analyses of the ACTRII gene locus in human colorectal tumors

The activin type II receptorgene (ACTRII) is mutated in 58.1% of microsatellite-unstable (MSI-H) colorectal cancers and is a close relative of the TGFbeta-1 type II receptor, which is known to be involved in both MSI-H and non-MSI-H colorectal carcinogenesis. We therefore sought to determine whether ACTRII was involved in non-MSI-H colorectal cancers. We evaluated ACTRII inactivation by allelic deletion, loss of mRNA expression, or somatic mutation in 51 non-MSI-H colon cancers. Loss of heterozygosity (LOH) at the ACTRII locus (2q23.1) was found in 9 (17.6%) of 51 primary tumors. Loss of ACTRII mRNA expression was seen in one (14.3%) of the seven LOH-positive primary tumors from which total RNA was available. We also performed DNA sequencing analysis of tumors showing LOH. One LOH-positive primary tumor exhibited a novel germline missense sequence alteration (amino acid substitution, 117 Ile to Phe) that was not found in 23 additional normal individuals, implying that this alteration is not a frequent polymorphism. We conclude that ACTRII is probably involved in both non-MSI-H and MSI-H colorectal carcinogenesis, but more frequently in the latter subgroup.     

Increased levels of endothelin-1 and differential endothelin type A and B receptor expression in scleroderma-associated fibrotic lung disease.

In addition to their vasoactive action, endothelins are potent peptides in the regulation of both cell proliferation and the turnover of extracellular matrix. Using immunohistochemical, autoradiographic, and molecular analyses, we have studied the localization and expression of endothelin-1 and endothelin A (ETA) and B (ETB) receptors in scleroderma-associated fibrotic lung disease. Increased ET-1 immunoreactivity was found in sclerotic tissue compared with control and was associated with the vasculature, pulmonary interstitium, and bronchial and alveolar epithelium. Microautoradiographic analysis after 125I-labeled ET-1 binding showed a two- to threefold increase in the expression of total ET-1 receptors in scleroderma lung tissue localized to the alveolar epithelium and the pulmonary interstitium which was composed of mainly fibroblastic cells with macrophages and some microvessels. RNAse protection assay revealed significantly reduced ETA receptor and slightly raised ETB message levels in systemic sclerosis lung. Surface expression of functional ET receptors was examined by targeted receptor blocking using mixed and receptor-subtype-selective ligands. A consistent decrease in ETA receptor binding sites was noted primarily within the interstitium and vasculature, in contrast to a slight increase in ETB receptors. Elevated ET-1 and the cell-specific pattern of endothelin receptor expression suggest that the endothelins may represent important mediators that influence the pathology of scleroderma-associated lung disease and other fibrotic conditions.     

Sequential Measurements of Human Decidua-Associated Protein (hDP) 200 in the Uterine Fluid During the Menstrual Cycle

PROBLEM: To examine the relationship between the concentration of uterine fluid human decidua-associated protein (hDP) 200, identified as a monoclonal rheumatoid factor, and different phases of the menstrual cycle. METHODS: Sequential measurements of hDP 200 concentration in uterine fluid were performed in 11 normal ovulatory women, aged 22–36 years. The samples were collected in early proliferative phase, late proliferative phase, periovulatory period, early secretory phase, and late secretory phase. RESULTS: Consistent fluctuations of hDP 200 levels in uterine fluid were found throughout the menstrual cycle. High levels were found during early proliferative phase and periovulatory period related to significantly lower levels during late proliferative and early luteal phases. CONCLUSION: There is menstrual phase dependent variation in the uterine fluid levels of hDP 200.     

The PapG tip adhesin of P fimbriae protects Escherichia coli from neutrophil bactericidal activity.

Compared with Escherichia coli ORN103, a nonfimbriated K-12 strain, P-fimbriated E. coli ORN103/pPAP5 was found to interact poorly with human neutrophils and resist their bactericidal activity in vitro. PapG, the Gal alpha(1-->4)Gal binding moiety located at the distal end of the P fimbrial filament, appeared to be responsible for this effect because an isogenic PapG- mutant, E. coli ORN103/pPAP24, exhibited binding interactions with neutrophils that were similar to nonfimbriated E. coli ORN103. Although no direct evidence is available, the poor adherence mediated by PapG could be related to its electrostatic properties because the isolated PapG protein had a pI of 5.2, which indicated that in the physiological pH range it possessed a net negative charge. Antibodies against PapG overcame the protective effect of PapG and markedly enhanced the interactions of P-fimbriated E. coli with neutrophils resulting in bacterial killing. When a P-fimbriated clinical E. coli strain or its isogenic PapG- derivative was injected into the peritoneal cavities of mice, a similar number of neutrophils was recruited to the site of injection. After 2 h, the number of P-fimbriated E. coli organisms that survived the neutrophil influx in the mouse peritoneum was approximately four times more than the number of surviving PapG- bacteria. This result demonstrates that the PapG protein, which is strategically located at the distal region of the P-fibrillum structure, protects E. coli from the bactericidal action of neutrophils.     

Modeling pandemic preparedness scenarios: health economic implications of enhanced pandemic vaccine supply

Influenza pandemic planning is a complex, multifactorial process, which involves public health authorities, regulatory authorities, academia and industry. It is further complicated by the unpredictability of the time of emergence and severity of the next pandemic and the effectiveness of influenza epidemic interventions. The complexity and uncertainties surrounding pandemic preparedness have so far kept the various stakeholders from joining forces and tackling the problem from its roots. We developed a mathematical model, which shows the tangible consequences of conceptual plans by linking possible pandemic scenarios to health economic outcomes of possible intervention strategies. This model helps to structure the discussion on pandemic preparedness and facilitates the translation of pandemic planning concepts to concrete plans. The case study for which the model has been used shows the current level of global pandemic preparedness in an assumed pandemic scenario, the health economic implications of enhanced pandemic vaccine supply and the importance of cell culture-based influenza vaccine manufacturing technologies as a tool for pandemic control.