The heparan sulfate on the surface of all adherent cells modulates the actions of a large number of extracellular ligands. Members of both cell surface heparan sulfate proteoglycan families, the transmembrane syndecans and the glycosylphosphoinositide-linked glypicans, bind these ligands and enhance formation of their receptor-signaling complexes. These heparan sulfate proteoglycans also immobilize and regulate the turnover of ligands that act at the cell surface. The extracellular domains of these proteoglycans can be shed from the cell surface, generating soluble heparan sulfate proteoglycans that can inhibit interactions at the cell surface. Recent analyses of genetic defects in Drosophila melanogaster, mice, and humans confirm most of these activities in vivo and identify additional processes that involve cell surface heparan sulfate proteoglycans. This chapter focuses on the mechanisms underlying these activities and on the cellular functions that they regulate.

Functions of Cell Surface Heparan Sulfate Proteoglycans

Exosomes are lipid-bilayer-enclosed extracellular vesicles that contain proteins and nucleic acids. They are secreted by all cells and circulate in the blood. Specific detection and isolation of cancer-cell-derived exosomes in the circulation is currently lacking. Using mass spectrometry analyses, we identify a cell surface proteoglycan, glypican-1 (GPC1), specifically enriched on cancer-cell-derived exosomes. GPC1(+) circulating exosomes (crExos) were monitored and isolated using flow cytometry from the serum of patients and mice with cancer. GPC1(+) crExos were detected in the serum of patients with pancreatic cancer with absolute specificity and sensitivity, distinguishing healthy subjects and patients with a benign pancreatic disease from patients with early- and late-stage pancreatic cancer. Levels of GPC1(+) crExos correlate with tumour burden and the survival of pre- and post-surgical patients. GPC1(+) crExos from patients and from mice with spontaneous pancreatic tumours carry specific KRAS mutations, and reliably detect pancreatic intraepithelial lesions in mice despite negative signals by magnetic resonance imaging. GPC1(+) crExos may serve as a potential non-invasive diagnostic and screening tool to detect early stages of pancreatic cancer to facilitate possible curative surgical therapy.

/pdf/glypican-1-identifies-cancer-exosomes-and-detects-early-4lejrakyfc.pdf

Glypican-1 identifies cancer exosomes and detects early pancreatic cancer

Proteins and peptides can enter cells by receptor-mediated endocytosis, a coupled process by which selected extracellular proteins or peptides are first bound to specific cell surface receptors and then rapidly internalised by the cell. Internalisation follows clustering of the receptors in specialised regions of the cell surface called coated pits that invaginate to form intracellular coated vesicles. It is now recognised that receptor-mediated endocytosis has a fundamental role in the growth, nutrition and differentiation of animal cells.

Coated pits, coated vesicles, and receptor-mediated endocytosis

Many neurodegenerative diseases, including Alzheimer's and Parkinson's and the transmissible spongiform encephalopathies (prion diseases), are characterized at autopsy by neuronal loss and protein aggregates that are typically fibrillar. A convergence of evidence strongly suggests that protein aggregation is neurotoxic and not a product of cell death. However, the identity of the neurotoxic aggregate and the mechanism by which it disables and eventually kills a neuron are unknown. Both biophysical studies aimed at elucidating the precise mechanism of in vitro aggregation and animal modeling studies support the emerging notion that an ordered prefibrillar oligomer, or protofibril, may be responsible for cell death and that the fibrillar form that is typically observed at autopsy may actually be neuroprotective. A subpopulation of protofibrils may function as pathogenic amyloid pores. An analogous mechanism may explain the neurotoxicity of the prion protein; recent data demonstrates that the disease-associated, infectious form of the prion protein differs from the neurotoxic species. This review focuses on recent experimental studies aimed at identification and characterization of the neurotoxic protein aggregates.

Protofibrils, pores, fibrils, and neurodegeneration: separating the responsible protein aggregates from the innocent bystanders.

Advanced Statistical Methods in Biometric Research.

The cellular distribution of a highly antigenic fibroblast glycoprotein, identified previously as aminopeptidase M, was studied in vitro by immunofluorescence and immuno-electron microscopy. The antigen was found to be localized at the surface of live and fixed fibroblasts in suspension and in cell layer; clustering or patching on live cells could be observed. Immunofluorescence after permeabilization of the cells with acetone showed distribution of the antigen in cytoplasmic granules. The tissue distribution of this antigen was examined by immunofluorescence on frozen sections of various human organs. In addition to fibroblasts, renal tubules, liver, pancreas and gut were found to react selectively with the antibody preparation. Moreover, a specific localization of the reaction product was observed. In the kidney the epithelium and brush border of the proximal convoluted tubules were stained. In the liver, the bile canaliculi reacted; in the pancreas the acinar cells and in the gut the brush border of the mucosal cells of the villi and crypts were stained. The same cells did not stain with an antiserum prepared in a similar fashion against another fibroblast component, or with preimmune IgG. In most sections of the selectively stained tissues, a predominant localization at the apical pole of the cells was observed. The localization of the antigen in different tissues corresponds with the distribution of aminopeptidase M, thereby confirming its identity and the antigenic cross-reaction between the fibroblast and tissue enzyme.

Localization of a highly antigenic human fibroblast surface glycoprotein (FSG) on fibroblasts and on epithelia involved in secretion or resorption

Application of crossed immunoelectrophoresis to the visualization and characterization of human fibroblast antigens.

phi1 count (ANC) between 300 and 500 x 10y/l. However. signs of bone marrow failure recurred with a decline in haemoglobin, platelets and granulocytes to 10.8 g/dl, 2 3 x 10y/l and 0 . 3 x 10y/l, respectively. Cyclosporin A (CSA) was initiated at a dose of 10 mg/kg/d orally. CSA was adjusted according to serum creatinine. bilirubin and plasma CSA levels. Haematological response was observed 6 weeks following administration of CSA. and she maintained adequate blood counts (Hb 14-16 g/dl, plt 80-100 000 x 1OY/l. ANC > 500 x 1 Oy/l) for a total of 18 months. However, her renal status became seriously compromised. CSA was tapered to a lower dose to prevent renal toxicity. With this lower dose, her haematological response deteriorated (plt < 2 0 0 0 0 x 1OY/1, ANC< 500 x lOY/ l , H b < 9 g/dl). A bone marow transplant was performed using her 18-yearold human lymphocyte antigen (HLA) identical and mixed lymphocyte culture (MLC) compatible brother. The cytoreductive regimen consisted of total lymphoid irradiation (TLI) and cyclophosphamide. TLI consisted of three fractions of 1 2 0 rads. administered at 4 h intervals, in 1 d (total 360 rads). It was administered through mantel and inverted Y fields (including the spleen) using a linear accelerator at a dose rate of 100-1 30 rads/min (Day 6). This was followed by 4 d of i.v. cyclophosphamide (40 mg/kg/d) (Days 5, -4. 3 and 2). Bone marrow was harvested from the donor on Day 0. The patient received 3.8 x 10\" nucleated cells/kg. For prevention of graft-versus-host disease (GVHD) the patient was given methotrexate ( 1 5 mg/m2 i.v.) on Day 1 , 10 m g / d on Days 3. 7. 1 1 . and CSA ( 1 5 mg/kg/day i.v.) on Day 1 . Both medications were adjusted according to serum creatinine. bilirubin and CSA levels. During cytoreductive treatment with cyclophosphamide the patient developed severe watery diarrhoea. She was placed on (i.v.) octreotideacetate (Sandostatin) a t a dose of 50 pg t.i.d.. with good response. Diarrhoea gradually resolved by Day 7. On Day 1 1, the patient became febrile to 38.5OC and was placed on broad-spectrum antibiotics. She subsequently became afebrile within 4 8 h, and antibiotics were discontinued soon thereafter. All cultures were negative. On Day 1 0 she developed acute GVHD manifested by a generalized maculopapular rash and confirmed histologically. Accordingly. she was placed on methylprednisolone. Her skin rash subsequently resolved and she was taken off steroid therapy on Day 1 5 . During steroid therapy. the patient required an increase in her insulin dose. This was resolved following discontinuation of steroids. The patient's peripheral white count began to rise by Day 14 and a n absolute neutrophil count of 1 .O x 1 0 y / I was attained by Day 2 7. Heparinized samples of the patient's bone marrow and peripheral blood obtained on Day 2 7 revealed the presence of male (donor) karyotype with identical results on Day 100. Her last platelet transfusion was administered on Day 29 and her last red cell transfusion was given on Day 3 0 . Cyclosporin was discontinued on Day 100. As with patients with Fanconi anaemia, in SDS. the defect appears to involve the pluripotent stem cell and BMT offers an opportunity for cure provided that there is an HLA identical donor. Nine months following BMT the patient is without evidence of chronic GVHD. marrow rejection. or interstitial pulmonary disease. These results differ from previous BMT reports in SDS (Tsai et al. 1990). Their patient expired secondary to congestive heart failure, attributed to a similar dose of cyclophosphamide (48.6 mg/kg/d). We conclude that BMT offers a potential cure as a means of reconstituting haematopoiesis in patients with SDS and bone marrow aplasia. Cyclophosphamide and TLI should be considered as pre-BMT conditioning regimen in patients with SDS.

Alteration of N-ras Mutation in a Patient With Aml M4 and Trilineage Myelodysplasia

The statistical problems in evaluating the results of sister chromatid exchange (SCE) scores can be solved by fitting log-linear models to the number of SCE scores (2). In each single test all measurements from all chromosome groups, both from the control and from the experimental sets are utilized. Previously, we analyzed for each chromosome group the number of metaphases with specified total SCE score for the group. We have refined this method to an analysis of the total number of chromosomes with specified SCE score over all metaphases. The results show that the refined method uses more information and can lead to conclusions which are at variance with the conclusions of the first method. By proceeding in this way, better use is made of all available information on the SCE scores and the shortcomings of the classical Student's t-test and chi-square (X2) tests are avoided.

Jj. Cassiman

Papers

Localization of a highly antigenic human fibroblast surface glycoprotein (FSG) on fibroblasts and on epithelia involved in secretion or resorption

Application of crossed immunoelectrophoresis to the visualization and characterization of human fibroblast antigens.

Alteration of N-ras Mutation in a Patient With Aml M4 and Trilineage Myelodysplasia

Statistical evaluation of sister chromatid exchanges: refined method.

Genetic analysis of the 12Q candidate region for bipolar disorder