Maternal serum ADAM12 levels in Down and Edwards' syndrome pregnancies at 9-12 weeks' gestation

BACKGROUND: Maternal serum ADAM12 is reduced, on average, in early first-trimester Down and Edwards' syndrome pregnancies but the extent of reduction declines with gestation. Here we study levels at 9-12 weeks when the marker might be used concurrently with other established markers. METHODS: Samples from 16 Down and 2 Edwards' syndrome cases were retrieved from storage and tested together with 313 unaffected singleton pregnancies using a semi-automated time-resolved immuno-fluorometric assay. Results were expressed in multiples of the gestation-specific median (MoM) based on regression. RESULTS: The median in Down syndrome was 0.94 MoM with a 10th-90th centile range of 0.22-1.63 MoM compared with 1.00 and 0.33-2.24 MoM in unaffected controls (P = 0.21, one-side Wilcoxon Rank Sum Test). The two Edwards' syndrome cases had values 0.31 and 2.17 MoM. CONCLUSIONS: ADAM12 cannot be used concurrently with other markers in the late first trimester. However, it does have the potential to be used earlier in pregnancy either concurrently with other early markers or in a sequential or contingent protocol. More data will be required to reliably predict the performance of either approach.     

Improving outcome of malignant brain tumours in very young children: a population-based study in Finland during 1975-93

Abstract Sixty-four children with malignant brain tumours diagnosed at less than 3 years of age were reported to the Finnish Cancer Registry from 1975 to 1993. The survival rate has improved significantly: the 5-year survival rate was 26% for all children, 13% for children diagnosed during 1975-85 ( n = 30) and 40% for those diagnosed during 1986-93 ( n = 4). Of the surviving children in 1986-93, 43% were categorized in Bloom's group I or II and could lead active lives without major disabilities. The remaining children had severe neurologic late complications, such as hemiplegia, intractable seizures, and mental retardation.     

CD63 is a component of Weibel-Palade bodies of human endothelial cells

Weibel-Palade bodies are secretory granules of vascular endothelial cells specialized in the storage of von Willebrand factor (vWF) and P- selectin, two adhesion proteins that can be rapidly mobilized to the cell surface by exocytosis in response to thrombin or other agonists. In this study, we attempted to identify additional components of Weibel- Palade bodies by raising monoclonal antibodies to these granules, purified by cell fractionation. One antibody, 2C6, was found to be specific for CD63, a membrane glycoprotein previously described in the lysosomes of platelets and other cell types. The immunopurified 2C6 antigen was recognized by an anti-CD63 reference antibody, 2.28, by Western blotting. Also, the biosynthetic profile of the 2C6 antigen in endothelial cells showed a nascent molecular mass and a glycosylation pattern identical to that of CD63. Immunofluorescence staining with 2C6 showed the lysosomes, and also elongated structures identified as Weibel-Palade bodies by their shape, distribution, and positive staining with anti-vWF antibodies, CD63 was also found by Western blotting of subcellular fractions highly enriched in Weibel-Palade bodies. Our results indicate that CD63 colocalizes with vWF and P- selectin in the Weibel-Palade bodies of endothelial cells, and together with these adhesion proteins it could be rapidly expressed on the cell surface in areas of vascular injury and inflammation.     

ADAM12 alleviates the skeletal muscle pathology in mdx dystrophic mice

Muscular dystrophy is characterized by muscle degeneration and insufficient regeneration and replacement of muscle fibers by connective tissue. New therapeutic strategies directed toward various forms of muscular dystrophy are needed to preserve muscle mass and promote regeneration. In this study we examined the role of the transmembrane ADAM12, a disintegrin and metalloprotease, which is normally associated with development and regeneration of skeletal muscle. We demonstrate that ADAM12 overexpression in the dystrophin-deficient mdx mice alleviated the muscle pathology in these animals, as evidenced by less muscle cell necrosis and inflammation, lower levels of serum creatine kinase, and less uptake of Evans Blue dye into muscle fibers. These studies demonstrate that ADAM12 directly or indirectly contributes to muscle cell regeneration, stability, and survival.     

De novo deposition of laminin-positive basement membrane in vitro by normal hepatocytes and during hepatocarcinogenesis

De novo formation of laminin-positive basement membranes was found to be a distinct morphologic feature of diethylnitrosamine/phenobarbital-induced hepatocellular carcinomas of the rat. The first appearance of extracellularly located laminin occurred in the preneoplastic liver lesions (corresponding to neoplastic nodules), and this feature became successively more prominent during the course of hepatocellular carcinoma development. Most groups of tumor cells were surrounded by laminin-positive basement membrane material. The laminin-positive material was also deposited along the sinusoids, a location where no laminin was seen in normal rat liver. The amount of extractable laminin from hepatocellular carcinomas was significantly higher (approximately 100 ng per mg tissue) than that of normal liver tissue (less than 20 ng per mg). In vitro experiments demonstrated that normal and preneoplastic rat hepatocytes had the capacity to lay down basement membrane-like material. This occurred, however, only when the hepatocytes were cocultured with certain feeder cells or when grown in the presence of their conditioned media. These results indicate that during experimental hepatocarcinogenesis in the rat some as yet undefined humoral factor(s) might influence the hepatocytes to turn on genes encoding the basement membrane components and further stimulate the assembly and deposition of basement membranes.     

Regulation of platelet production and function by megakaryocyte growth and development factor in nonhuman primates

The primary physiologic regulator of platelet production, Mpl ligand, has recently been cloned and characterized. To define the regulatory role of Mpl ligand on platelet production and function we measured the effects of a recombinant truncated human Mpl ligand, megakaryocyte growth and development factor (rHu-MGDF) on megakaryocytopoiesis, platelet function, and thrombogenesis in nonhuman primates. rHu-MGDF was administered to 10 baboons for 28 days while performing pharmacokinetics and repeated measurements of the following: (1) platelet count, volume, turnover, and function ex vivo and in vitro; (2) marrow megakaryocyte number, volume, and ploidy; and (3) platelet deposition and fibrin accumulation on segments of vascular graft and endarterectomized aorta in vivo. Daily subcutaneous injections of rHu- MGDF (5 microgram/kg/d) attained plasma concentrations averaging 1,300 +/- 300 pg/mL 2 hours after injection with trough levels of 300 +/- 65 pg/mL before the next dose. These levels of rHu-MGDF incrementally increased the peripheral platelet concentration threefold by day 7 and fivefold by day 28 (P < 10(-4)) associated with a reciprocal decrease of 25% in mean platelet volumes (P < 10(-3)). Platelet mass turnover, a steady-state measure of platelet production, increased fivefold (P < 10(-4)). Platelet morphology, life span, and recovery were normal. No significant change occurred in peripheral leukocyte, neutrophil, or erythrocyte counts (P > .1 in all cases). The platelet count gradually returned to baseline within 2 weeks after discontinuing rHu-MGDF infections. Marrow megakaryocyte volume doubled (P < 10(-3)) three days after initiating rHu-MGDF therapy and the modal ploidy shifted from 16N to 64N (P < 10(-4)). Marrow megakaryocyte number increased twofold by day 7, and nearly fourfold by day 28 (P < 10(-4)), resulting in a 6.5- fold increase in marrow megakaryocyte mass (P < 10(-3)). The effects of rHu-MGDF on thrombosis were determined by comparing baseline, day 5, and day 28 rHu-MGDF-treatment measurements of 111In-platelet deposition and 125I-fibrin accumulation on segments of homologous endarterectomized aorta (EA) and vascular graft (VG) interposed in arteriovenous femoral shunts. rHu-MGDF increased 111In-platelet deposition in direct proportion to the circulating concentration of platelets for both EA and VG (r=.98 in both cases), without significant changes in fibrin accumulation (P > .5 in both cases). During the first week of rHu-MGDF treatment ex vivo platelet aggregatory responsiveness was enhanced to physiologic agonists (adenosine diphosphate, collagen, and thrombin receptor agonist peptide, TRAP1-6) (P < .05 in all cases). Although in vitro platelet aggregation was not induced by any concentration of rHu-MGDF tested (P > .5), rHu-MGDF enhanced aggregatory responses to low doses of physiologic agonists, effects that were maximal at 10 ng/mL for baboon platelets and 100 ng/mL for human platelets, and were blocked by excess soluble c-Mpl receptor. Flow cytometric expression of platelet activation epitopes was not increased on resting platelets (ligand-induced binding sites, P- selectin, or Annexin V binding sites; P > .1 in all cases). Megakaryocyte growth and development factor regulates platelet production and function by stimulating endoreduplication and megakaryocyte formation from marrow progenitor cells, and transiently enhancing platelet functional responses ex vivo. rHu-MGDF has the potential for achieving platelet hemostatic protection with minimal thrombo-occlusive risk.     

Demonstration of single chain urokinase-type plasminogen activator on human platelet membrane

Fibrinolytic activity was found to be associated with sonicated platelet membranes after separation from cytosol by differential centrifugation. This fibrinolytic activity was attributed to the presence of a plasminogen activator, which was immunochemically identified as urinary-type plasminogen activator (uPA) by antibody neutralization assay, immunoblotting, and immunofluorescence. The molecular weight (mol wt) of this uPA was 54,000 and was present as the single chain form, although a small amount was detected in a higher mol wt complex indicative of a uPA-inhibitor complex. Treatment of membrane preparations with Triton X-100, 3 mol/L KCl, and 0.1 mol/L glycine, (pH 2.3), but not 10 mmol/L ethylenediamine tetraacetic acid (EDTA), removed the uPA from the membrane. This suggests that uPA is a peripheral membrane protein and that metal ions do not mediate protein- membrane association. Immunofluorescent staining revealed the presence of uPA on the outer surface of the platelet in preparations of intact unstimulated platelets. Thus, uPA is associated with the outer leaflet of the platelet membrane and may be involved with the acceleration of thrombus degradation observed with platelet-rich thrombi.     

Distinct signaling by insulin and IGF-1 receptors and their extra- and intracellular domains

Insulin and insulin-like growth factor 1 (IGF-1) receptors share many downstream signaling pathways but have unique biological effects. To define the molecular signals contributing to these distinct activities, we performed global phosphoproteomics on cells expressing either insulin receptor (IR), IGF-1 receptor (IGF1R), or chimeric IR-IGF1R receptors. We show that IR preferentially stimulates phosphorylations associated with mammalian target of rapamycin complex 1 (mTORC1) and Akt pathways, whereas IGF1R preferentially stimulates phosphorylations on proteins associated with the Ras homolog family of guanosine triphosphate hydrolases (Rho GTPases), and cell cycle progression. There were also major differences in the phosphoproteome between cells expressing IR versus IGF1R in the unstimulated state, including phosphorylation of proteins involved in membrane trafficking, chromatin remodeling, and cell cycle. In cells expressing chimeric IR-IGF1R receptors, these differences in signaling could be mapped to contributions of both the extra- and intracellular domains of these receptors. Thus, despite their high homology, IR and IGF1R preferentially regulate distinct networks of phosphorylation in both the basal and stimulated states, allowing for the unique effects of these hormones on organismal function.

Insulin and insulin-like growth factor 1 (IGF-1) act through their cognate receptors to regulate a wide variety of biological processes. The insulin and IGF-1 receptors (IR and IGF1R) are highly homologous and share many overlapping signaling pathways (1). Activation of both receptors results in stimulation of two major canonical pathways of signaling: the PI 3-kinase/Akt pathway, which is linked to most metabolic actions of these hormones, and the Ras/MAP kinase pathway, which is linked to regulation of cell and organismal growth and differentiation (2–5). Perturbations in these pathways can lead to insulin resistance, as manifested by glucose intolerance, dyslipidemia, and increased risk of cardiovascular disease or alterations in growth at both the cellular and organismal level (1, 3). Due to the high degree of homology between these receptors and shared downstream signaling, defining differential molecular signatures has been challenging. This is further complicated by the fact that although insulin and IGF-1 preferentially bind to their own receptors, both ligands can also bind to the alternate receptor with lower affinity (6, 7). Many studies have tried to identify the differences between IR and IGF1R which account for their differences in vivo (2, 8). Some of the differential effects between insulin and IGF-1 have been ascribed to differences in the relative concentration of the hormones, the presence of IGF-1 binding proteins, the relative expression level of receptors in different tissues and the affinity of the two ligands for the two different receptors (1, 9).Some insight has come from the use of chimeric receptors. For example, activation of chimeric receptors containing the extracellular domains (ECDs) of the neurotrophin receptor (TrkC) fused to the intracellular domains (ICDs) of the insulin or IGF-1 receptors have suggested that the IGF1R intracellular domain (IGF1R-ICD) is more important for mitogenic response, while the IR-ICD is more strongly related to metabolic effects (10, 11). Similar conclusions have been reached using chemical and antibody-based inhibitors of these receptors (12). More recently, using chimeric receptors containing the ECD of IR fused to the ICD of IGF1R and vice versa, we showed that both the intracellular and extracellular domains of IR and IGF1R contribute to their effects on gene expression and that this is in part due to differences in their intracellular juxtamembrane regions and ability to engage IRS proteins versus Shc as substrates of the receptor (2).In addition to these ligand-stimulated and tyrosine-kinase-dependent events, previous studies from our laboratory have demonstrated a class of ligand and tyrosine-kinase-independent effects mediated by the unoccupied IR and IGF1R. Thus, cells lacking both IR and IGF1R are resistant to apoptosis (13) and show altered expression of a network of imprinted genes and miRNAs associated with changes in DNA methylation (14). These findings suggest that these receptors have receptor-dependent but ligand-independent signaling events, but how these are mediated is unknown.Intracellular signal transduction by membrane receptors such as IR and IGF1R is primarily mediated by the reversible phosphorylation of networks of signaling molecules. While IR and IGF1R are tyrosine kinases with a limited number of primary substrates, these in turn activate downstream kinases that lead to much broader networks of phosphorylation on serine and threonine residues. The resultant phosphorylated proteins may have altered activity levels, tertiary structure, and/or subcellular localization. To define the molecular differences in signaling between IR and IGF1R that create their unique physiological actions and to determine to what extent these differences are contributed by the ECDs vs. ICDs of these receptors, we performed global phosphoproteomic analysis of preadipocytes in which both endogenous receptors had been genetically inactivated to create double knock out (DKO) cells after which the cells were reconstituted with IR, IGF1R, and chimeric receptors consisting of the ECD of IR and the ICD of IGF1R (named hereafter as IR_IGF1R) and vice versa (i.e., IGF1R_IR) and studied both with and without ligand stimulation to understand signaling by the unoccupied receptor. We show that despite many similarities in signaling, there are major differences in the phosphoproteome mediated by activated IR versus IGF1R which contribute to their specific patterns of action. In addition, there are important and previously unrecognized differences in the basal phosphoproteome in cells expressing IR versus IGF1R. Using chimeric receptors we show that these differences in both the basal and stimulated states are contributed to by both the ICD and ECD of these receptors. Together, these unique signaling networks lead to the important differential effects of insulin and IGF-1 at the postreceptor level.