An ultrasensitive time-resolved immunofluorometric assay of human epidermal growth factor

We have developed a sandwich-type time-resolved immunofluorometric assay (TR-IFMA) for human epidermal growth factor (hEGF) in body fluids. A two-step solid-phase technique was used. The assay utilizes a polyclonal anti-hEGF attached to the solid phase, and a monoclonal anti-hEGF labeled with Europium (III) as a tracer. The sensitivity of the assay (2.5 pg/ml) is at least 20 times better than what has been achieved by radioimmunoassay (RIA), and the measuring range is much wider: 2.5-5000 pg/ml. The feasibility of TR-IFMA was tested by assaying urine containing large amounts and amniotic fluid containing small amounts (mostly undetectable by RIA) of immunoreactive hEGF. The correlation between urine hEGF concentrations (1-100 ng/ml) measured by RIA and TR-IFMA was good: r = 0.96.     

Serum complex of trypsin 2 and alpha 1 antitrypsin as diagnostic and prognostic marker of acute pancreatitis: clinical study in consecutive patients.

OBJECTIVE--To estimate the usefulness of serum concentrations of the complex of trypsin 2 and alpha 1 antitrypsin in diagnosing and assessing the severity of acute pancreatitis in comparison with serum C reactive protein, amylase, and trypsinogen 2 concentrations (reference markers). DESIGN--Markers were measured in consecutive patients admitted with acute abdominal pain that was either due to pancreatitis or to other disease unrelated to the pancreas (controls). SETTING--Department of surgery of a teaching hospital in Helsinki. SUBJECTS--110 patients with acute pancreatitis and 66 with acute abdominal diseases of extrapancreatic origin. On the basis of the clinical course, acute pancreatitis was classified as mild (82 patients) or severe (28 patients). MAIN OUTCOME MEASURES--Clinical diagnosis of acute pancreatitis and severity of the disease. RESULTS--At admission all patients with acute pancreatitis had clearly raised concentrations of trypsin 2-alpha 1 antitrypsin complex (32 micrograms/l), whereas only three of the controls had such values. Of the markers studied, trypsin 2-alpha 1 antitrypsin complex had the largest area under the receiver operating curve, both in differentiating acute pancreatitis from extrapancreatic disease and in differentiating mild from severe disease. CONCLUSIONS--Of the markers studied, trypsin 2-alpha 1 antitrypsin complex was the most accurate in differentiating between acute pancreatitis and extrapancreatic disease and in predicting a severe course for acute pancreatitis.     

A novel protease homolog differentially expressed in breast and ovarian cancer.

BACKGROUND: Using differential display (DD), we discovered a new member of the serine protease family of protein-cleaving enzymes, named protease M. The gene is most closely related by sequence to the kallikreins, to prostate-specific antigen (PSA), and to trypsin. The diagnostic use of PSA in prostate cancer suggested that a related molecule might be a predictor for breast or ovarian cancer. This, in turn, led to studies designed to characterize the protein and to screen for its expression in cancer. MATERIALS AND METHODS: The isolation of protease M by DD, the cloning and sequencing of the cDNA, and the comparison of the predicted protein structure with related proteins are described, as are methods to produce recombinant proteins and polyclonal antibody preparations. Protease M expression was examined in mammary, prostate, and ovarian cancer, as well as normal, cells and tissues. Stable transfectants expressing the protease M gene were produced in mammary carcinoma cells. RESULTS: Protease M was localized by fluorescent in situ hybridization analysis to chromosome 19q13.3, in a region to which other kallikreins and PSA also map. The gene is expressed in the primary mammary carcinoma lines tested but not in the corresponding cell lines of metastatic origin. It is strongly expressed in ovarian cancer tissues and cell lines. The enzyme activity could not be established, because of difficulties in producing sufficient recombinant protein, a common problem with proteases. Transfectants were selected that overexpress the mRNA, but the protein levels remained very low. CONCLUSIONS: Protease M expression (mRNA) may be a useful marker in the detection of primary mammary carcinomas, as well as primary ovarian cancers. Other medical applications are also likely, based on sequence relatedness to trypsin and PSA.     

Immunochemical characterization of human plasma fibronectin.

Human plasma fibronectin has been purified by a non-denaturing affinity chromatography procedure [Vuento & Vaheri, (1979) Biochem.J. 183, 331--337], and antisera have been raised by immunizing rabbits with the native protein. The antisera reacted strongly with native fibronectin, but only weakly with reduced and alkylated fibronectin or with heat-denaturated fibronectin. Denaturation also affected the haemagglutinating and gelatin-binding activities of fibronectin and increased its susceptibility to proteolytic degradation. The antisera reacted with fragments of fibronectin obtained by proteolysis with plasmin. Large fragments (mol.wt. 180000--200000), lacking the region harbouring the interchain disulphide bridges but containing the sites responsible for gelatin-binding and haemagglutinating activity, showed as intense a reaction with the antisera as intact fibronectin. Smaller peptides showed a weaker reaction. All fragments tested showed sensitivity to denaturation in their reaction with the antisera. The results were interpreted as showing that: (1) native fibronectin has an ordered conformation that is easily perturbed by denaturation; (2) most of the antigenic determinants of the protein are dependent on conformation; (3) the region of the fibronectin molecule containing the interchain disulphide bridges has only few antigenic determinants; and (4) covalent interaction of the two subunits does not contribute to the antigenic structure recognized by rabbit antisera. The observed correlation between the antigenic activity and a structural and functional intactness of fibronectin suggests that the antibodies to native fibronectin could be used as a conformational probe in studies on this protein.     

Fibronectin expression is determined by the genotype of the transformed parental cells in heterokaryons between normal and transformed fibroblasts

The expression of fibronectin, a cell surface-associated transformation-sensitive glycoprotein, was studied in hetero- and homokaryons of normal and SV40-transformed human fibroblasts. In immunofluorescence, fibroblast homokaryons had an intense surface-associated and intracelluar fibronectin fluorescence similar to that of normal fibroblasts. Transformed cells and their homokaryons had a minimal surface-associated and a weak intracellular fibronectin fluorescence. In heterokaryons formed between transformed and normal fibroblasts, the expression of fibronectin fell within 24 h to the level of the transformed cell homokaryons. The change was detectable already at 3 h after fusion and was gene-dose dependent. These results show that the transformed genotype determines fibronectin expression in the heterokaryons.     

The human islet amyloid polypeptide (IAPP) gene. Organization, chromosomal localization and functional identification of a promoter region

We report the isolation and characterization of the human gene encoding islet amyloid polypeptide (IAPP). Previously characterized cDNA sequences correspond to three exons of which the first is noncoding. A functional promoter region was identified in the 5' flanking DNA; however, this was farther upstream than expected. Northern blot analysis of human insulinoma RNA revealed three IAPP mRNAs of sizes 1.2, 1.8 and 2.1 kb, in agreement with three polyadenylation signals present in the 3' end of the gene. In situ hybridization to metaphase chromosomes resulted in two distinct peaks on chromosome 12, at 12p12-p13 and 12q13-q14. Southern blot analysis of genomic DNA suggested a single IAPP locus but also indicated the presence of additional homologous sequences in human genomic DNA.     

Essential charged amino acids in the binding of fibronectin to gelatin.

The binding of fibronectin to gelatin-agarose was strictly dependent on pH, having a pH optimum of 7-9. The binding was strongly inhibited by increasing ionic strength. A chemical modification of lysyl and arginyl groups of fibronectin abolished the binding activity. The anionic detergents sodium dodecyl sulphate and sodium deoxycholate in concentrations of 10-100mM had the same effect. The binding was not affected by the non-ionic detergents Triton X-100, Tween 20 or Lubrol WX. The results demonstrate an important role of ionic interactions in the binding of fibronectin to gelatin. Absence of inhibition by non-ionic detergents suggests that hydrophobic interactions contribute relatively little to the binding of fibronectin to gelatin.     

Sequential phases of cortical specification involve Neurogenin-dependent and -independent pathways

Neocortical projection neurons, which segregate into six cortical layers according to their birthdate, have diverse morphologies, axonal projections and molecular profiles, yet they share a common cortical regional identity and glutamatergic neurotransmission phenotype. Here we demonstrate that distinct genetic programs operate at different stages of corticogenesis to specify the properties shared by all neocortical neurons. Ngn1 and Ngn2 are required to specify the cortical (regional), glutamatergic (neurotransmitter) and laminar (temporal) characters of early-born (lower-layer) neurons, while simultaneously repressing an alternative subcortical, GABAergic neuronal phenotype. Subsequently, later-born (upper-layer) cortical neurons are specified in an Ngn-independent manner, requiring instead the synergistic activities of Pax6 and Tlx, which also control a binary choice between cortical/glutamatergic and subcortical/GABAergic fates. Our study thus reveals an unanticipated heterogeneity in the genetic mechanisms specifying the identity of neocortical projection neurons.     

Endostatin inhibits human tongue carcinoma cell invasion and intravasation and blocks the activation of matrix metalloprotease-2, -9, and -13

Endostatin, a 20-kDa collagen XVIII fragment, inhibits angiogenesis and tumor growth in vivo, but the mechanisms are still unclear. Matrix metalloproteases (MMPs), a family of extracellular and membrane-associated endopeptidases, collectively digest almost all extracellular matrix and basement membrane components, and thus play an important role in tumor progression. We studied the effects of recombinant human endostatin on human MMP-2, -9, -8, and -13. We found that endostatin inhibited the activation and catalytic activity of pro-MMP-9 and -13 as well as recombinant pro-MMP-2. It prevented the fragmentation of pro-MMP-2 that was associated with reduction of catalytic activity. Endostatin had no effect on MMP-8 as shown by collagenase activity assays. An in vitro migration assay and an in vivo chicken chorioallantoic membrane intravasation assay with the human tongue squamous cell carcinoma cell line HSC-3 revealed the biphasic nature of endostatin; low endostatin concentrations inhibited intravasation and migration of these cells in a dose-dependent manner, but at increased concentrations, the inhibitory effect was far less efficient. The results show that endostatin blocks the activation and activities of certain tumor-associated pro-MMPs, such as pro-MMP-2, -9, and -13, which may explain, at least in part, the antitumor effect of endostatin. Our results also suggest that endostatin inhibits tumor progression by directly affecting the tumor cells and not just acting via endothelial cells and blockage of angiogenesis.     

Identification and functional characterization of a human GalNAc [alpha]2,6-sialyltransferase with altered expression in breast cancer

BACKGROUND: We sought to identify genes with altered expression during human breast cancer progression by applying mRNA comparisons of normal and tumor mammary cell lines with increasingly malignant phenotypes. The gene encoding a new sialyltransferase (STM) was found to be down-regulated in tumor cells. Abnormal expression and enzymatic activities of sialyltransferases in tumor cells result in the formation of tumor-associated carbohydrate antigens that can be used for the better understanding of the disease process and are applied for tumor diagnosis and immunotherapy. Altered glycosylation patterns of the MUC1 mucin, in particular, is a target antigen for immunotherapy of breast and other cancers. MATERIALS AND METHODS: Total RNAs from multiple normal mammary epithelial cell strains and tumor cell lines were compared by differential display and the differential expression of selected cDNAs was confirmed by Northern analyses. Recombinant STM was expressed in COS-7 cells. The substrate and linkage specificity of STM was examined using various oligosaccharides and O-glycosylated proteins as acceptor substrates. The chromosomal localization of the SIATL1 gene was assigned by somatic cell hybrid analysis. RESULTS: A human sialyltransferase gene was identified by differential display as being down-regulated in breast tumor cell lines as compared to normal mammary epithelial cell strains, and the corresponding full-length cDNA (stm) was cloned. The encoded protein of 374 amino acid residues contained the L- and S-sialylmotifs, two catalytic regions conserved in all functional sialyltransferases. Recombinant STM is an active GalNAc alpha2,6-sialyltransferase with Gal beta 1,3 GalNAc-O-Ser/Thr and (+/- Neu5Ac alpha 2,3) Gal beta 1,3GalNAc-O-Ser/Thr acceptor specificity. The SIATL1 gene, encoding STM, was mapped to the long arm of human chromosome 17 at q23-qter, a region that is nonrandomly deleted in human breast cancers. However, Southern analyses indicated that SIATL1 is usually not grossly rearranged in breast tumors. Northern analyses showed that the gene was widely expressed in normal human tissues, as well as in normal breast and prostate epithelial cell lines, but significantly down-regulated or absent in corresponding tumor cell lines. CONCLUSIONS: Our findings suggest that aberrant expression of STM sialyltransferase in tumors could be a feature of the malignant phenotype. In breast cancers, the MUC1 mucin is overexpressed and contains shorter O-glycans as compared to the normal mucin. Because STM catalyzes the synthesis of O-glycans, cloning and characterization of its substrate specificity will contribute to the understanding of the molecular mechanisms underlying the aberrant glycosylation patterns of O-glycans and the formation of mucin-related antigens in human breast cancers.