Transformation by v-sis occurs by an internal autoactivation mechanism.

Transformation by the v-sis oncogene appears to require an interaction of its protein product, p28v-sis, with the receptor for the platelet-derived growth factor (PDGF). However, this interaction may not occur at the cell surface as predicted by the autocrine hypothesis because phenotypic transformation was not reversed by incubation of SSV-NRK cells with antisera to PDGF and because morphological transformation did not occur when nontransformed NRK cells were cultured continuously with p28v-sis. A mutant of the wild-type v-sis gene was constructed that encodes a v-sis protein targeted for retention within the endoplasmic reticulum and Golgi. NRK cells expressing the mutant v-sis gene did not secrete any detectable v-sis protein but were as fully transformed as wild-type v-sis transfectants. The results support a mechanism of transformation by v-sis in which internal activation of the PDGF receptor occurs before expression of either p28v-sis or the PDGF receptor at the cell surface.

Development and characterization of three recombinant single chain antibody fragments (scFvs) directed against the CD19 antigen.

Antibodies that recognize antigens restricted to leukemia, lymphoma, and normal hematopoietic cells represent a unique opportunity to develop therapeutics, because they have the potential for relatively selective treatment of these diseases. Antibodies that recognize the CD19 antigen found on normal and malignant B cells, but not on stem cells, have been used to develop immunoconjugates. However, these conjugates are large and might be suboptimal in tumor penetration when compared to molecules using smaller single chain Fv (scFv) antibody fragments. scFv has the advantage of being a molecularly engineered homogeneous molecule. In this report, we demonstrate the cloning, expression, and binding of three anti-CD19 antibodies as scFvs. All three scFvs were successfully cloned and expressed. FVS191, derived from cell line B43, and FVS192, derived from SJ25C1, were properly refolded and bound CD19 antigen in FACS competition assays. These anti-CD19 scFv should be useful in the further development of diagnostic and therapeutic molecules.

Identification of the 1.4 kb and 4.0 kb messages for the lipoprotein associated coagulation inhibitor and expression of the encoded protein.

Lipoprotein-Associated Coagulation Inhibitor (LACI) is a factor Xa dependent inhibitor of the factor VII(a)/Tissue Factor catalytic complex. Deduced from partial cDNA sequence, LACI's amino acid sequence has recently been reported. Northern blot analysis showed LACI cDNA hybridizes to RNAs of 1.4 and 4.0 kb in size. To complete the characterization of the LACI message(s), overlapping LACI cDNAs were isolated from a human endothelial cell library. Sequence analysis revealed the clones' inserts span 4023 bases of sequence, consisting of 381 bases of 5' untranslated sequence, an open reading frame of 912 bases, 2682 bases of 3' untranslated sequence and 48 bases of poly(A) sequence. In addition, a short 1.4 kb insert which encodes for LACI was found to contain 49 bases of 3' untranslated sequence and a 3' poly(A) tail. The 1.4 kb of sequence is contained in the 4.0 kb sequence, except for 14 bases of 5' sequence, suggesting that the LACI messages arise by the use of alternative termination and polyadenylation signals during processing. Northern blot analysis of RNA isolated from cells treated with actinomycin D showed both RNA species appear to be relatively stable. Using a bovine papilloma virus vector, LACI cDNA was transfected into mouse C127 fibroblasts. The recombinant LACI is recognized by polyclonal anti-LACI IgG, binds to factor Xa and inhibits VII(a)/Tissue Factor activity in a similar fashion as LACI purified from HepG2 cell conditioned media.

Improved detection limits of toxic biochemical species based on impedance measurements in electrochemical biosensors.

An impedance based electrochemical biosensor was designed and fabricated for the detection of various chemical and biological species, with glass as substrate material and gold interdigitated electrodes. A flow cell with inlet and outlet ports for the microfluidic chamber was designed and fabricated using acrylic material with a reservoir volume of 78 µl. The feasibility of the fabricated sensor for detecting very low concentration of chemical and biological species was demonstrated. Electrochemical impedance spectroscopy (EIS) was employed as the detection technique. The impedance based response of the two-terminal device revealed a very high sensitivity with low concentrations of mouse monoclonal IgG, sarcosine, cadmium sulphide (CdS) and potassium chloride (KCl) at pico mole levels.

A cryptic plasmid from Pasteurella multocida has a predicted protein nearly identical to a transport protein from Actinobacillus actinomycetemcomitans.

Several plasmids from Pasteurella multocida have been shown to carry antibiotic resistance genes but no other genes possibly related to the organism's pathogenesis. We report here that sequence from the plasmid pLEM from a fowl isolate of P. multocida, strain 1059, contained one open reading frame that had significant identity with a predicted protein from pVT745, a plasmid that was isolated from a human oral isolate of Actinobacillus actinomycetemcomitans. This predicted protein had significant homology at the amino acid level to cation transport proteins.

A mapping study of 13 genes on human chromosome bands 4q11-->q25.

A chromosome band 4q21 gene (MLLT2, formerly called AF-4/FEL) involved in a reciprocal translocation with chromosome band 11q23 in t(4;11) acute leukemia has been cloned. To provide better definition of gene order and relationships in this region where MLLT2 resides, we used pulsed field gel electrophoresis (PFGE) to investigate 13 genes (including MLLT2) with physical locations in bands 4q11-->q25. Somatic cell hybrids derived from RS4;11, a leukemic cell line carrying the t(4;11)(q21;q23), were also used to localize genes in relation to MLLT2. Linkage of the interleukin 8 (IL8), albumin (ALB), and platelet factor 4 (PF4) genes was confirmed by NotI, SalI and SacII digests. The maximum distance between PF4 and ALB is 210 kb and between ALB and IL8 is 420 kb. The alcohol dehydrogenase, class I (ADH2, ADH3) gene cluster can be linked to the alcohol dehydrogenase, class III gene (ADH5) by SacII, NruI, and EagI digests. The maximum distance between them is 590 kb. Our study indicated that ALB, alpha-fetoprotein (AFP), PF4, beta-thromboglobulin (PPBP), GRO1 (encoding a cytokine also called melanoma growth-stimulatory activity), and IL8 genes can be physically linked. In this study the gamma-interferon induced protein 10 (INP10), bone morphogenetic protein 3 (BMP3), annexin III (ANX3), KIT, amphiregulin (AREG), immunoglobulin J polypeptide (IGJ), deoxycytidine kinase (DCK) and MLLT2 genes were not linked to one another or to the above two groups of genes. Our analysis using somatic cell hybrids combined with previous reports demonstrated that the ADH gene cluster is telomeric to MLLT2 and KIT, ALB, AFP, PF4, beta TG, GRO1, IL8, ANX3, AREG and DCK are centromeric to MLLT2.

Biosynthesis and processing of the platelet derived growth factor type alpha receptor.

The homodimers (AA, BB) of the platelet derived growth factor (PDGF) differentially interact with two highly related PDGF receptors (alpha, beta) that appear to mediate different functional responses in different cell types. To seek a basis for these apparent functional differences, we investigated the processing of the PDGF alpha-receptor. The PDGF alpha-receptor is rapidly glycosylated to a 160 kD form and undergoes a number of intermediate glycosylation steps that result in a mature form of 185 kD that appears at the cell surface within 60-90 minutes. The alpha receptor has a half-life of approximately 4 1/2 hours without and approximately 20 minutes in the presence of ligand. The processing steps of the alpha-receptor are similar to the processing of the PDGF beta receptor, suggesting that differential binding of signalling molecules to activated receptors may be responsible for the apparent functional differences in cellular responses to PDGF AA and PDGF BB.

SHP-2 can suppress transformation induced by platelet-derived growth factor.

Signaling by either the type alpha or type beta receptors of platelet-derived growth factor occurs by phosphorylation of at least 10 intra-cytoplasmic tyrosine residues and their subsequent association of secondary signaling molecules with Src homology 2 (SH2) domains. Although the role of several of these secondary signaling molecules in mitogenesis has become increasingly clear, their roles in morphological transformation are not as well defined. Here we present evidence that the SHP-2 phosphatase which associates with Tyr 1009 of the type beta receptor and Tyr 720 of the type alpha receptor may suppress transformation induced by the PDGF B chain. Cotransfection of a dominant negative mutant of the SHP-2 gene and the PDGF B chain gene into mouse fibroblasts that only poorly formed foci with the PDGF B chain alone resulted in larger and more prominent foci. Furthermore, introduction of a wild-type copy of the SHP-2 gene into a tumor cell line, U-87MG, which relies on PDGF expression to form foci in vitro, caused a reversion of phenotype.

The v-sis oncogene product but not platelet-derived growth factor (PDGF) A homodimers activate PDGF alpha and beta receptors intracellularly and initiate cellular transformation.

The v-sis oncogene product p28v-sis and the platelet-derived growth factor (PDGF) B chain share 92% homology with each other and over 50% homology with the PDGF A chain. Exogenously added homodimers of PDGF A and PDGF B and of p28v-sis are potent mitogens but only PDGF B and p28v-sis induce transformation when endogenously expressed with a strong promoter. Because exogenous PDGF AA and PDGF BB both initiate a full mitogenic response, understanding the mechanisms underlying the difference in their transforming potential may clarify how growth factor genes act as oncogenes. In this work, we compared cells expressing high levels of PDGF A and v-sis. We observed that transformation by v-sis correlated directly with the rapid degradation (t1/2 approximately 20 min) of the alpha and beta PDGF receptors, with a failure of either the alpha or beta receptor to be fully processed and with the association of high levels of phosphatidylinositol (PI) 3-kinase with immunoprecipitates of the PDGF receptors. In contrast, in cells expressing essentially equal levels of PDGF A, transformation was not detected, alpha and beta PDGF receptor processing was normal, and association of PI 3-kinase with receptors in immunoprecipitates was not found above control values. The ability of v-sis to autoactivate PDGF receptors within processing compartments and to initiate activation of the PI 3-kinase signaling pathway coupled with the failure of PDGF A to activate its receptor intracellularly and to induce transformation when endogenously expressed at high levels suggests that the internal autoactivation of PDGF receptors may be essential for transformation by v-sis.