Safety study and characterization of E1A-liposome complex gene-delivery protocol in an ovarian cancer model

A phase I clinical trial of E1A-liposome complex is currently ongoing in patients with HER-2/neu-overexpressing breast or ovarian cancers. To optimize the E1A-liposome complex for a further stage of clinical trial, several aspects of the current protocol have been examined in an animal model. In the orthotopic ovarian cancer model, different doses of lipid in the the E1A-liposome complex, which is currently used in clinical trials, were tested for the in vivo gene-transfer efficacy and tumor-suppression function. A lowered lipid dose--1/13 of the previous amount--produced gene expression level and E1A tumor-suppression efficacy similar to that of the original protocol. Mini-E1A, an E1A construct without its immortalization domain and yet capable of repressing HER-2/neu, was proved to be as potent as E1A in suppressing tumor development in vivo. These changes in the E1A-liposome complex will significantly reduce any potential adverse effects caused by lipid vector and E1A DNA. To examine further whether residual E1A DNA may still exist in normal organs after the E1A-liposome treatment, PCR was used to detect E1A DNA in mice that survived for 1 1/2 years after the last treatment. E1A DNA was detected only in the lungs and kidneys, but not in livers, hearts, spleens, brains, uterus or the ovaries. Furthermore, resistance of the E1A DNA extracted from tissues to the digestion of Dpnl restriction enzyme, which can cleave the methylated E1A plasmid DNA generated by methylation-competent bacteria, suggested integration of E1A DNA into the chromosome of the lungs and kidneys. Experimental results presented here provide important information for safety concerns and for the design of future phase II and phase III trials.     

Ndd, the bacteriophage T4 protein that disrupts the Escherichia coli nucleoid, has a DNA binding activity

Early in a bacteriophage T4 infection, the phage ndd gene causes the rapid destruction of the structure of the Escherichia coli nucleoid. Even at very low levels, the Ndd protein is extremely toxic to cells. In uninfected E. coli, overexpression of the cloned ndd gene induces disruption of the nucleoid that is indistinguishable from that observed after T4 infection. A preliminary characterization of this protein indicates that it has a double-stranded DNA binding activity with a preference for bacterial DNA rather than phage T4 DNA. The targets of Ndd action may be the chromosomal sequences that determine the structure of the nucleoid.     

Continuous separation of serum proteins using a stirred cell charged with carboxylated and sulfonated microspheres

We contrived a new separation system using a stirred cell charged with uncoupled microsphere similar to the chromatographic separation. Microspheres, carboxylated PS/PMAA and sulfonated PS/PNaSS, were prepared by emulsifier-free emulsion polymerization. To complement the submicron size weakness and the absence of ligands, we employed the latex form, the dispersion of microsphere, and took advantage of interaction relationships between proteins and microspheres. Adsorption isotherm is contemplated to investigate continuous separation behaviours of serum proteins. Selectivity of separation is in the following order: PS/PNaSS-2.0 (high sulfonated) < PS/PNaSS-0.3 (low sulfonated) < PS/PMAA-0.5 (low carboxylated). Unlike previous works on batch separation, not only the adsorbed amount in equilibrium (Cm), but also adsorption coefficient (K), played an important role in continuous separation. Functional groups (carboxyl and sulfonate), induced from the co-monomer, also affected the adsorption behaviours.     

CD164, a novel sialomucin on CD34(+) and erythroid subsets, is located on human chromosome 6q21

CD164 is a novel 80- to 90-kD mucin-like molecule expressed by human CD34(+) hematopoietic progenitor cells. Our previous results suggest that this receptor may play a key role in hematopoiesis by facilitating the adhesion of CD34(+) cells to bone marrow stroma and by negatively regulating CD34(+) hematopoietic progenitor cell growth. These functional effects are mediated by at least two spatially distinct epitopes, defined by the monoclonal antibodies (MoAbs), 103B2/9E10 and 105A5. In this report, we show that these MoAbs, together with two other CD164 MoAbs, N6B6 and 67D2, show distinct patterns of reactivity when analyzed on hematopoietic cells from normal human bone marrow, umbilical cord blood, and peripheral blood. Flow cytometric analyses revealed that, on average, 63% to 82% of human bone marrow and 55% to 93% of cord blood CD34(+) cells are CD164(+), with expression of the 105A5 epitope being more variable than that of the other identified epitopes. Extensive multiparameter flow cytometric analyses were performed on cells expressing the 103B2/9E10 functional epitope. These analyses showed that the majority (>90%) of CD34(+) human bone marrow and cord blood cells that were CD38(lo/-) or that coexpressed AC133, CD90(Thy-1), CD117(c-kit), or CD135(FLT-3) were CD164(103B2/9E10)+. This CD164 epitope was generally detected on a significant proportion of CD34(+)CD71(lo/-) or CD34(+)CD33(lo/-) cells. In accord with our previous in vitro progenitor assay data, these phenotypes suggest that the CD164(103B2/9E10) epitope is expressed by a very primitive hematopoietic progenitor cell subset. It is of particular interest to note that the CD34(+)CD164(103B2/9E10)lo/- cells in bone marrow are mainly CD19(+) B-cell precursors, with the CD164(103B2/9E10) epitope subsequently appearing on CD34(lo/-)CD19(+) and CD34(lo/-)CD20(+) B cells in bone marrow, but being virtually absent from B cells in the peripheral blood. Further analyses of the CD34(lo/-)CD164(103B2/9E10)+ subsets indicated that one of the most prominent populations consists of maturing erythroid cells. The expression of the CD164(103B2/9E10) epitope precedes the appearance of the glycophorin C, glycophorin A, and band III erythroid lineage markers but is lost on terminal differentiation of the erythroid cells. Expression of this CD164(103B2/9E10) epitope is also found on developing myelomonocytic cells in bone marrow, being downregulated on mature neutrophils but maintained on monocytes in the peripheral blood. We have extended these studies further by identifying Pl artificial chromosome (PAC) clones containing the CD164 gene and have used these to localize the CD164 gene specifically to human chromosome 6q21.     

Identification of an NAD+ binding site of brain glutamate dehydrogenase isoproteins by photoaffinity labeling

Photoaffinity labeling with [32P]nicotinamide 2-azidoadenosine dinucleotide (2N3NAD+) was used to identify the NAD+ binding site within two types of glutamate dehydrogenase isoproteins (GDH I and GDH II) isolated from bovine brain. In the absence of photolysis, 2N3NAD+ is a substrate for the GDH isoproteins. When the enzymes were covalently modified by photolysis in the presence of saturating amounts of photoprobe, about 50% inhibition of the GDH activities was observed. Photoinsertion of probe was increased by GTP or glutarate and decreased by NAD+ or ADP. With the combination of immobilized boronate affinity chromatography and reversed-phase HPLC, photolabel-containing peptides generated with trypsin were isolated. This identified a portion of the adenine ring binding domain of GDH isoproteins as the region containing the sequence, CIAVGXSDGSIWNPDGIDPK for both GDH isoproteins, corresponding to Cys270 through Lys289 of the amino acid sequence of well known bovine liver GDH. The X indicates a position for which no phenylthiohydantoin-derivative could be assigned. The missing residue, however, can be designated as a photolabeled glutamate since the sequences including the glutamate residue in question have a complete identity with those of the other GDH species known. Photolabeling of these peptides was prevented by the presence of NAD+ during photolysis. These results demonstrate selectivity of the photoprobe for the NAD+ binding site and suggest that the peptide identified using the photoprobe is located in the NAD+ binding domain of the brain GDH isoproteins. Both amino acid sequencing and compositional analysis identified Glu275 as the site of photoinsertion.