The relationship between HLA-B45 and B* 5002 in the five major U.S. population groups.

The antigen encoded by B*5002 differs in sequence from that encoded by B*5001 only at amino acid residue 167 (consensus tryptophan vs. serine) which results in B45 serologic reactivity. To search for B*5002, the frequencies of alleles encoding the serologically defined B45 antigen were determined by sequence-based typing in 5 major U.S. populations: Caucasians, African Americans, Asians/Pacific Islanders, Hispanics, and Native Americans. The percent of serologically defined B45-positive individuals in the 5 populations ranged from 0.7-9.0%. Thirty-two B45-positive individuals were randomly chosen, when available, for sequence-based typing from each ethnic group from a database of 82,979 consecutively typed unrelated individuals. The B*5002 allele was most prevalent in Hispanic (22%) and Caucasian (9%) individuals, while conspicuously absent in African Americans. In addition, a new allele associated with the B45 antigenic specificity, B*4502, has been identified from an African American individual of Middle Eastern descent. In light of the continuing need to reconcile differences between relationships determined by the sequence homologies among alleles and relationships based on the serologic determinants carried by allelic products when determining the level of HLA match for hematopoietic stem cell transplantation, it is suggested that B*5002 be recognized individually from other B*50 alleles when reporting HLA-B typings for clinical purposes.

PNA openers as a tool for direct quantification of specific targets in duplex DNA.

We report a new approach for target quantification directly within DNA duplex. Our assay is based on the formation of a new biomolecular structure, the PD-loop. The approach takes advantage of a selective hybridization of a probe to double-stranded DNA (dsDNA), which is locally opened by a pair of bis-PNA oligomers. To optimize the technique, several experimental formats are tested with the use of PNA and oligonucleotide probes. The highest sensitivity is achieved when the hybridized probe is extended and multiply labeled with 125I-dCTP by DNA polymerase via strand displacement in the presence of single-strand binding (SSB) protein. In this case, the PNA-assisted probe hybridization combined with the method of multiphoton detection (MPD) allows to monitor sub-attomolar amounts of the HIV-1 target on the background of unrelated DNA at sub-nCi level of radioactivity. The developed robust methodology is highly discriminative to single mutations, thus being of practical use for DNA analysis.

Membrane fusion mediated by baculovirus gp64 involves assembly of stable gp64 trimers into multiprotein aggregates.

The baculovirus fusogenic activity depends on the low pH conformation of virally-encoded trimeric glycoprotein, gp64. We used two experimental approaches to investigate whether monomers, trimers, and/or higher order oligomers are functionally involved in gp64 fusion machine. First, dithiothreitol (DTT)- based reduction of intersubunit disulfides was found to reversibly inhibit fusion, as assayed by fluorescent probe redistribution between gp64-expressing and target cells (i.e., erythrocytes or Sf9 cells). This inhibition correlates with disappearance of gp64 trimers and appearance of dimers and monomers in SDS-PAGE. Thus, stable (i.e., with intact intersubunit disulfides) gp64 trimers, rather than independent monomers, drive fusion. Second, we established that merger of membranes is preceded by formation of large (greater than 2 MDa), short-lived gp64 complexes. These complexes were stabilized by cell-surface cross-linking and characterized by glycerol density gradient ultracentrifugation. The basic structural unit of the complexes is stable gp64 trimer. Although DTT-destabilized trimers were still capable of assuming the low pH conformation, they failed to form multimeric complexes. The fact that formation of these complexes correlated with fusion in timing, and was dependent on (a) low pH application, (b) stable gp64 trimers, and (c) cell-cell contacts, suggests that such multimeric complexes represent a fusion machine.

SPARC/osteonectin induces matrix metalloproteinase 2 activation in human breast cancer cell lines.

Activation of the matrix metalloproteinase 2 (MMP-2) has been shown to play a major role in the proteolysis of extracellular matrix (ECM) associated with tumor invasion. Although the precise mechanism of this activation remains elusive, levels of the membrane type 1-MMP (MT1-MMP) at the cell surface and of the tissue inhibitor of MMP-2 (TIMP-2) appear to be two important determinants. Induction of MMP-2 activation in cells cultivated on collagen type I gels indicated that the ECM is important in the regulation of this process. In this study, we show that SPARC/osteonectin, a small ECM-associated matricellular glycoprotein, can induce MMP-2 activation in two invasive breast cancer cell lines (MDA-MB-231 and BT549) but not in a noninvasive counterpart (MCF-7), which lacks MT1-MMP. Using a set of peptides from different regions of SPARC, we found that peptide 1.1 (corresponding to the NH2-terminal region of the protein) contained the activity that induced MMP-2 activation. Despite the requirement for MT1-MMP, seen in MCF-7 cells transfected with MT1-MMP, the activation of MMP-2 by SPARC peptide 1.1 was not associated with increased steady-state levels of MT1-MMP mRNA or protein in either MT1-MMP-transfected MCF-7 cells or constitutively expressing MDA-MB-231 and BT549 cells. We did, however, detect decreased levels of TIMP-2 protein in the media of cells incubated with peptide 1.1 or recombinant SPARC; thus, the induction of MMP-2 activation by SPARC might be due in part to a diminution of TIMP-2 protein. We conclude that SPARC, and specifically its NH2-terminal domain, regulates the activation of MMP-2 at the cell surface and is therefore likely to contribute to the proteolytic pathways associated with tumor invasion.

Apoptosis induced by hyperthermia and verapamil in vitro in a human colon cancer cell line.

The aim of this study was to determine the mechanisms responsible for the growth inhibitory effect of hyperthermia and verapamil in human colon cancer cell line HT-29. Apoptotic cell death was verified by flow cytometry analysis. The effect of treatment with hyperthermia and verapamil on the expression of apoptosis-associated proteins including Bcl-2, p53, bax, and c-Myc was studied by Western blot analysis. Changes in intracellular calcium homeostasis was analysed by fluorescence microscopy. The combination of 42 degrees C hyperthermia and verapamil caused a significant delay of human colon cancer cell proliferation as a result of apoptosis. Administration of these agents alone did not cause any cell inhibitory effect. Our experiments have shown that HT-29 cells constitutively express apoptosis-promoting proteins, such as Bax and c-Myc, while they fail to produce Bcl-2. Therefore, we hypothesize that HT-29 cells must have Bcl-2 independent pathways to protect cells against death-inducing signals. Also, apoptosis of HT-29 cells produced by hyperthermia in the presence of verapamil is a p53-independent process. Verapamil, when it did not act as a calcium channel blocker or inhibitor of release from intracellular storages under hyperthermic conditions, accelerated the increase of [Ca2+]i in HT-29 cells which resulted in programmed cell death (apoptosis).

MT1-MMP correlates with MMP-2 activation potential seen after epithelial to mesenchymal transition in human breast carcinoma cells.

We have previously reported that human breast carcinoma (HBC) cell lines expressing the mesenchymal intermediate filament protein vimentin (VIM+) are highly invasive in vitro, and highly metastatic in nude mice when compared to their VIM- counterparts. Since only VIM+ cell lines can be induced to activate matrix metalloproteinase-2 (MMP-2) upon stimulation with Concanavalin A (Con A), we have examined here membrane type 1 MMP (MT1-MMP), a cell surface activator of MMP-2. Northern analysis reveals baseline expression of MT1-MMP in five of the six VIM+ cell lines studied (MDA-MB-231, MDA-MB-435, BT-549, Hs578T, MCF-7(ADR)), each of which showed variable activation of exogenous MMP-2 after treatment with Con A. In contrast, the four VIM-, poorly invasive HBC cell lines studied (MCF-7, T47D, MDA-MB 468, ZR-75-1) lacked baseline MT1-MMP mRNA expression, and showed no induction of either MT1-MMP expression or MMP-2-activation with Con A. Such differential MT1-MMP expression was confirmed in vivo using in situ hybridization analysis of nude mouse tumor xenografts of representative cell lines. Western analysis of the MDA-MB-231 cells revealed baseline membrane expression of a 60 kDa species, which was strongly induced by Con A treatment along with a weaker band co-migrating with that from MT1-MMP-transfected COS-1 cells (63 kDa), presumably representing latent MT1-MMP. MT1-MMP immunofluorescence strongly decorated Con A-stimulated MDA-MB-231 cells in a manner consistent with membranous staining, but did not decorate the unstimulated MDA-MB-231 cells or MCF-7 cells under either condition. Collectively, the results suggest the constitutive production of active MT1-MMP which is unavailable for either MMP-2 activation or immuno-decoration until Con A treatment. Since VIM expression arises by virtue of the so-called epithelial to mesenchymal transition (EMT) in invasive embryonic epithelia, we propose that this represents a major metastasis mechanism in breast carcinomas. MT1-MMP on the surface of such 'fibroblastoid' carcinoma cells may mediate a paracrine loop for the utilization of stromally produced MMP-2, and contribute to the poorer survival associated with VIM+ breast carcinomas.

Detection of a single base mismatch in double-stranded DNA by electrophoresis on uncrosslinked polyacrylamide gel.

Uncrosslinked polyacrylamide forms gels in the concentration range of 15-40% acrylamide. Electrophoresis in these gels of a commercially available 350 bp heteroduplex DNA preparation separates it from the homoduplex DNA of the same size. The separation is qualitatively equivalent to that previously achieved in a commercial proprietary gel ("Mutation Detection Gel" of AT-Biochem), or in an equivalent 14% T, 0.15% C (N,N'-methylenebisacrylamide) gel, but the mechanical stability of mutation detection electrophoresis (MDE) gels or 0.15% C gels is better than that of uncrosslinked polyacrylamide gels. The separation in any of these three gel media can be carried out in short gel tubes within a few hours of electrophoresis time. In both uncrosslinked polyacrylamide and MDE gel media, the Ferguson plots [log(mobility) vs. gel concentration] and the plots of effective molecular radius vs. gel concentration ("T-plots") of both the heteroduplex and homoduplex DNA indicate an augmented size but similar flexibility upon passage through the gel than exhibited by the components of a DNA standard ladder. Homoduplex and heteroduplex DNA correspondingly exhibit a parallelism of their Ferguson curves in transverse MDE pore gradient gel electrophoresis, suggesting a surface net charge difference, possibly due to a conformational reorientation too subtle to be detected by a shift in the slope of the Ferguson plot, as has been observed once previously with a "kinked" DNA species. The gel fiber radius or length per unit volume of uncrosslinked polyacrylamide and MDE gels do not differ significantly within confidence limits, which are wide compared to unconventionally crosslinked gels, presumably because of their greater swelling.

Collagen induced MMP-2 activation in human breast cancer.

Matrix metalloproteinase-2 (MMP-2), a zymogen requiring proteolytic activation for catalytic activity, has been implicated broadly in the invasion and metastasis of many cancer model systems, including human breast cancer (HBC). MMP-2 has been immunolocalized to carcinomatous human breast, where the degree of activation of MMP-2 correlates well with tumor grade and patient prognosis. Using Matrigel assays, we have stratified HBC cell lines for invasiveness in vitro, and compared this to their potential for metastatic spread in nude mice. HBC cell lines expressing the mesenchymal marker protein vimentin were found to be highly invasive in vitro, and tended to form metastases in nude mice. We have further discovered that culture on collagen-I gels (Vitrogen; Vg) induces MMP-2-activator in highly invasive but not poorly invasive HBC cell lines. As seen for other MMP-2-activator inducing regimens, this induction requires protein synthesis and an intact MMP-2 hemopexin-like domain, appears to be mediated by a cell surface activity, and can be inhibited by metalloproteinase inhibitors. The induction is highly specific to collagen I, and is not seen with thin coatings of collagen I, collagen IV, laminin, or fibronectin, or with 3-dimensional gels of laminin, Matrigel, or gelatin. This review focuses on collagen I and MMP-2, their localization and source in HBC, and their relationship(s) to MMP-2 activation and HBC metastasis. The relevance of collagen I in activation of MMP-2 in vivo is discussed in terms of stromal cell: tumor cell interaction for collagen I deposition, MMP-2 production, and MMP-2-activation. Such cooperativity may exist in vivo for MMP-2 participation in HBC dissemination. A more complete understanding of the regulation of MMP-2-activator by type I collagen may provide new avenues for improved diagnosis and prognosis of human breast cancer.

Capillary zone electrophoresis of large DNA.

Capillary zone electrophoresis (CZE) of DNA 23.1 to 48.5 kb in length in polyacrylamide solutions of several concentrations provides evidence for polymer concentration and DNA length-dependent stretching and orientation of these species and suggests an effective separation at a polymer concentration of about 0.6%. Applying a 0.1% polyacrylamide concentration to the lambda-phage DNA ladder, at least 5 components are separated; separation improves with lowering of the field strength to 2 V/cm and, correspondingly, extended duration of CZE. Saccharomyces pombe chromosomal DNA separates into 3 major components on CZE at high field strength (270 V/cm) in 0.9% polyacrylamide solution, confirming a previous finding made on electrophoresis in a 1.1 mm ID tube at low field strength. However, the finding is limited to one source of the DNA plug, and the chromosomal identity of the components remains unknown. Methodological problems in the CZE of large DNA relate to the need for extended duration of pressure injection if absorbance detection is applied, the need to define the starting zone after extended pressure injection, the need to melt and digest agarose plugs prior to loading, and related needs for thermostating of the sample chamber and for software compatible with low voltage operation.

Advances in DNA electrophoresis in polymer solutions.

DNA electrophoresis in gels and solutions of agarose and polyacrylamide was objectively evaluated with regard to separation efficiency at optimal polymer concentrations. In application to DNA fragments, polyacrylamide gels were superior for separating fragments of less than 7800 bp, and agarose gels are the best choice for larger fragments. Agarose solutions are nearly as good as polyacrylamide gels for small DNA (< 300 bp). Agarose solutions have a higher efficiency than polyacrylamide solutions for DNA of less than 1200 bp. Separation efficiency sharply decreases with increasing length of DNA. Retardation in polyacrylamide solutions was found to depend on polymer length in a biphasic fashion. The choice of resolving polymer concentrations depends on the progressive stretching of DNA in proportion to polymer concentration. The rate of that stretching appears higher in polyacrylmide solution than in gels or in liquid or gelled agarose. Application of polymer solutions to capillary electrophoresis raises further problems concerning agarose plugs, DNA interactions with the polymers, operation at low field strength and long durations as well as detection sensitivity.

Molecular sieving of lambda phage DNA in polyacrylamide solutions as a function of the molecular weight of the polymer.

Electrophoresis of lambda phage DNA was carried out in solutions at various concentrations of uncrosslinked polyacrylamide of 0.6, 1, 5 and 9 x 10(6) molecular weight (Mw) with narrow Mw distribution. By inspection of mobilities in the various concentration ranges, it appears that mobilities decrease, and retardation increases, with increasing Mw. The relation between electrophoretic retardation and the Mw of the polymer was also interpreted (i) in the manner previously applied to nonlinear Ferguson plots and compatible with the Ogston model; and (ii) empirically, on the basis of the first derivatives of the functions describing the Ferguson plots at the polymer concentrations used. Interpretation (i) shows that the retardation increases linearly in the order of 0.6, 1, 5 and 9 x 10(6) Mw of polyacrylamide. Interpretation (ii) shows a nonlinear increase of retardation in the Mw range 5 to 9 x 10(6), and a decrease in retardation as Mw is raised from 0.6 to 5.0 x 10(6). Hypothetically, interpretation (ii) can be explained mechanistically by a progressive change, as the polymer size is increased, from a collision with the surface of the polymer fiber to one occurring after permeation in the interior of a random-coiled fiber. Interpretation (i) may fail to detect that change due to the large difference between DNA mobility in solutions of the smallest polymer and the free mobility. DNA peak detection in all of the four size classes of polyacrylamide in solution is limited to relatively narrow ranges of polymer concentration.(ABSTRACT TRUNCATED AT 250 WORDS)