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Biomedical subjects

D L Taylor

Publications and source records attributed to D L Taylor.

At least 55 records · Page 3Linked to original sources
PubMed1995

Apoptosis induction mediated by wild-type p53 adenoviral gene transfer in squamous cell carcinoma of the head and neck.

Cancer gene therapy strategies for inducing apoptosis in solid tumors may allow contemporary medicine to reassess its management of these cancers. We demonstrated previously that overexpression of the wild-type p53 gene in squamous cell carcinoma of the head and neck cell lines via adenovirus-mediated gene transfer suppressed growth both in vitro and in vivo. Here, we characterize the mechanism of the growth suppression by the exogenous p53 gene as a consequence of programmed cell death (apoptosis). One of the cell lines used in this study, Tu-138, harbors a mutated p53 gene, whereas the other cell line, MDA 686LN, possesses a wild-type p53 gene. DNA fragmentation was detected by electrophoresis in both cell lines after infection with the wild-type p53 adenovirus, Ad5CMV-p53. With the use of the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling method, 4.4% of the remaining viable Tu-138 cell population was identified as apoptotic as early as 15 h after inoculation with Ad5CMV-p53. The percentage of apoptotic cells increased to 31% at 22 h. In contrast, only 10% of the viable MDA 686LN cells (wt-p53) had undergone apoptosis 30 h after Ad5CMV-p53 infection, although the percentage of apoptotic cells rapidly increased to 60% at 48 h after infection. For in vivo analysis of apoptosis, nude mice in which squamous cell carcinoma of the head and neck cell lines had been implanted s.c. had exogenous wt-p53 transiently introduced to the tumor cells via Ad5CMV-p53 2 days later. In situ end labeling clearly illustrated apoptosis in the tumor cells. These results suggest that wt-p53 plays an important role in the induction of apoptosis in human head and neck cancer cell lines and that selective induction of apoptosis in cancer cells can be further explored as a strategy for cancer gene therapy.

Adenoviridae↗
PubMed1995

Expression of basic fibroblast growth factor in squamous cell carcinoma of the head and neck is associated with degree of histologic differentiation.

Basic fibroblast growth factor (bFGF) is a potent nitogen and angiogenic protein that may function as an autocrine growth regulator in a variety of malignancies. Expression of bFGF in squamous cell carcinoma of the head and neck (SCCHN) was characterized by Western blot and immunohistochemical analyses. We found that the levels of bFGF in tumors were the same or reduced relative to non-malignant adjacent mucosa. Reverse transcription-polymerase chain reaction and Southern blot transfer of mRNA derived from 7 SCCHN cell lines showed that the IIIb isoform of FGF-receptor 2 (FGFR2) was expressed at high levels, whereas the IIIc isoform and FGFRI were weakly expressed or not detected. No correlation was observed between levels of bFGF revealed by immunohistochemical staining and vascular counts in frozen sections derived from 11 different SCCHN tumors. Immunohistochemical analysis demonstrated that all differentiated tumors exhibited high levels of bFGF immunoreactivity, while all poorly differentiated tumors exhibited low to nondetectable levels. This expression pattern is consistent with that observed in non-tumoral mucosa and suggests that other angiogenic factors must play a predominant role in the development of poorly differentiated SCCHN.

Antibodies, Monoclonal↗
PubMed1995

Antiviral activity and metabolism of the castanospermine derivative MDL 28,574, in cells infected with herpes simplex virus type 2.

The 6-O-butanoyl derivative of castanospermine (MDL 28,574: BUCAST), an inhibitor of glycoprotein processing, blocked the growth of herpes simplex virus type-2 with the effect markedly enhanced by exposure of cells to the compound pre- as well as post-infection. The effectiveness of the derivative corresponded to an increased uptake with greatest accumulation after virus infection. Gas chromatography/mass spectrometry identified the predominant component in MDL 28,574 treated cells as castanospermine, an inhibitor of alpha-glucosidase 1. The effects of this compound on the synthesis of viral glycoprotein, gB, was determined with the increased molecular weight of the mannose-rich precursor evidence for the modulation of glycoprotein processing.

Animals↗
PubMed1995

In vivo molecular therapy with p53 adenovirus for microscopic residual head and neck squamous carcinoma.

Developing gene therapy strategies may allow contemporary medicine to reassess its management of solid malignancies. We have demonstrated previously that the wild-type p53 adenovirus (Ad5CMV-p53) suppressed the growth of established tumors of the head and neck. In this paper we develop a microscopic residual model which mimics the postsurgical environment of head and neck cancer patients with advanced disease. Using this squamous cell carcinoma of the head and neck model, we prevented the establishment of tumors in nude mice in which tumor cells had been s.c. implanted by transiently introducing exogenous wild-type p53 via an adenoviral vector 2 days following tumor cell implantation. These effects were vector dose dependent but independent on the endogenous wild-type or mutated p53 status of the cells. Importantly, karyotypically normal and nontumorigenic fibroblast cell lines are inert to the p53 adenovirus treatment. These results pave the ground work for further development of molecular therapy for head and neck cancer and other solid malignancies.

Adenoviridae↗
PubMed1995

The inhibition of human immunodeficiency virus type 1 in vitro by a non-nucleoside reverse transcriptase inhibitor MKC-442, alone and in combination with other anti-HIV compounds.

MKC-442, a derivative of the non-nucleoside reverse transcriptase (RT) inhibitor 1-[(2-hydroxyethoxy)methyl)-6-(phenylthio)thymidine (HEPT), showed potent and selective inhibition of human immunodeficiency virus type 1 (HIV-1) replication in vitro, using a range of host-cell/virus systems including human peripheral blood mononuclear cells infected with primary clinical isolates. MKC-442 was evaluated in combination with the nucleoside analogues AZT, ddI and ddC, the non-nucleoside RT inhibitor nevirapine, the HIV-1 proteinase inhibitor Ro-31-8959, and the alpha-glucosidase 1 inhibitor, MDL-28,574, using a cell viability assay. Drug interactions were evaluated by the isobologram technique and by calculating combination indices. Notable synergistic inhibition of HIV-1 replication was observed when MKC-442 was combined with AZT and MDL-28,574 and moderate synergy with ddI. In combination with ddC, nevirapine or Ro-31-8959, only a slightly better than additive effect was observed. Impressive synergy was seen using the three-drug combinations of MKC-442, AZT and MDL-28,574 or MKC-442, AZT and Ro-31-8959. No additional cytotoxicity was observed as measured by [3H]thymidine incorporation by concanavalin A-stimulated peripheral blood mononuclear cells, when MKC-442 was combined with any of the above-mentioned compounds. The use of MKC-442 in a two- or three-drug combination regimen with other RT inhibitors, a proteinase inhibitor or an alpha-glucosidase 1 inhibitor should be considered for HIV-1-related chemotherapy.

Antiviral Agents↗
PubMed1995

Potent inhibition of human immunodeficiency virus by MDL 101028, a novel sulphonic acid polymer.

MDL 101028, a novel biphenyl disulphonic acid urea co-polymer was designed and synthesised as a heparin mimetic. This low molecular weight polymer showed potent inhibition of human immunodeficiency virus type 1 (HIV-1) replication in a number of host-cell/virus systems, including primary clinical isolates of the virus cultured in human peripheral blood mononuclear cells (PBMCs). When compared with the heterogeneous polysulphated molecules, heparin and dextran sulphate, this chemically defined compound showed equivalent antiviral activity with 50% inhibitory concentrations (IC50s) in the range 0.27-3.0 micrograms/ml in the host-cell/virus systems tested. MDL 101028 also inhibited the replication of HIV type 2 and the simian immunodeficiency virus (SIV), as well as HIV-1 variants resistant to reverse transcriptase inhibitors. Virus growth was blocked when exposure of T-lymphocytes to MDL 101028 was restricted to the virus absorption stage, or even in whole blood conditions. MDL 101028 did not irreversibly inactivate virions, and in contrast to heparin, did not inhibit the attachment of radiolabelled HIV-1 to CD4+ T-cells. MDL 101028 blocked HIV-induced cell-to-cell fusion and this activity appears to explain the mechanism of its antiviral action. The antiviral evaluation of discrete oligomer molecules of MDL 101028 showed that a polymer chain length of six repeating units had optimal potency. The lack of anticoagulant properties and significant antiviral activity in whole blood may allow the development of MDL 101028 as a treatment of HIV infections.

Animals↗
PubMed1995

Measurement and manipulation of cytoskeletal dynamics in living cells.

A new ear in cell biology is at hand with the development of tools for imaging molecular functions in living cells and tissues. Specific chemical and molecular events can now be measured and manipulated in cells in order to explore the mechanisms of cell functions. In particular, cytoskeletal processes are being dissected temporally and spatially in single cells from lower eukaryotes, plants, and animals using light-based reagents and electronic light microscopy.

Animals↗
PubMed1995

Investigation into the role of N-acetylaspartate in cerebral osmoregulation.

Marked abnormalities of the magnetic resonance intensity of N-acetylaspartate (NAA) have been reported in patients with various neurological disorders, but the neurochemical consequences of these alterations are difficult to assess because the function of NAA remains speculative. The purpose of this study was to examine whether NAA plays a role in protecting neurons against osmotic stress. Intracerebral microdialysis was used to expose a small region of the rat dorsolateral striatum to an increasingly hyposmotic environment and to measure resulting changes in NAA extracellular concentrations. NAA changes in the extracellular fluid (ECF) were compared with those of the amino acids, in particular, taurine, known to be involved in brain osmoregulation. Stepped increases in cellular hydration produced by hyposmotic perfusion media induced a marked increase in ECF NAA, reflecting a redistribution of NAA from intra-to extracellular space. Parallel experiments showed that, of all the extracellular amino acids measured, only taurine markedly increased with hyposmolar perfusion medium, indicating that the ECF NAA increase associated with hyposmotic stress was a specific response and not passive leakage out of the cells. As NAA is predominantly neuronal, it may contribute to the protection of neurons against swelling (i.e., regulatory volume decrease). In conditions with impaired blood-brain barrier and cytotoxic oedema, efflux of intracellular NAA subsequent to sustained cellular swelling might lead to a reduction in total brain NAA detectable by magnetic resonance spectroscopy. Alternatively, redistribution of NAA from intra-to extracellular space implies changes in its chemical environment that may alter its magnetic resonance visibility.

Amino Acids↗
PubMed1995

A fluorescent protein biosensor of myosin II regulatory light chain phosphorylation reports a gradient of phosphorylated myosin II in migrating cells.

Phosphorylation of the regulatory light chain by myosin light chain kinase (MLCK) regulates the motor activity of smooth muscle and nonmuscle myosin II. We have designed reagents to detect this phosphorylation event in living cells. A new fluorescent protein biosensor of myosin II regulatory light chain phosphorylation (FRLC-Rmyosin II) is described here. The biosensor depends upon energy transfer from fluorescein-labeled regulatory light chains to rhodamine-labeled essential and/or heavy chains. The energy transfer ratio increases by up to 26% when the regulatory light chain is phosphorylated by MLCK. The majority of the change in energy transfer is from regulatory light chain phosphorylation by MLCK (versus phosphorylation by protein kinase C). Folding/unfolding, filament assembly, and actin binding do not have a large effect on the energy transfer ratio. FRLC-Rmyosin II has been microinjected into living cells, where it incorporates into stress fibers and transverse fibers. Treatment of fibroblasts containing FRLC-Rmyosin II with the kinase inhibitor staurosporine produced a lower ratio of rhodamine/fluorescein emission, which corresponds to a lower level of myosin II regulatory light chain phosphorylation. Locomoting fibroblasts containing FRLC-Rmyosin II showed a gradient of myosin II phosphorylation that was lowest near the leading edge and highest in the tail region of these cells, which correlates with previously observed gradients of free calcium and calmodulin activation. Maximal myosin II motor force in the tail may contribute to help cells maintain their polarized shape, retract the tail as the cell moves forward, and deliver disassembled subunits to the leading edge for incorporation into new fibers.

3T3 Cells↗
PubMed1995

Treatment with the alpha-glucosidase 1 inhibitor 6-O-butanoyl castanospermine reduces the detection of LFA-1 (CD18/CD11a) by monoclonal antibodies.

The antiviral clinical candidate 6-O-butanoyl castanospermine (MDL 28,574), an alpha-glucosidase 1 inhibitor, was examined for its effect on elementary parameters of immune function. It did not affect the mitogenic response of uninfected human mononuclear leukocytes or the detection of a range of cell surface markers, with the exception of the integrin LFA-1 (CD18/CD11a), which was reduced, after cell growth in vitro. The detection of LFA-1 was also reduced on both human and murine cells after oral administration of the compound to xenochimaeric or normal mice, respectively. Altered LFA-1 expression or function may contribute to reduced cell adhesion and the observed reduction in the in vitro allogeneic response by uninfected cells, as well as the previously described prevention of cell conjugate and HIV-induced syncytium formation.

Animals↗
PubMed1995

An algorithm for improving the accuracy of discrete ROI integrals.

Many problems in the analysis of medical digital images, e.g., digitized x-ray radiograph, computed tomography (CT), magnetic resonance imaging (MRI), or positron emission tomography (PET), require a detailed and precise analysis of user chosen regions of interest (ROIs). Examples of their use include calculating integrals of area, volume, mass, structural moments, and statistical measures for either organs, tumors, or the musculoskeletal system. Among various ROI scan conversion schemes, binary approximate scan conversion is usually preferred due to its efficiency. In this paper, geometric area error is tabulated for typical scan conversion techniques, including whole pixel (WP) approximation and subpixel (SP) approximation methods, and compared to exact pixel (EP) coverage methods for medical ROIs. A new efficient and general EP method for scan conversion of these ROIs is presented. The algorithm traverses the boundary of the polygon while simultaneously scan converting the ROI, and calculates the fractional area of each pixel covered at the perimeter. The resultant geometric area is substantially more accurate than the SP or WP methods, without a significant loss of speed. The numerical results for a ROI with a large ratio of boundary to polygon area demonstrated that the geometric error for a SP method was 40% of the total polygon area, and 150% of the total polygon area for a WP method. The new algorithm could "exactly" calculate the pixel coverage area, in addition to being four times faster than the widely used EP method of Catmull. Efficient and accurate calculation of ROI integrals is essential for comparative analysis.

Algorithms↗
PubMed1995

Fluorescent protein biosensors: measurement of molecular dynamics in living cells.

A new generation of reagents that report on specific molecular events in living cells, called fluorescent protein biosensors, has evolved from in vitro fluorescence spectroscopy and fluorescent analogue cytochemistry. Creative designs of fluorescent protein biosensors to measure the molecular dynamics of macromolecules, metabolites, and ions in single cells emerge from the integrative use of contemporary synthetic organic chemistry, biochemistry, and molecular biology. Future advances in fluorescent probe design, computer-driven optical instrumentation, and software will allow us to engineer endogenous cellular components that localize and function as reporters of their activities, thus moving molecular measurement beyond the single cell to living tissues and the whole organism.

Biosensing Techniques↗
PubMed1995

Cytogenetic response to alpha-interferon is predicted in early chronic phase chronic myeloid leukemia by M-bcr breakpoint location.

Alpha-interferon (alpha-IFN) therapy is an effective agent in early chronic phase (ECP) chronic myeloid leukemia (CML), achieving hematologic control in the majority and major cytogenetic response (MCR) (reduction in Ph' +ve metaphases to < 35%) in a substantial minority. Currently no pretreatment markers exist to ascertain likelihood of meaningful response. The site of breakpoint in M-bcr and relationship to prognosis is controversial. Studies have been hampered by variation in definition of breakpoint and difference in treatment protocols. In this study of ECP CML patients, Southern analysis and reverse transcription polymerase chain reaction (RT-PCR) were used to determine breakpoint location. Patients received alpha-IFN (9 x 10(6) units/day) and dose-adjusted hydroxyurea (HU) to maintain granulocyte count between 1.0-2.0 x 10(9)/l for 6 months or more. Twelve of 31 patients entered on the study achieved a MCR. The Sokal index did not predict for cytogenetic response to alpha-IFN. Eight of 11 patients with 5' breakpoint achieved MCR compared to only four of 20 patients with 3' breakpoint (P = 0.007). These results suggest site of M-bcr rearrangement may be predictive of response to alpha-IFN therapy. If verified by further study, this may allow more appropriate use of alpha-IFN with respect to other modalities such as allogeneic transplant.

Base Sequence↗
PubMed1994

Growth suppression of human head and neck cancer cells by the introduction of a wild-type p53 gene via a recombinant adenovirus.

Mutations of the p53 gene constitute one of the most frequent genetic alterations in squamous cell carcinoma of the head and neck (SCCHN). In this study, we introduced wild-type p53 into two separate SCCHN cell lines via a recombinant adenoviral vector, Ad5CMV-p53. Northern blotting showed that following infection by the wild-type p53 adenovirus (Ad5CMV-p53), cells produced up to 10-fold higher levels of exogenous p53 mRNA than cells treated with vector only (without p53). Western blotting showed that the increased levels of p53 protein produced in the Ad5CMV-p53-infected cells were a reflection of p53 mRNA expression. In vitro growth assays revealed growth arrest following Ad5CMV-p53 infection as well as cell morphological changes consistent with apoptosis. In vivo studies in nude mice with established s.c. squamous carcinoma nodules showed that tumor volumes were significantly reduced in mice that received peritumoral infiltration of Ad5CMV-p53. These data suggest that Ad5CMV-p53 may be further developed as a potential novel therapeutic agent for SCCHN since introduction of wild-type p53 into SCCHN cell lines attenuates their replication and tumor growth.

Adenoviridae↗
PubMed1994

A genetically engineered, protein-based optical biosensor of myosin II regulatory light chain phosphorylation.

Myosin II is an important motor in the contraction of smooth and striated muscle as well as in a variety of non-muscle cell motile events including cytokinesis, cortical contractions during migration of fibroblasts, and capping of receptors. Phosphorylation of the 20-kDa light chain by myosin light chain kinase is part of the regulation of smooth muscle and mammalian nonmuscle myosin II. We designed, characterized, and tested the use of a protein-based optical biosensor to monitor this phosphorylation "switch." A regulatory light chain was genetically engineered to contain a single cysteine at amino acid position 18. The mutant light chain (Cys18.LC20), reacted with the fluorophore acrylodan, responded to phosphorylation of serine 19 with a fluorescence emission quenching of 60% and a 28-nm red-shift. When the acrylodan-labeled mutant light chain (AC-Cys18.LC20) was exchanged into turkey gizzard myosin II, it exhibited a 25% fluorescence emission quenching and a 10-nm red-shift upon phosphorylation of serine 19. The myosin II optical biosensor exhibited nearly control levels of the rate of phosphorylation, K+ATPase activity, and in vitro motility. The acrylodan-labeled light chain was exchanged into the A-bands of chicken pectoralis myofibrils in situ to demonstrate the localization and activity of the biosensor in a highly ordered contractile system. Fluorometry and quantitative fluorescence microscopic imaging experiments demonstrated that AC-Cys18.LC20 exchanged myofibrils expressed a phosphorylation-dependent fluorescence change. Labeled light chains were also incorporated into stress fibers of living fibroblasts and smooth muscle cells. This general approach of combining molecular biology and fluorescence spectroscopy to create novel protein-based optical biosensors should provide valuable tools for investigations with model systems and solution studies and ultimately yield important information about temporal-spatial chemical and molecular changes in live cells.

2-Naphthylamine↗
PubMed1994

Antigen presentation and cytotoxic T lymphocyte killing studied in individual, living cells.

Interactions between individual, living fibroblasts and cytotoxic T lymphocyte (CTL) clones were analyzed by using video-enhanced differential interference contrast and fluorescence microscopy in a multimode configuration. Fibroblasts expressing known major histocompatibility complex I alleles (MC57: H-2b; Balb: H-2d) were sensitized for killing by incubating or microinjecting them with peptide fragments of lymphocytic choriomeningitis virus. Previous determination of the CTL clones' specificity for these peptides and MHC-I alleles enabled us to study CTL killing of fibroblasts, and nonlethal CTL interaction with targets due to "mismatches" of the CTL, target, and/or peptide. During viral peptide-specific MHC-restricted CTL killing, distinct morphological alterations were observed (CTL shape changes, movements of granules in CTL cytoplasm, and target cell contraction and blebbing). When no killing occurred, CTL engaged in prolonged, nonrandom movement on the target cells. Alloreactive and virus-specific CTL displayed the same morphology during killing. To study antigen presentation further within individual, living cells, a LCMV glycoprotein peptide (aa 272-286, LSDSSGVENPGGYCL) was covalently labeled with tetramethylrhodamine. In 51Cr release assays, the labeled peptide specifically induced potent CTL killing, but neither labeled nor unlabeled peptide proved toxic for unsensitized targets. Microinjection of the labeled peptide into the cytoplasm of fibroblast cells led to CTL killing of those cells, yet nearby uninjected cells contacted by CTL were not killed, indicating that killing was due to presentation of microinjected peptide rather than binding of extracellular peptide to cell surface MHC. Peptide-injected target cells were killed only when combined with CTL specific for the peptide and for the MHC allele of the injected cell.

Amino Acid Sequence↗