PubMed Health⌕ Search

Biomedical subjects

R Shoemaker

Publications and source records attributed to R Shoemaker.

At least 19 recordsLinked to original sources
PubMed1992

Characterization of silkworm chlorophyll metabolites as an active photosensitizer for photodynamic therapy.

Silkworm excreta containing chlorophyll metabolites have long been used for their medicinal activities in the Far East. The major chlorophyll derivative (CpD) fraction in the Me2CO extract from the silkworm excreta exhibits strong photodynamic action on mice and human tumor cells in vitro. The main CpD component was isolated, purified, and subjected to extensive structural and characterization studies to assess its photodynamic therapeutic activity. According to the spectral analyses, the structure of the major unknown CpD was determined to be 10-hydroxypheophytin a [1]. The CpD component possessed a high quantum efficiency (50%) for the photosensitized production of singlet molecular oxygen, thus providing a molecular basis for its photodynamic therapeutic efficiency. A further advantage is that the "photooxidation/degradation" product(s) from this CpD retains all the characteristics of a photodynamic sensitizer.

Animals↗
PubMed1992

Immunocytochemical and functional characterization of Na+ conductance in adult alveolar pneumocytes.

The purpose of this study was to document the existence, assess the spatial localization, and characterize some of the transport properties of proteins antigenically related to epithelial Na+ channels in freshly isolated rabbit and rat alveolar type II (ATII) cells. ATII cells, isolated by elastase digestion of lung tissue and purified by density-gradient centrifugation, were incubated with polyclonal antibodies raised against Na+ channel protein purified from beef kidney papilla (NaAb), followed by a secondary antibody (goat antirabbit immunoglobulin G conjugated to fluorescein isothiocyanate). Rat ATII cells exhibited specific staining with NaAb at the level of the plasma membrane, which, in most cells, colocalized with that of the lectin Maclura pomiferra agglutinin, an apical surface marker. In Western blots, NaAb specifically recognized a 135 +/- 10-kDa protein in rat ATII membrane vesicles. When patch clamped in the whole cell mode using symmetrical solutions (150 mM Na+ glutamate), ATII cells exhibited outwardly rectified Na+ currents that were diminished by amiloride (10-100 microM) instilled into the bath solution. Ion substitution studies showed that the conductive pathways were three times more permeable to Na+ than K+. Amiloride, benzamil, and 5-(N-ethyl-N-isopropyl)-2',4'-amiloride were equally effective in diminishing 22Na+ flux into rabbit and rat ATII cells (45% inhibition at 100 microM, with IC50 of approximately 1 microM for all inhibitors). Tetraethylammonium chloride (10 mM) or BaCl2 (2 mM), well-known K+ channel blockers, had no effect on 22Na+ uptake. These results indicate that ATII cells express an amiloride-sensitive Na+ conductance, probably a channel, with a lower affinity for amiloride and its structural analogues than the well-established amiloride-sensitive Na+ channels found in bovine renal papila and cultured amphibian A6 kidney cells.

Amiloride↗
PubMed1991

Induction of multiple-drug resistance during anti-neoplastic chemotherapy in vitro.

Induction of P-glycoprotein-related multi-drug-resistance (MDR) has been shown in normal and malignant tissues to result from environmental stresses such as heat shock, exposure to carcinogens or X-ray irradiation. To identify conditions under which MDR is enhanced during anti-neoplastic chemotherapy, a cell line showing low-level intrinsic MDR was investigated. In the pleural mesothelioma cell line, PXF1118, less than 1% of cells expressed P-glycoprotein (P-gp), as shown by immunocytochemical staining with monoclonal antibody (MAb) MRK16. Exposure of PXF1118 to vincristine, vindesine, vinblastine or doxorubicin for 2-3 weeks led to an increase in the MDR cell fraction of up to 15-28% during 2 to 3 weeks. For doxorubicin and vindesine, dose-dependence was observed: drug concentrations not capable of eliciting cytotoxicity failed to induce significant P-gp expression. Nutrient starvation in aging medium, exposure to activated cyclophosphamide (even at high concentrations) or cisplatin caused only negligible MDR induction. After exposure to vindesine for 6 weeks, tumor colonies exhibited highly enhanced resistance to Vinca alkaloids, doxorubicin, etoposide and dacarbacine, whereas their sensitivity to mitomycin, activated cyclophosphamide or cisplatin remained unchanged. As determined by [3H]-thymidine uptake and proliferation antigen expression, induction of MDR phenotype was observed at minimal proliferative activity with no change in cell count during exposure to anti-cancer drugs, thus suggesting that the drug treatments changed the phenotype of the cells rather than selecting for a resistant sub-population. In addition, changes in cell differentiation were observed during MDR induction. Induction of P-gp during exposure to anti-cancer drugs thus provides a model for MDR development during initially successful chemotherapy. of P-gp during exposure to anti-cancer drugs thus provides

Adult↗
PubMed1991

Feasibility of a high-flux anticancer drug screen using a diverse panel of cultured human tumor cell lines.

We describe here the development and implementation of a pilot-scale, in vitro, anticancer drug screen utilizing a panel of 60 human tumor cell lines organized into subpanels representing leukemia, melanoma, and cancers of the lung, colon, kidney, ovary, and central nervous system. The ultimate goal of this disease-oriented screen is to facilitate the discovery of new compounds with potential cell line-specific and/or subpanel-specific antitumor activity. In the current screening protocol, each cell line is inoculated onto microtiter plates, then preincubated for 24-28 hours. Subsequently, test agents are added in five 10-fold dilutions and the culture is incubated for an additional 48 hours. For each test agent, a dose-response profile is generated. End-point determinations of the cell viability or cell growth are performed by in situ fixation of cells, followed by staining with a protein-binding dye, sulforhodamine B (SRB). The SRB binds to the basic amino acids of cellular macromolecules; the solubilized stain is measured spectrophotometrically to determine relative cell growth or viability in treated and untreated cells. Following the pilot screening studies, a screening rate of 400 compounds per week has been consistently achieved.

Cell Division↗
PubMed1991

The genetic locus controlling supernodulation in soybean (Glycine max L.) co-segregates tightly with a cloned molecular marker.

The genetic locus (nts) controlling nitrate-tolerant nodulation, supernodulation, and diminished autoregulation of nodulation of soybean (Glycine max (L.) Merill) was mapped tightly to the pA-132 molecular marker using a restriction fragment length polymorphism (RFLP) detected by subclone pUTG-132a. The nts (nitrate-tolerant symbiotic) locus of soybean was previously detected after its inactivation by chemical mutagenesis. Mutant plant lines were characterized by abundant nodulation (supernodulation) and tolerance to the inhibitory effects of nitrate on nodule cell proliferation and nitrogen fixation. The large number of RFLPs between G. max line nts382 (homozygous for the recessive nts allele) and the more primitive soybean G. soja (PI468.397) allowed the detection of co-segregation of several diagnostic markers with the supernodulation locus in F2 families. We located the nts locus on the tentative RFLP linkage group E about 10 cM distal to pA-36 and directly next to marker pA-132. This very close linkage of the molecular marker and the nts locus may allow the application of this clone as a diagnostic probe in breeding programs as well as an entry point for the isolation of the nts gene.

Chromosome Mapping↗
PubMed1990

Feasibility of cellular microencapsulation technology for evaluation of anti-human immunodeficiency virus drugs in vivo.

We investigated the feasibility of micro-encapsulation technology for the evaluation of anti-human immunodeficiency virus (HIV) drugs in vivo. The ability to place human cells in microcapsules with semipermeable membranes for implantation into test animals led to the development of this assay. The anti-HIV activity assay involves microencapsulating human T-lymphoblastoid cells sensitive to the cytopathic effects of HIV; the encapsulated cells are then implanted into athymic nude mice and recovered after drug treatment in vivo. A positive antiviral effect of the test substance is indicated by growth or survival of the virus-infected cells in the microcapsules. Several HIV-sensitive cell lines of T-lymphocyte, monocyte, and nonlymphocyte origin were examined for growth in microcapsules in vitro and in vivo. Light and electron microscopic analysis of the capsules and the human cells contained therein revealed the invasion of mouse immune cells and other adverse effects that could not be overcome by any of numerous technical modifications attempted. We conclude that cellular microencapsulation technology is not feasible for in vivo drug-testing protocols because of immunogenic reactions.

Animals↗
PubMed1989

Machine vision system for diagnostic histopathology.

In a machine vision system for the diagnostic assessment of histopathologic sections, human diagnostic knowledge and human ability to recognize components in a complex image need to be emulated. This is attempted by three integrated expert systems, supported by a multiprocessor computer with data-driven, dynamically reconfigurable architecture.

Adenocarcinoma↗
PubMed1988

A multidrug-resistant MCF-7 human breast cancer cell line which exhibits cross-resistance to antiestrogens and hormone-independent tumor growth in vivo.

MCF-7 human breast cancer cells provide a useful in vitro model system to study hormone-responsive breast cancer as they contain receptors for estrogen and progesterone, and estrogen both induces the synthesis of specific proteins in these cells and increases their rate of proliferation. An MCF-7 cell line which was selected for resistance to adriamycin (MCF-7/AdrR) exhibits the phenotype of multidrug resistance (MDR), and displays multiple biochemical changes. MDR in MCF-7/AdrR is also associated with a loss of mitogenic response to estrogen and the development of cross-resistance to the antiestrogen 4-hydroxytamoxifen. In addition, while the parental MCF-7 cell line responds to estrogen with increased levels of progesterone receptors and the secretion of specific proteins, these estrogen responses are lost in MCF-7/AdrR. Furthermore, while the formation of tumors in nude mice by wild-type MCF-7 cells is dependent upon the presence of estrogen, MCF-7/AdrR cells form tumors in the absence of exogenous estrogen administration. These changes in hormonal sensitivity and estrogen-independent tumorigenicity of the multidrug-resistant MCF-7 cell line are associated with a loss of the estrogen receptor and a concomitant increase in the level of receptors for epidermal growth factor. Thus, in MCF-7/AdrR cells, the development of MDR is associated with alterations in the expression of both cytosolic and membrane receptors, resulting in resistance to hormonal agents and the expression of hormone-independent tumor formation.

Animals↗
PubMed1987

Microencapsulated tumor assay: new short-term assay for in vivo evaluation of the effects of anticancer drugs on human tumor cell lines.

A new in vivo has been developed for evaluating the antitumor activity of chemotherapeutic drugs. The assay is based on a microencapsulation technology developed by Damon Biotech, Inc., Boston, MA, which makes it possible to encapsulate human tumor cells in small (about 1 mm in diameter) microcapsules with semipermeable membranes. Microcapsules containing human tumor cells were injected i.p. into nude or C57BL/6 mice and drugs were administered i.v. The microcapsules were recovered at various intervals following treatment and determinations of drug effects were made based on the differences in the number of tumor cells recovered from the treated and nontreated animals. Using this assay we found that (a) encapsulated tumor cells grew better in the in vivo system than in vitro under the conditions tested; (b) drugs crossed the capsular membrane and killed or inhibited the proliferation of tumor cells; and (c) the antitumor effect was consistent with the relative therapeutic efficacy of drugs or level of resistance of tumor cells detected by other in vitro or in vivo tests. The tumor microencapsulation assay offers several properties which make it attractive for use in new drug development: (a) the antitumor activity of drugs can be tested against human tumor cells under conditions which provide for three-dimensional growth and in vivo supply of nutrients; (b) the sensitivity of tumor cells can be assessed following exposure to drugs at concentrations which are achievable in vivo; (c) compounds requiring in vivo metabolic activation can be tested; (d) the effect of each drug injection can be quickly evaluated; (e) inhibition of tumor cell proliferation versus cytoreductive effects of drugs can be discriminated; (f) the test is applicable to virtually all histological types of human tumor cells; and (g) the tumor microencapsulation assay is a short-term, simple, and relatively inexpensive assay.

Animals↗
PubMed1987

Metastasis models for human tumors in athymic mice: useful models for drug development.

Although human tumor xenografts have been extensively used for preclinical evaluation of antitumor agents, most of this work has utilized subcutaneous or subrenal capsule assays based on change in tumor size. To obtain experimental models more reflective of the human clinical situations, we have developed several metastatic models that are based on and complement a panel of cell strains used in large-scale in vitro drug screening. One melanoma and four lung tumors produced metastatic lesions in the lung within 60 days following subcutaneous, intraperitoneal, or intrasplenic inoculation of BALB/C athymic nude mice. Several tumors also produced liver lesions, and one lung tumor strain showed metastasis to the brain. The metastatic lesions histologically resembled the tumors that grew at the inoculation site. In vitro and in vivo cell strains were rederived from the metastatic lesions. These systems may provide practical models for experimental drug and immunotherapeutic trials.

Animals↗
PubMed1986

Use of nude mouse xenografts as preclinical drug screens. Further studies on in vitro growth of xenograft tumor colony-forming cells.

Previous studies suggested tumor colony-forming cells (CFC) grown from xenografts might be useful as a preclinical, in vitro drug screen. To further evaluate this possibility, eight melanoma and six ovarian carcinoma xenografts were established from untreated patients and tested for in vitro CFC growth. For each tumor, linear relationships between cells plated and colony (30 cells or greater than 75 micron diameter) and cluster (10-30 cells or 50-75 micron) growth were observed. All eight melanomas grew sufficient colonies (greater than or equal to 30) for in vitro drug assessment, although four required hypoxic (pO2 = 40) incubation to reliably attain this level of growth. Only one in six of the ovary xenografts consistently grew enough colonies, and growth was not significantly improved by hypoxic incubation, or addition of luteinizing hormone, follicle-stimulating hormone, or steroid hormones. Cloning efficiencies (colonies + clusters/cells plated) for tumors demonstrating adequate growth ranged from 0.01% to 0.3%. For most tumors, no direct relationship was observed between characteristics of xenograft tumors (size) or their resulting cell suspensions (viabilities, cell yield) and CFC growth. Cell suspensions were incubated with a 3 log concentration of nine established chemotherapeutic agents. Resulting dose-effect curves were linear and showed no plateaus of drug effect. Analyzing 447 in vitro drug trials on six melanomas and one ovarian carcinoma, interexperiment variability was high. Cell lines were established from three xenografts using a low concentration of fetal bovine serum (1%), and also examined for in vitro drug sensitivity. Using both liquid culture isotope incorporation and a colony-forming assay, drug sensitivity profiles for the cell lines were nearly identical to those for parent xenograft CFC. However, assays performed using the cell lines were more reproducible than those using xenograft tissue. The authors conclude that tumor CFC can be reliably grown from melanoma xenografts, but in vitro drug assays using these xenografts are poorly reproducible. The xenografts are a resource for establishing cell lines, and drug assays performed using these lines are highly reproducible. Similarities in drug sensitivity profiles for parent xenograft CFC and derived cell lines suggest that, despite poor reproducibility, repetitive assays using melanoma CFC accurately reflect some properties of cells which sustain tumor cell growth.

Animals↗
PubMed1986

Metabolic stability of experimental chemotherapeutic agents in hepatocyte:tumor cell co-cultures.

A U.S. National Cancer Institute screening program for new anticancer drugs, based on the growth of primary human tumor cells in an in vitro soft agar colony formation assay, has resulted in the identification of a number of compounds that have cytotoxic activity against primary human tumor cells in vitro but are inactive in the conventional in vivo murine P388 leukemia animal model pre-screen. To investigate whether metabolic inactivation ov the compounds might be a factor in the lack of in vivo cytotoxicity we have co-cultured rat hepatocytes with A204 rhabdomyosarcoma and murine P388 leukemia cell lines in the soft agarose colony formation assay for 24 h during exposure to the compounds. Twenty compounds with a range of in vitro activities were studied. Thirteen compounds exhibited cytotoxicity against A204 cells in culture; nine of them were less active when co-cultured with hepatocytes, two were activated by hepatocyte co-culture, and two showed no effect of hepatocyte co-culture. P388 cells were more sensitive to the antiproliferative effects of the compounds than A204 cells. Two compounds that were not active against A204 cells exhibited cytotoxicity against P388 cells. One compound was inactivated by hepatocyte co-culture and one showed no effect. Five compounds showed no cytotoxicity toward either A204 cells or P388 cells. Two of the compounds showing hepatocyte inactivation in vitro possess activity in one or more in vivo tumor models. Thus, evidence for metabolic inactivation in hepatocyte co-culture is not always an indication for lack of in vivo antitumor activity. Hepatocyte co-culture methodology provides a simple and objective means, amenable to large-scale screening, of distinguishing metabolic activation or inactivation of a given compound from other pharmacokinetic and pharmacodynamic factors with a minimum of material.

Animals↗
PubMed1979

Ultrafast laser scanner microscope.

Advances in monolayer deposition of cervical cells have removed one of the last serious obstacles to the design of high-resolution automated diagnostic assessment systems. In this article, we describe the design considerations for a system that is capable of acquiring, within 60 sec, a 0.5 micron digitized image of a 4 cm2 area on a standard glass slide. The most feasible approach is found to be a system using a rotating polygon to sweep the focused spot from a laser across a 2-mm scan line while the slide is uniformly translated perpendicular to the scan direction the use of laser sources (a helium-neon laser at 632 nm and a krypton ion laser at 568 and/or 476 nm) as compared to the incoherent light sources used in conventional microscope systems alleviates many of the optical design problems and provides the proper wavelengths needed for recognition of Papanicolaou stained cells. We also find that focus control of the scanning spot should be achievable using a technique involving a holographic grating. Other relevant considerations such as sample heating problems, multiphoton absorption by the sample, detector signal-to-noise ratios, laser amplitude noise control, and the digitization and buffering of the data stream are also discussed.

Computers↗