Direct communication of homologous and heterologous endocrine islet cells in culture

The transfer of 6-carboxyfluorescein between islet cells in monolayer culture was observed by fluorescence microscopy, and the endocrine cells involved in this transfer were identified by immunohistochemistry and electron microscopy. The results show that carboxyfluorescein was directly exchanged between homologous B-cells and also between B- and A- or D-cells. Successive microinjections of the probe into different cells of the same cluster showed the existence of separate territories, each formed by 2-8 communicating cells. Intercellular communication was not observed after every dye microinjection, and communicating and noncommunicating islet cells were found to coexist within the same cluster. The data indicate that the exchange of exogenous cytoplasmic molecules occurs between different types of endocrine islet cells. However, within a single cluster, all islet cells are not metabolically coupled to one another, at a given time.     

Interactions of competent Streptococcus sanguis (Wicky) cells with native or denatured, homologous or heterologous deoxyribonucleic acids.

Competent cell-deoxyribonucleic acid (DNA) interactions were examined using tritium-labeled homologous or heterologous native or denatured DNAs and competent Streptococcus sanguis Wicky cells (strain WE4). The DNAs used were extracted from WE4 cells, Escherichia coli B cells, and E. coli bacteriophages T2, T4, T6, and T7. The reactions examined were: (i) total DNA binding, (ii) deoxyribonuclease-resistant DNA binding, and (iii) the production of acid-soluble products from the DNA. Optimal temperatures for the reactions were as follows: reaction (i), between 30 and 40 degrees C; reaction (ii), 30 degrees C; and reaction (iii), greater than 40 degrees C. The rates for the reactions (expressed as molecules of DNA that reacted per minute per colony-forming unit) did not vary greatly from one DNA source to another. With a constant competent cell concentration and differing DNA concentrations below a saturation level (from a given source), a different but constant fraction of the added DNA was cell bound, deoxyribonuclease resistant, and degraded to acid-soluble products. In experiments where the number of competent cells was varied and the DNA concentration was held constant, again essentially the same result was obtained. The extent of reactions (i), (ii), and (iii) depended upon the numbers as well as the source of DNA molecules applied to competent cells. Calcium ion essential for native DNA-cell reactions was also found essential for denatured DNA-cell reactions. Data obtained from competition experiments lead to the conclusion that competent WE4 cells contain specific sites for native as well as denatured DNAs.     

CHSMiner: a GUI tool to identify chromosomal homologous segments

Background  

The identification of chromosomal homologous segments (CHS) within and between genomes is essential for comparative genomics. Various processes including insertion/deletion and inversion could cause the degeneration of CHSs.     

The incorporation of homologous and heterologous hypoxanthine-guanine phosphoriboxyltransferase into mutant cells

Experiments are described leading to partial compensation of a deficiency in the enzyme hypoxanthine-guanine phosphoribosyltransferase in mutant cells by supplying the cells with exogenous purified enzymes. DEAE-dextran is an effective helper agent, whereas poly (L-lysine), lysolecithin and amphotericin B seem to inhibit the entry of the enzymes of their activity. Enzyme preparation from Chinese hamster was found to have different effects in different mutant cell lines. In mutant Chinese hamster cells, the electrophoretic activity pattern remains unchanged for the Chinese hamster enzyme, but changes progressively to faster-moving activity peaks for the human enzyme after several hours. The metabolic effect of the incorporated enzyme is in the range between 3 and 4% of the normal cellular enzyme activity which corresponds to a 10--20 fold increase of hypoxanthine-guanine phosphoribosyltransferase activity in the mutant cells.     

The incorporation of homologous and heterologous hypoxanthine-guanine phosphoribosyltransferase into mutant cells

Experiments are described leading to partial compensation of a deficiency in the enzyme hypoxanthine-guanine phosphoribosyltransferase in mutant cells by supplying the cells with exogenous purified enzymes. DEAE-dextran is an effective helper agent, whereas poly(L-lysine, lysolecithin and amphotericin B seem to inhibit the entry of the enzymes or their activity. Enzyme preparation from Chinese hamster was found to have different effects in different mutant cell lines. In mutant Chinese hamster cells, the electrophoretic activity pattern remains unchanged for the Chinese hamster enzyme, but changes progressively to faster-moving activity peaks for the human enzyme after several hours. The metabolic effect of the incorporated enzyme is in the range between 3 and 4% of the normal cellular enzyme activity which corresponds to a 10–20 fold increase of hypoxanthine-guanine phosphoribosyltransferase activity in the mutant cells.     

SP analysis may be used to identify cancer stem cell populations

Side populations (SP), as defined by Hoechst exclusion in flow cytometry, have been described a few years ago. While they represent only a small fraction of the whole cell population, their properties confer an important place in several investigations. SP cells express high levels of various members of ABC transporters family, such as MDR1 and BCRP, which are responsible for drug resistance. Targeting SP could improve cancer therapy by blocking these transporters. In addition, SP appear to be enriched in stem cells, cells that play a pivotal role in normal development and cancer biology. Thus, they could provide a useful tool and a readily accessible source for stem cell studies in both the normal and cancerous settings. However, these cells are poorly defined and pose challenges in their identification and isolation, particularly since they are few in number. Thus, better characterization of SP will advance our understanding of stem cells and will provide us an accessible target for drug resistance in cancer therapy.     

Chemotherapy sorting can be used to identify cancer stem cell populations

Cancer stem cells (CSCs) of bladder transitional cell cancers (BTCC) had not been identified by the reported common methods. According to the phenomenon that CSCs were resistant to chemotherapy, BTCC cell lines T24 and 5637 were cultured with mitomycin C respectively. Cell inhibition assay revealed an increased population of drug resistant cancer cells with a concentration gradient of mitomycin C. The maximal and minimal cell inhibition rate in cell line T24 was 92.5?%?±?1.0 versus 64.1?%?±?1.4 (P?相似文献   

Platelet-activating factor-induced homologous and heterologous desensitization in cultured vascular smooth muscle cells

Intracellular free Ca2+ concentrations were monitored in vascular smooth muscle cells (VSMC) using the Ca2+-sensitive dye fura II. Superfusion of VSMC with platelet-activating factor (S-PAF; 1-100 nM) increased cytosolic Ca2+ in a dose-dependent manner. The response was transient and returned to base line even though the agonist was still present. A second, higher dose of PAF did not elicit a response. The inactive optical isomer, R-PAF, was ineffective suggesting that the S-PAF response is specific and receptor-mediated. Pretreatment of VSMC with PAF attenuated angiotensin II-stimulated Ca2+ mobilization but not vasopressin-stimulated Ca2+ mobilization. Treatment of VSMC with PAF (10 nM) stimulated inositol trisphosphate and inositol tetrakisphosphate formation above control by 260 +/- 15% and 195 +/- 11%, respectively. Diacylglycerol levels also rose during PAF stimulation and remained increased over 15 min. Pretreatment of VSMCs with phorbol-12,13-myristate acetate (10 nM) for 30 min abolished both the PAF- and angiotensin II-induced increases in cytosolic Ca2+, but not the vasopressin-induced increase. Pretreatment of VSMC with dioctanoylglycerol (10 microM) abolished the S-PAF-, angiotensin II-, and vasopressin-induced elevation in cytosolic Ca2+. We propose that this desensitization is possibly mediated by diacylglycerol formed in response to PAF.     

Interferon and butyrate treatment leads to a decreased sensitivity of NK target cells to lysis by homologous but not by heterologous effector cells

Human K-562 and HHMS cells were pretreated with human recombinant interferon (IFN)-gamma and used as targets in NK assays against human and murine effector cells. A protective effect against NK lysis was observed only in the homologous assay, whereas no change or even a slight increase in NK sensitivity against heterologous effector cells was found. In cold target inhibition experiments IFN-treatment of K-562 cells led to a decrease in their capacity to act as competitors in the homologous NK assay, leaving their inhibitory capacity unaltered in the heterologous assay. In accordance with results observed using human NK targets, murine YAC-1 cells treated with mouse recombinant IFN-gamma did not lose their susceptibility to human NK cells. However, they were markedly less susceptible to lysis mediated by murine effectors. Butyrate, another compound causing decreased sensitivity of K-562 cells for human natural killing, also failed to reduce the susceptibility against murine NK cells. The results indicate that the NK-resistant tumor target phenotype caused by IFN or differentiation-inducing agents can only be detected by homologous but not by heterologous effector cells. This suggests that major differences exist between the inter- and intraspecies NK killing mechanisms.     

Use of pancreatic beta cells in culture to identify diabetogenic N-nitroso compounds

Epidemiological observations suggest that environmental factors play a role in the pathogenesis of insulin dependent diabetes mellitus (1). Several chemicals have been identified as specific beta cell toxins (2-4). We report here studies to determine the feasibility of using monolayer cultures of pancreatic beta cells from neonatal rat to screen potential diabetogenic chemicals. Cytotoxicity was monitored both by phase microscopy and the release of insulin into the culture medium. In comparative studies, cellular protein and release of 51chromium (51Cr) were measured after addition of test compounds to cultures of fibroblasts derived from pancreatic tissue. The nitrosoamides 1 methyl-l-nitrosourea (MNU), 1,3 bis (2-choroethyl) nitrosourea (BCNU), chlorozotocin (CLZ), and the beta cell toxin, streptozotocin (SZ), were examined. CLZ and SZ were more toxic to pancreatic beta cells than to fibroblasts. In contrast, MNU and BCNU damaged both beta cells and fibroblasts at identical concentrations. These results suggest that in vitro techniques can be used to identify chemicals that selectively injure beta cells. Although SZ-induced toxicity was ameliorated with addition of nicotinamide to cultures of beta cells, nicotinamide did not prevent damage caused by CLZ. This observation indicates different mechanisms of drug-induced cytotoxicity.     

Centrifugal separation of irradiated cultures of Escherichia coli cells into viable and nonviable populations.

Incubation of ultraviolet-irradiated Escherichia coli B/r cultures with 0.7% Triton X-100 resulted in a large decrease in turbidity. Under phase-contrast optics, most of the irradiated detergent-treated cells were smaller than normal and of low phase density; only a small percentage were normal or larger than normal and of normal phase density. Irradiated cells not treated with detergent showed fewer pronounced morphological changes. Irradiated cells treated with detergent lost large amounts of proteins and ribonucleic acid, but not of deoxyribonucleic acid. Such cultures could be separated by centrifugation into populations of (i) slowly sedimenting cells consisting of small, phase-light cells of low viability and (ii) large cells of normal phase density and high viability (100%). A similar separation was effected in gamma-irradiated cultures.     

Enhancement of the antibody response to flavivirus B-cell epitopes by using homologous or heterologous T-cell epitopes.

We have been investigating the T-helper (Th)-cell response to the flavivirus envelope (E) glycoprotein. In our studies with Murray Valley encephalitis (MVE) virus, we previously identified synthetic peptides capable of priming Th lymphocytes for an in vitro antivirus proliferative response (J. H. Mathews, J. E. Allan, J. T. Roehrig, J. R. Brubaker, and A. R. Hunt, J. Virol. 65:5141-5148, 1991). We have now characterized in vivo Th-cell priming activity of one of these peptides (MVE 17, amino acids 356 to 376) and an analogous peptide derived from the E-glycoprotein sequence of the dengue (DEN) 2, Jamaica strain (DEN 17, amino acids 352 to 368). This DEN peptide also primed the Th-cell compartment in BALB/c mice, as measured by in vitro proliferation and interleukin production. The failure of some MVE and DEN virus synthetic peptides to elicit an antibody response in BALB/c mice could be overcome if a Th-cell epitope-containing peptide was included in the immunization mixture. A more detailed analysis of the structural interactions between Th-cell and B-cell epitope donor peptides revealed that the peptides must be linked to observe the enhanced antibody response. Blockage or deletion of the free cysteine residue on either peptide abrogated the antibody response. The most efficient T-B-cell epitope interaction occurred when the peptides were colinearly synthesized. These Th-cell-stimulating peptides were also functional with the heterologous B-cell epitope-containing peptides. The Th-cell epitope on DEN 17 was more potent than the Th-cell epitope on MVE 17.