Organic cations (OC's) are actively secreted by the renal proximal tubules in a number of species. The transepithelial transport of OC's involves a secondary active OC/H+ exchange process at the brush border (luminal) membrane. This study employed rabbit renal brush border membrane vesicles (BBMV) to investigate the structural requirements associated with substrate recognition at the OC transporter. A number of compounds (an N-alkylammonium series, an N1-alkylpyridinium series and some clinically important organic bases) were tested for their ability to competitively block the uptake of radioactively labelled tetraethylammonium (TEA) into BBMV. The inhibitory effectiveness of these compounds was correlated to the degree of hydrophobicity surrounding the positively-charged nitrogenous nucleus common to all the inhibitors. Preloading BBMV with N1-substituted pyridines trans-stimulated the uptake of TEA, suggesting that these compounds are translocated substrates for the OC transporter. The activity of the OC transport inhibitor and neurotoxin 1-methyl-4-phenylpyridinium was of special interest, and thus its transport characteristics were fully evaluated.

To study adaptation of pancreatic amylase to diet, an affinity adsorbent, alpha-GHI-AH-Sepharose 4B, was used to determine amylase synthesis in cultured pancreatic acinar cells. This adsorbent exhibited a consistent binding capacity and was specific for amylase. Acinar cells from rats fed high fat (HF) or carbohydrate (HC) diets for 7 d were cultured 1-48 h in serum-free medium. Amylase activity remained significantly higher in HC cells than in HF cells through 24 h in culture, despite its decrease with time in culture. The relative synthesis of amylase (3H-phe amylase/3H-phe total protein x 100) was significantly higher in HC than in HF cells at isolation and remained higher during culture. These results demonstrate that this affinity adsorbent can be used to determine amylase synthesis and suggest that the effect of diet on amylase activity and relative synthesis persists in cultured pancreatic acinar cells.

No description available.

No description available.

No description available.

No description available.

Master of Science / Biochemistry and Molecular Biophysics Interdepartmental Program / Anna Zolkiewska / Cellular senescence is a state of irreversible cell cycle arrest in response to non-lethal stress. In cancer cells, senescence can be induced by chemotherapy, radiation, or signals from the tumor microenvironment, such as transforming growth factor β (TGFβ). Senescent cells are metabolically active and have altered gene expression compared to their non-senescent counterparts. Senescent cells release a wide variety of factors, including extracellular domains of transmembrane proteins that require proteolytic cleavage by specific proteases. ADAMs (A Disintegrin and Metalloprotease domain-containing proteins) are enzymes that cleave many transmembrane proteins, such as growth factor precursors or adhesion molecules, and thus may act as sheddases in senescent cells. Here, we investigate ADAM expression levels during TGFβ- induced cellular senescence. SUM149PT and SUM102PT breast cancer cells were incubated with TGFβ, followed by treatment with high doses of paclitaxel to remove actively proliferating, non-senescent cells. Induction of cellular senescence was examined by evaluating changes in cell size and granularity, and by β-galactosidase staining. ADAM mRNA levels were measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Among several ADAMs tested, ADAM12 mRNA was significantly upregulated in senescent cells. In addition, we demonstrated that ADAM12 knock-down leads to decreased activation of epidermal growth factor receptor (EGFR), an important modulator of cancer cell growth, survival, and metastasis. This effect of ADAM12 knock-down was likely due to a diminished release of soluble EGF or EGF-like ligands from cells. Since senescent cells often release increased amounts of these ligands, ADAM12 may modulate the senescence secretome in senescent breast cancer cells.

Thesis (Ph.D.)--Massachusetts Institute of Technology, Dept. of Biology, 1982. / MICROFICHE COPY AVAILABLE IN ARCHIVES AND SCIENCE / Vita. / Bibliography: leaves 211-223. / by Douglas Elliott Koshland. / Ph.D.

by Grant Richardson Stenton. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (leaves 259-287). / Abstract --- p.i / Acknowledgements --- p.iii / Publications --- p.iv / Abbreviations --- p.v / Contents --- p.vii / Chapter Chapter 1 --- Introduction / Chapter 1 1.1. --- Historical Background --- p.2 / Chapter 1.2. --- Origin and distribution of mast cells --- p.2 / Chapter 1.3. --- Mast cell heterogeneity --- p.3 / Chapter 1.4. --- Mast cell mediators --- p.5 / Chapter 1.4.1. --- Preformed mast cell mediators --- p.6 / Chapter 1.4.2. --- Newly synthesised mast cell mediators --- p.7 / Chapter 1.5. --- Mast cell activation --- p.11 / Chapter 1.5.1. --- Antigenic pathway of mast cell activation --- p.11 / Chapter 1.5.1.1. --- Antigen binding and receptor aggregation --- p.12 / Chapter 1.5.1.2. --- Early events following FcεRI aggregation --- p.13 / Chapter 1.5.1.3. --- Antigenic induction of mast cell second messenger production --- p.15 / Chapter 1.5.1.4. --- Phospholipase C activation and mast cells --- p.16 / Chapter 1.5.1.5. --- Phospholipase A2 activation and mast cells --- p.17 / Chapter 1.5.1.6. --- Intracellular calcium and mast cells --- p.18 / Chapter 1.5.1.7. --- Calcium and calmodulin --- p.21 / Chapter 1.5.1.8. --- Adenylate cyclase activation and mast cells --- p.21 / Chapter 1.5.2. --- Non-antigenic pathway of mast cell activation --- p.22 / Chapter 1.6. --- Aims of the study --- p.25 / Chapter 1.6.1. --- Diuretics --- p.26 / Chapter 1.6.2. --- Histamine receptor directed compounds --- p.27 / Chapter 1.6.3. --- Cyclo-oxygenase inhibitors --- p.28 / Chapter 1.6.4. --- Immunosuppressive compounds --- p.29 / Chapter Chapter 2 --- Materials and Methods --- p.31 / Chapter 2.1. --- Materials and methods --- p.32 / Chapter 2.1.1. --- Secretagogues --- p.32 / Chapter 2.1.2. --- Anti-allergic compounds --- p.32 / Chapter 2.1.3. --- Diuretics --- p.32 / Chapter 2.1.4. --- Immunosuppressants --- p.33 / Chapter 2.1.5. --- Histamine agonists and antagonists --- p.33 / Chapter 2.1.6. --- Cyclo-oxygenase inhibitors --- p.33 / Chapter 2.1.7. --- Materials for buffers --- p.34 / Chapter 2.1.8. --- Materials for rat sensitization --- p.34 / Chapter 2.1.9. --- Materials for histamine assay --- p.35 / Chapter 2.1.10. --- Materials for calcium measurement --- p.35 / Chapter 2.1.11. --- Materials for prostaglandin D2 measurement --- p.35 / Chapter 2.1.12. --- Materials for leukotriene C4 measurement --- p.36 / Chapter 2.1.13. --- Materials for cyclic AMP measurement --- p.36 / Chapter 2.1.14. --- Miscellaneous --- p.36 / Chapter 2.2. --- Buffers and stock solutions --- p.37 / Chapter 2.2.1. --- Buffer ingredients --- p.37 / Chapter 2.2.2. --- Stock solutions --- p.38 / Chapter 2.3. --- Animals and cell isolation --- p.39 / Chapter 2.3.1. --- Animals --- p.39 / Chapter 2.3.2. --- Sensitization of animals --- p.39 / Chapter 2.3.3. --- Cell isolation --- p.40 / Chapter 2.3.4. --- Cell washing and purification --- p.41 / Chapter 2.3.5. --- Preparation of cells for counting --- p.42 / Chapter 2.3.6. --- Cell counting on a haemocytometer --- p.42 / Chapter 2.4. --- General protocol for histamine release and histamine measurement --- p.43 / Chapter 2.4.1. --- Histamine release --- p.43 / Chapter 2.4.2. --- Spectroflurometric determination of histamine contents --- p.44 / Chapter 2.4.3. --- Calculation of histamine levels --- p.45 / Chapter 2.5. --- Protocol for cellular calcium measurement --- p.47 / Chapter 2.5.1. --- 45Ca2+ influx measurement --- p.47 / Chapter 2.5.2. --- Calculation of 45Ca2+ influx --- p.48 / Chapter 2.5.3. --- Fura-2 fluorescence measurement of intracellular calcium --- p.48 / Chapter 2.5.4. --- Fura-2 cell loading --- p.48 / Chapter 2.5.5. --- Fura-2 fluorescence parameters --- p.49 / Chapter 2.5.6. --- Calculation of basal calcium levels --- p.50 / Chapter 2.6. --- Protocol for prostaglandin D2 (PGD2) measurement --- p.52 / Chapter 2.6.1. --- PGD2 production --- p.52 / Chapter 2.6.2. --- Enzyme Immunosorbent Assay (EIA) method of PGD2 measurement --- p.52 / Chapter 2.6.3. --- Calculation of (EIA) PGD2 production --- p.53 / Chapter 2.6.4. --- Radio Immunosorbent Assay (RIA) method of PGD2 measurement --- p.53 / Chapter 2.6.5. --- Calculation of (RIA) PGD2 concentration --- p.54 / Chapter 2.7. --- Protocol for leukotriene C4 (LTC4) measurement --- p.54 / Chapter 2.7.1. --- LTC4 production --- p.54 / Chapter 2.7.2. --- Enzyme Immunosorbent Assay (EIA) method of LTC4 measurement --- p.55 / Chapter 2.7.3. --- Calculation of (EIA) LTC4 concentration --- p.55 / Chapter 2.8. --- Protocol for cyclic adenosine monophosphate (cAMP) measurement --- p.56 / Chapter 2.8.1. --- cAMP production --- p.56 / Chapter 2.8.2. --- Radio Immunosorbent Assay (RIA) method of cAMP measurement --- p.56 / Chapter 2.8.3. --- Calculation of cAMP concentration --- p.57 / Chapter 2.9. --- Statistical analysis --- p.57 / Chapter Chapter 3 --- "Frusemide, Bumetanide and DSCG" --- p.58 / Chapter 3.1. --- Introduction --- p.59 / Chapter 3.1.1. --- Frusemide and bumetanide as loop diuretics --- p.59 / Chapter 3.1.2. --- Effects of frusemide and bumetanide on the airways --- p.59 / Chapter 3.1.3. --- Effects of frusemide on mast cells --- p.60 / Chapter 3.1.4. --- Experimental aims --- p.61 / Chapter 3.2. --- Materials and methods --- p.62 / Chapter 3.3. --- Results --- p.63 / Chapter 3.3.1 --- "Effects of frusemide, bumetanide and DSCG on immunologically induced histamine release from rat peritoneal mast cells" --- p.63 / Chapter 3.3.2. --- "Effects of frusemide, bumetanide and DSCG on compound 48/80 induced histamine release from rat peritoneal mast cells" --- p.64 / Chapter 3.3.3. --- "Effects of frusemide, bumetanide and DSCG on compound 48/80 induced histamine release from rat peritoneal mast cells suspended in calcium free buffer" --- p.65 / Chapter 3.3.4. --- "Effects of frusemide, bumetanide and DSCG on ionophore A23187 and thapsigargin induced histamine release from rat peritoneal mast cells" --- p.65 / Chapter 3.3.5. --- Cross-tachyphylaxis effects of frusemide and bumetanide --- p.66 / Chapter 3.3.6. --- Effects of DSCG on the inhibition of anaphylactic histamine release due to frusemide --- p.67 / Chapter 3.3.7. --- Effects of frusemide and DSCG on immunologically and non-immunologically induced 45Ca2+ uptake --- p.67 / Chapter 3.3.8. --- Effects of frusemide and DSCG on immunologically and non-immunologically induced changes in the free intracellular calcium concentration of rat peritoneal mast cells --- p.68 / Chapter 3.3.9. --- Effects of frusemide and bumetanide on the spontaneous and secretagogue induced PGD2 production from rat peritoneal mast cells --- p.69 / Chapter 3.3.10. --- Effects of frusemide and DSCG on cellular cAMP levels --- p.70 / Chapter 3.4. --- Discussion --- p.101 / Chapter 3.5. --- Summary --- p.111 / Chapter 3.6. --- Conclusion --- p.114 / Chapter 3.7. --- Future studies --- p.114 / Chapter Chapter 4 --- Histamine Receptor Directed Compounds --- p.115 / Chapter 4.1. --- Introduction --- p.116 / Chapter 4.1.1. --- Histamine receptor subtypes --- p.116 / Chapter 4.1.2. --- Histamine effects on the airways --- p.117 / Chapter 4.2. --- Signal transduction mechanisms --- p.118 / Chapter 4.2.1. --- H1-receptors --- p.118 / Chapter 4.2.2. --- H2-receptors --- p.119 / Chapter 4.2.3. --- H3-receptors --- p.120 / Chapter 4.3. --- Histamine receptors and mast cells --- p.120 / Chapter 4.3.1. --- Effects of histamine agonists and antagonists on mast cells --- p.120 / Chapter 4.3.2. --- Experimental aims --- p.122 / Chapter 4.3.3. --- Materials and methods --- p.123 / Chapter 4.4. --- Results --- p.123 / Chapter 4.4.1. --- Effects of the test compounds on the spontaneous histamine release from rat peritoneal mast cells --- p.123 / Chapter 4.4.2. --- Effects of the test compounds on anti-IgE induced histamine release from rat peritoneal mast cells --- p.125 / Chapter 4.4.3. --- Effects of the test compounds on compound 48/80 induced histamine release from rat peritoneal mast cells --- p.126 / Chapter 4.4.4. --- Effects of the test compounds on anti-IgE and compound 48/80induced histamine release from rat peritoneal mast cells in calcium free buffer --- p.126 / Chapter 4.4.5. --- Effects of the test compounds on ionophore A23187 induced histamine release from rat peritoneal mast cells --- p.127 / Chapter 4.4.6. --- "Effects of histamine antagonists on dimaprit, imetit and impromidine induced histamine release from rat peritoneal mast cells" --- p.128 / Chapter 4.4.7. --- "Effects of anti-IgE, dimaprit and imetit on PGD2 production from rat peritoneal mast cells" --- p.128 / Chapter 4.4.8. --- "Effects of benzalkonium chloride (BAC) on dimaprit, imetit, compound 48/80 and anti-IgE induced histamine release from rat peritoneal mast cells" --- p.129 / Chapter 4.4.9. --- "Effects of pertussis toxin on dimaprit, imetit, compound 48/80and anti-IgE induced histamine release from rat peritoneal mast cells" --- p.129 / Chapter 4.4.10. --- "Effects of dimaprit, imetit, compound 48/80 and anti-IgE on the free intracellular calcium concentration of rat peritoneal mast cells" --- p.130 / Chapter 4.5. --- Discussion --- p.171 / Chapter 4.5.1. --- The possible existence of histamine receptors on rat peritoneal mast cells --- p.171 / Chapter 4.5.2. --- "Possible mechanism of action for the histamine releasing actions of dimaprit, imetit and impromidine on rat peritoneal mast cells" --- p.174 / Chapter 4.6. --- Conclusion --- p.181 / Chapter 4.7. --- Future studies --- p.182 / Chapter Chapter 5 --- Cyclo-oxygenase Inhibitors --- p.184 / Chapter 5.1. --- Introduction --- p.185 / Chapter 5.1.1. --- Cyclo-oxygenase isozymes --- p.185 / Chapter 5.1.2. --- Cyclo-oxygenase inhibitors and mast cells --- p.186 / Chapter 5.1.3. --- Experimental aims --- p.190 / Chapter 5.2. --- Materials and methods --- p.190 / Chapter 5.3. --- Results --- p.191 / Chapter 5.3.1. --- Effects of cyclo-oxygenase inhibitors on immunologically and non-immunologically induced histamine release from rat peritoneal mast cells - --- p.191 / Chapter 5.3.2. --- Effects of cyclo-oxygenase inhibitors on immunologically and non-immunologically induced PGD2 production from rat peritoneal mast cells --- p.192 / Chapter 5.3.3. --- Effects of cyclo-oxygenase inhibitors on immunologically and non-immunologically induced LTC4 production from rat peritoneal mast cells --- p.192 / Chapter 5.3.4. --- Effects of cyclo-oxygenase inhibitors on immunologically and non-immunologically induced 45Ca uptake by rat peritoneal mast cells --- p.193 / Chapter 5.4. --- Discussion --- p.221 / Chapter 5.5. --- Summary and Conclusion --- p.225 / Chapter Chapter 6 --- Immunosuppressive Drugs --- p.228 / Chapter 6.1. --- Introduction --- p.229 / Chapter 6.1.1. --- CsA and FK506 binding proteins --- p.230 / Chapter 6.1.2. --- Distribution of CyPA and FKBP12 --- p.231 / Chapter 6.1.3 --- Mechanism of immunosuppression --- p.232 / Chapter 6.1.4. --- The role of calcineurin in IL-2 promoter induction --- p.233 / Chapter 6.2. --- Immunosuppressive agents and mast cells --- p.234 / Chapter 6.2.1. --- Introduction --- p.234 / Chapter 6.2.2. --- CsA and FK506 inhibit mast cell cytokine production --- p.235 / Chapter 6.2.3. --- "CsA mediated inhibition of mediator release from, and calcium uptake by mast cells and basophils" --- p.236 / Chapter 6.2.4. --- Inhibition of mediator release from mast cells and basophils by FK506 --- p.239 / Chapter 6.2.5. --- Aim of this study --- p.240 / Chapter 6.2.6. --- Materials and methods --- p.241 / Chapter 6.3. --- Results --- p.241 / Chapter 6.3.1. --- Effects of CsA and FK506 on immunologically and non-immunologically induced histamine release from rat peritoneal mast cells --- p.241 / Chapter 6.3.2. --- Effects of CsA and FK506 on immunologically and non-immunologically induced PGD2 production from rat peritoneal mast cells --- p.242 / Chapter 6.3.3. --- Effects of CsA and FK506 on immunologically and non-immunologically induced 45Ca uptake by rat peritoneal mast cells --- p.243 / Chapter 6.4. --- Discussion --- p.254 / Chapter 6.4.1. --- "Effects of CsA on histamine release from, and 45Ca uptake by rat peritoneal mast cells, following immunological and non-immunological activation" --- p.254 / Chapter 6.4.2. --- Effects of CsA on PGD2 production from rat peritoneal mast cells --- p.256 / Chapter 6.4.3. --- "Effects of FK506 on histamine release from, and 45Ca uptake by rat peritoneal mast cells, following immunological and non-immunological activation" --- p.256 / Chapter 6.4.4. --- Effects of FK506 on immunological PGD2 production from rat peritoneal mast cells --- p.257 / Chapter 6.5. --- Summary --- p.257' / Chapter 6.6. --- Future work --- p.258 / References

The obligate intracellular protozoan, Leishmania infantum chagasi (Lic) is the causative agent of visceral leishmaniasis in South America. The flagellated promastigote life stage of the parasite undergoes receptor-mediated phagocytosis by macrophages. This process is followed by a transient delay in phagolysosome maturation that allows for conversion into the amastigotes, a stage that is resistant to degradation inside host cells. We hypothesized that events occurring early during parasite-host interaction influence whether the pathogen ultimately survives or is eliminated in the intracellular environment, and that these processes are facilitated by determinants from both the macrophage and the incoming Leishmania. We found differences in the pathway through which virulent Lic metacyclic promastigotes or avirulent logarithmic promastigotes are phagocytosed by human monocyte-derived macrophages (MDMs). The macrophage surface receptors that ligated the two forms of promastigotes differed, guiding metacyclic promastigotes into a compartment that supported their replication and logarithmic promastigotes into a vacuole that rapidly assembled its microbicidal machinery. Survival of metacyclic promastigotes following their phagocytosis also varied greatly on characteristics of the host macrophage. U937 cells, a model monocytic cell line lacking the third complement receptor (CR3) on their surface, took up parasites via a unique "coiling" mode of pseudopod extension, leading to a formation of a phagosome that did not fully mature. Since the parasites never demonstrated escape into the macrophage cytosol, it is logical to predict that they synthesize and release virulence factors that localize within the parasitophorous vacuole (PV) in order to establish communication with the host cell. Using a previously assembled bioinformatic catalogue of putatively secreted or excreted (E/S) proteins encoded in the Leishmania infantum genome, we chose four candidate proteins for further analysis. Two of these, serine carboxypeptidase (CBP) and a flavodoxin domain-containing protein (HP) coding sequences, were overexpressed or removed in Lic. Parasites lacking one allele of either CBP or HP were defective in survival within MDMs. Furthermore, recombinant overexpressed HP was detected from parasite lysate in a stage-specific manner, paralleling expression in wild type Lic. This implies that the regulatory elements within the protein coding sequence remain functional outside of their native locus. Taken together, our study shows that quiescent entry of virulent Leishmania spp. into macrophages is accounted for by i) the ability of metacyclic promastigotes to selectively bypass macrophage components leading to deleterious pathways, as well as ii) tightly regulated parasite virulence factors for deliberately enhancing intracellular survival.