Nucleoside analogs are important components of treatment regimens for acute leukemia in adults. Plasma membrane permeation of the nucleoside analog molecules, the initial event in the cellular conversion of nucleosides to active agents, is mediated by nucleoside-specific membrane transporters. The widely-expressed es nucleoside transporter accepts as substrates diverse nucleoside analogs, including cytarabine (araC), 2-chlorodeoxyadenosine, and fludarabine. The cellular content of es transporter sites has been measured in blasts from patients with acute lymphoblastic leukemia and acute myelogenous leukemia, by a sensitive, quantitative flow cytometry assay that employs the tightly-bound es ligand, SAENTA fluorescein. Values for es transporter expression varied ten-fold among samples from patients with acute myelogenous leukemia. In this article, we review current findings that document, in confocal fluorescence microscopy images and in flow cytometry assays of SAENTA fluorescein-stained cells, the patient-to-patient variance of es transporter expression in leukemic blasts from patients. Our data show a correlation between the expression of es transporters and the in vitro sensitivity to nucleoside drugs of blasts from acute leukemia patients. These findings show that the flow cytometry assay of es expression provides a facile means of predicting resistance of leukemia cells to the cytotoxicity of araC and other nucleosides.
We have analyzed the kinetics of synthesis, phosphorylation, and stability of the soluble and insoluble plakoglobin (PG) and their interactions with Dsg1 and E-cadherin in Madin-Darby canine kidney (MDCK) epithelial cells in the absence of cell adhesion and after the induction of cell-cell contact. Using a combination of biochemical and morphological approaches, we show that newly synthesized PG enters a soluble:insoluble pool of proteins in a 60:40 ratio regardless of cell-cell contact. Following synthesis, PG is increasingly found in the insoluble pool. Although cell-cell contact does not effect either the size of each pool or the rate or efficiency of the transfer from the soluble into the insoluble pool, it results in a significant increase in the metabolic stability of the newly synthesized insoluble PG. The soluble PG initially forms separate complexes with E-cadherin and Dsg1. PG-Dsg1 complexes become insoluble and localize to the desmosome. PG-E-cadherin complexes remain soluble and are distributed intracellularly. The insoluble PG and E-cadherin detected at the cell periphery remain distinctly separate, as demonstrated previously [Hinck et al., 1994: J. Cell Biol. 125:1327-1340; Nathke et al., 1994: J. Cell Biol. 125:1341-1352]. In addition, we detected a separate pool of PG which is not associated with either Dsg1 or E-cadherin and after the induction of cell-cell contact becomes primarily insoluble and is distributed along the lateral membrane. Phosphorylation analysis showed that there is a significantly greater amount of phosphorylated PG in the soluble pool than in the insoluble pool. In addition the soluble pool is both serine and threonine phosphorylated, whereas the insoluble PG is primarily phosphorylated on serine residues.
Polyclonal antibodies raised against mouse 2.5S NGF (mNGF) and against synthetic peptides made from hydrophilic portions of mNGF have been used to compare the immunological properties of mNGF, human recombinant brain-derived neurotrophic factor (hrBDNF), and human recombinant neurotrophin-3 (hrNT-3). Affinity-isolated antibodies raised against intact mNGF reacted with all three neurotrophins when tested by ELISA and totally or partially blocked the bioactivities of the proteins in survival assays of embryonic chicken sensory and sympathetic neurons. On Western blots, mNGF antibodies reacted with all three neurotrophins but less well with hrBDNF and hrNT-3 than with mNGF. Antibodies to hydrophilic peptides within NGF (amino acids 23-35, 59-67, 69-79, and 91-100) showed partial reactivity with some but not all of the neurotrophins when tested by ELISA and on Western blots. The peptide antibodies were also selectively effective in reducing the survival-promoting activity of the neurotrophins on sensory neurons. Results show that mNGF, hrBDNF, and hrNT-3 are immunologically related proteins and that mNGF antibodies react also with other members of the neurotrophin family.
Hypocrellins are naturally occurring compounds with photosensitizing properties in biological systems. We have prepared synthetic derivatives of hypocrellin B, which have promise as photosensitizers in the clinical application of photodynamic therapy. The intracellular localization and uptake kinetics of hypocrellin B and several selected hypocrellin congeners were determined semiquantitatively by fluorescence confocal microscopy in monolayer cultures of EMT6/Ed murine tumor cells. Each compound had unique uptake kinetics. Although no compound tested to date has demonstrated nuclear labeling, most could be detected in lysosomes, Golgi, endoplasmic reticulum and, to a minor extent, in cellular membranes. No two compounds gave identical labeling distributions. The differences are assumed to originate in physicochemical properties characteristic of each compound, which may ultimately impact upon the primary modality of phototoxicity.
The prevalence of celiac disease (CD) was determined in healthy blood donors and in high-risk groups of adults (a total of 1835 adults--randomly selected 1312 healthy blood donors, 102 patients with primary osteoporosis, 58 patients with autoimmune diseases and 365 infertile women). It was calculated on the basis of a two-step serologic screening method--in the first step IgA and IgG antigliadin antibodies (AGA) and IgA anti-gamma-glutamyltransferase ('transglutaminase') antibodies (ATG) were estimated, in the second step sera positive for IgA AGA and/or IgA ATG were examined for antiendomysial IgA (AEA) antibodies. Immunoenzymic assay (ELISA) was used for determining of AGA and ATG antibodies; immunofluorescence method, performed on human umbilical cord tissue, was used for assaying of AEA antibodies. Total serum IgA level in only IgG AGA positive subjects was measured by routine turbidimetric method. 0.45% of healthy blood donors, 0.98% of osteoporotic patients, 2.7% of patients suffering from autoimmune disease and 1.13% of women with infertility considered as immunologically mediated were found to be positive in both steps of serologic screening (AGA and/or ATG and antiendomysium positive). The presumed high prevalence of seropositivity for CD in apparently healthy Czech adult population was confirmed. In the high-risk groups, the prevalence of seropositivity for CD was approximately 2-4 times higher than in healthy blood donors. The real prevalence of CD in the tested groups, however, can be estimated after performing small intestinal biopsy in the seropositive patients.
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