Biomedical subjects
Publications and source records attributed to K D Lee.
[reaction: see text]. A new fluorescent chemosensor, anthryl tetra acid, was synthesized and showed large fluorescence quenching effects in 100% aqueous solution with metal ions via photoinduced electron transfer (PET). Chelatoselective fluorescence perturbation was observed with Cd(II) and resulted from electrophilic aromatic cadmiation.
Biodegradable microspheres containing recombinant human epidermal growth factor (rhEGF) were prepared using poly(L-lactic acid) by a solvent evaporation method based on multiple w/o/w emulsion. Encapsulation efficiency and initial release were influenced by the amount of polymer, inner water phase volume and osmotic pressure difference between inner water phase and outer water phase. The effect of osmotic pressure difference between inner water phase and outer water phase in w/o/w emulsion on particle size, porosity and in vitro release of rhEGF from microspheres were also studied. Microspheres prepared with the optimized osmotic pressure, polymer amount and inner water volume produced 21% initial release on the first day with 92% encapsulation efficiency. The blood concentration of rhEGF was maintained at constant levels for 9-11 days after a single subcutaneous (s.c.) administration of rhEGF microspheres. The gastric ulcer healing effect of a single s.c. administration of rhEGF microspheres was increased 1.44-fold compared with twice a day s.c. administration of rhEGF saline solution after 11 days. The enhanced curative ratio of rhEGF loaded microspheres may be due to the optimized osmotic pressure, high encapsulation efficiency and sustained release pattern.
Acori graminei Rhizoma (AGR) is shown to exhibit a number of pharmacological actions including sedation and anticonvulsive action. To further characterize its actions in the CNS, the present study evaluated the effects of essential oils (EO) from AGR on the excitotoxic neuronal cell death induced in primary rat cortical cell cultures. EO inhibited the glutamate-induced excitotoxicity in a concentration-dependent manner, with the IC50 of 0.241 mg/ml. EO exerted more potent neuroprotection against the toxicity induced by NMDA (IC50 = 0.139 mg/ml). In contrast, the AMPA-induced toxicity was not inhibited by EO. Receptor-ligand binding studies were performed to investigate the neuroprotective action mechanism. EO dramatically inhibited the specific bindings of a use-dependent NMDA receptorion channel blocker [3H]MK-801, indicating an NMDA receptor antagonist-like action. However, the bindings of [3H]MDL 105,519, a ligand selective for the glycine binding site of NMDA receptor, were not considerably inhibited. These results demonstrated that EO extracted from AGR exhibited neuroprotective effects on cultured cortical neurons through the blockade of NMDA receptor activity, and that the glycine binding site appeared not to be the major site of action.
We have cloned the genomic sequence coding for the high mobility group 1 (HMG1) protein in chickens. Multiple sequence alignment shows that the chicken HMG1 gene is highly homologous to the human and the mouse HMG1 genes. The gene structure of chicken HMG1 is similar to that of the mouse and the human HMG1 genes, with the same exon-intron boundaries. However, in contrast to other avian genes that have shorter introns, the chicken HMG1 gene has introns that are twice as long as their mammalian homologues. In addition to the functional, intron-containing HMG1 gene, all mammalian genomes contain more than 50 copies of HMG1 retropseudogenes each, while in the chicken genome there are no HMG1 retropseudogenes. This finding suggests that the HMG1 retropseudogenes arose in mammals after their divergence away from the birds.
Advances in our understanding of fundamental cell biological processes have facilitated an expansion of therapeutic approaches to altering cellular physiology and phenotype. As many of these methods involve macromolecular agents that act on targets within the nucleus or cytoplasm, achieving their full potential ultimately requires the efficient delivery of these agents across the cell membrane barrier into the cytosol. Various strategies have been employed to enhance cytosolic delivery. These include either directly penetrating the plasma membrane, or avoiding degradation within the hydrolytic environment of the endosomal/lysosomal pathway after endocytic uptake. Some of the more promising methods in this regard have exploited the mechanisms utilized by certain viruses and bacteria for escaping into their host cell's cytosol. In this review, we will discuss some of these methods with an emphasis on the use of pore-forming proteins from bacteria. Particular attention will be drawn to the pH-sensitive endosomolytic bacterial hemolysins, such as listeriolysin O, and the potentiol for their use in cytosolic drug delivery systems.
Acori graminei rhizoma (AGR) are reported to exhibit a number of pharmacological actions in the central nervous system. The effects of the methanol extract of AGR on excitotoxic neuronal death were evaluated in the present study using cultured rat cortical neurons. Based on the phase-contrast microscopic examinations of cultures and lactate dehydrogenase activities measured in the culture media, the glutamate-induced excitotoxicity was significantly inhibited by the extract. The inhibitory action of the extract was more potent and selective for the N-methyl-D-aspartate (NMDA) receptor-mediated toxicity. The AGR extract competed with [3H]MDL 105,519 for the specific binding to the glycine site of the NMDA receptor with the IC(50) value of 164.7 microg/ml. Modulation of the NMDA receptor activity by the extract was determined using [3H]MK-801 binding studies. The reduction of the binding in the presence of the extract indicated the receptor inactivation by AGR. These results demonstrated that the methanol extract of AGR exhibited protective action against excitotoxic neuronal death, and that the neuroprotective action was primarily due to the blockade of NMDA receptor function by the interaction with the glycine binding site of the receptor.
Aldosterone, a steroid hormone, regulates renal Na+ reabsorption and, therefore, plays an important role in the maintenance of salt and water balance. In a model renal epithelial cell line (A6) we have found that phosphoinositide 3-kinase (PI 3-kinase) activity is required for aldosterone-stimulated Na+ reabsorption. Inhibition of PI 3-kinase by the specific inhibitor LY-294002 markedly reduces both basal and aldosterone-stimulated Na+ transport. Further, one of the products of PI 3-kinase, phosphatidylinositol 3,4,5-trisphosphate, is increased in response to aldosterone in intact A6 monolayers. This increase occurs just before the manifestation of the functional effect of the hormone and is also inhibited by LY-294002. With the use of blocker-induced noise analysis, it has been demonstrated that inhibition of phosphoinositide formation causes an inhibition of Na+ entry in both control and aldosterone-pretreated cultures by reducing the number of open functional epithelial Na+ channels (ENaCs) in the apical membrane of the A6 cells. These novel observations indicate that phosphoinositides are required for ENaC expression and suggest a mechanism for aldosterone regulation of channel function.
The "evolution" of a thing, a custom, an organ is thus by no means its progressus toward a goal, even less a logical progressus by the shortest route and with the least expenditure of force, but a succession of more or less profound, mutually independent processes of subduing, plus the resistances they encounter, the attempts at transformation for the purpose of defense and reaction, and the results of successful counteractions. The form is fluid, but the "meaning" is even more so.
The pore-forming toxin listeriolysin O (LLO) is a major virulence factor implicated in escape of Listeria monocytogenes from phagocytic vacuoles. Here we describe the pH-dependence of vacuolar perforation by LLO, using the membrane-impermeant fluorophore 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) to monitor the pH and integrity of vacuoles in mouse bone marrow-derived macrophages. Perforation was observed when acidic vacuoles containing wild-type L. monocytogenes displayed sudden increases in pH and release of HPTS into the cytosol. These changes were not seen with LLO-deficient mutants. Perforation occurred at acidic vacuolar pH (4.9-6.7) and was reduced in frequency or prevented completely when macrophages were treated with the lysosomotropic agents ammonium chloride or bafilomycin A1. We conclude that acidic pH facilitates LLO activity in vivo.
Endothelial-selective delivery of therapeutic agents, such as drugs or genes, would provide a useful tool for modifying vascular function in various disease states. A potential molecular target for such delivery is E-selectin, an endothelial-specific cell surface molecule expressed at sites of activation in vivo and inducible in cultured human umbilical vein endothelial cells (HUVEC) by treatment with cytokines such as recombinant human interleukin 1beta (IL-1beta). Liposomes of various types (classical, sterically stabilized, cationic, pH-sensitive), each conjugated with mAb H18/7, a murine monoclonal antibody that recognizes the extracellular domain of E-selectin, bound selectively and specifically to IL-1beta-activated HUVEC at levels up to 275-fold higher than to unactivated HUVEC. E-selectin-targeted immunoliposomes appeared in acidic, perinuclear vesicles 2-4 hr after binding to the cell surface, consistent with internalization via the endosome/lysosome pathway. Activated HUVEC incubated with E-selectin-targeted immunoliposomes, loaded with the cytotoxic agent doxorubicin, exhibited significantly decreased cell survival, whereas unactivated HUVEC were unaffected by such treatment. These results demonstrate the feasibility of exploiting cell surface activation markers for the endothelial-selective delivery of biologically active agents via immunoliposomes. Application of this targeting approach in vivo may lead to novel therapeutic strategies in the treatment of cardiovascular disease.
Characterization of the 5.5 kb promoter of human thromboxane synthase (TS) gene revealed a proximal positive regulatory sequence (PPRS, -90 to -25 bp) and several distal repressive elements. The maximal promoter activity was found to reside within the first 285 bp, approximately 75% of which was contributed by the PPRS. The sequence between -365 and -665 bp exerted a strong repressive effect (approximately 55%) on reporter gene expression independent of orientation and position, consistent with properties expected for a silencer. The sequence upstream of -665 bp to -5.5 kb contains mainly repressive elements which further reduce the promoter activity by 30%. The 65 bp PPRS worked in an orientation-independent, but position-dependent, manner and could be further divided into two independent elements, PPRS1 (-90 to -50 bp) and PPRS2 (-50 to -25 bp). While similar nuclear factor(s) from different cell types interact with PPRS2, those interacting with PPRS1 exhibit cell specificity. Internal sequence deletion and oligonucleotide competition established that a binding sequence for NF-E2 in PPRS1 (-60 tgctgattcat -50) was important for enhancing TS promoter activity in HL-60 cells. The presence of NF-E2 mRNA in HL-60 cells was demonstrated by reverse-transcription PCR amplification of the cDNA and Northern blot analysis. A 9-fold transactivation of luciferase (luc) reporter gene expression had been detected when NF-E2 cDNA was co-expressed with a TS promoter/luc construct. Despite the fact that NF-E2 and the cis-elements could alter the efficiency of TS transcription, they were not sufficient for restricting cell-specific TS expression. Analysis of the methylation status at the TS promoter in several human cell lines reveals cell-specific patterns of methylation that might correlate with TS expression. Taken together, these results suggest that the expression of human TS gene is modulated by multiple factors including cis-elements, trans-activator(s), and possibly genomic methylation.
Thromboxane synthase (TS) is a cytochrome P-450 (CYP450) enzyme catalyzing the conversion of prostaglandin endoperoxide (PGH2) into thromboxane A2 (TxA2) which plays a crucial role in hemostasis and cardiovascular diseases. Twelve genomic clones containing the DNA encoding the human TS gene (hTS) were isolated and characterized to determine the exon/intron boundaries and restriction maps of the nearly contiguous structure of the gene. The hTS contains 13 exons spanning more than 150 kb. Its first five exons, divided by relatively large introns, spread over 100 kb, but encode less than one third of the full-length TS transcript. Southern analysis indicates that the human haploid genome contains a single copy of the TS gene. Although multiple transcription start points (tsp) are utilized, transcription of hTS is primarily TATA-independent, as determined by promoter-directed reporter gene expression in transfected cells. A dinucleotide (CA) repetitive sequence identified in the ninth intron of the gene exhibits allelic polymorphism. At least four distinctive alleles, containing from 13 to 20 copies of the CA repeats, have been detected.
The cytosolic space of cells is an important but relatively inaccessible target for the delivery of therapeutic macromolecules. Here we describe the efficient delivery of macromolecules into the cytosolic space of macrophages from liposomes that contain listeriolysin O (LLO), the hemolytic protein of Listeria monocytogenes that normally mediates bacterial passage from phagosomes into cytosol. LLO was purified and encapsulated inside pH-sensitive liposomes, along with other molecules to be delivered. When internalized by bone marrow-derived macrophages, these liposomes rapidly released encapsulated fluorescent dye, first into endosomes and then into the cytosol, without measurably harming the cells. Furthermore, these liposomes efficiently delivered encapsulated ovalbumin to the cytosolic pathway of antigen processing and presentation, as measured by the major histocompatibility complex (MHC) class I-restricted presentation of peptides derived from ovalbumin. Delivery was significantly better than that obtained by other currently available liposome formulations. LLO-containing liposomes should therefore provide an efficient vehicle for delivery of antigens or therapeutic molecules in vivo.
The messenger RNA level of several MAGE genes, some of which have been proven to encode tumor rejection antigens recognized by cytotoxic T lymphocytes, were examined in 41 benign and malignant lesions of the head and neck region. By a reverse transcription-polymerase chain reaction assay and Southern blot hybridization, MAGE-1, -2, -3, -4, and -6 genes were expressed in 25%, 41.7%, 33.3%, 8.3% and 33.3% of 12 non-squamous cell carcinomas, respectively. These tumors consisted of 6 papillary adenocarcinomas, 3 adenoid cystic carcinomas, 2 adenocarcinomas, and 1 mucoepidermoid tumor. Of 7 non-Hodgkin's lymphomas, one case from the oropharynx and 2 from the nasopharynx expressed for the MAGE-1 and MAGE-2 genes, respectively. In contrast, none of 12 benign tumors expressed any of these MAGE genes. Interestingly, of 10 other lesions including hyperplasia, keratosis, and ulcer, one histologically diagnosed as dysplasia expressed the MAGE-2, -3, -4, and -6 genes. These results suggest that the MAGE genes may be expressed in malignant tumors and precancerous lesions but not in benign tumors. In addition, non-squamous cell carcinomas may be suitable targets for specific immunotherapy against MAGE gene products.
The MAGE genes encode certain tumor-associated antigens recognized by cytotoxic T lymphocytes. We investigated the expression of the MAGE-1, -2, -3, -4, -41, and -6 genes in 88 head-and-neck squamous-cell carcinomas (83 fresh tumor samples and 5 cell lines), using a reverse-transcription-polymerase-chain-reaction assay, followed by dot-blot hybridization with sequence-specific oligonucleotides and/or restriction enzyme-pattern analysis. The MAGE-1, -2, -3, -4, -41 and -6 genes were expressed at the mRNA level in 27, 34, 36, 22, 16 and 35, respectively, of 83 fresh tumor samples. At least one of these genes was expressed in 59 of the 83 samples. Neither non-tumor inflammatory cells nor normal tissues were positive for these genes. The MAGE-1 gene was expressed relatively frequently in SCC of the oropharynx, hypopharynx and maxillary sinus, but at lower rates in SCC of the larynx and of the tongue and oral cavity. MAGE-1 was frequently expressed in poorly differentiated SCC, somewhat less frequently in moderately differentiated SCC, and only infrequently in well-differentiated SCC. The expression levels of the other MAGE genes also varied with the anatomic site as well as the degree of differentiation. Our results suggest that specific immunotherapy against MAGE gene products may be useful for patients with head-and-neck carcinomas.
Organelle acidification is an essential element of the endosomal-lysosomal pathway, but our understanding of the mechanisms underlying progression through this pathway has been hindered by the absence of adequate methods for quantifying intraorganelle pH. To address this problem in neurons, we developed a direct quantitative method for accurately determining the pH of endocytic organelles in live cells. In this report, we demonstrate that the ratiometric fluorescent pH indicator 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) is the most advantageous available probe for such pH measurements. To measure intraorganelle pH, cells were labeled by endocytic uptake of HPTS, the ratio of fluorescence emission intensities at excitation wavelengths of 450 nm and 405 nm (F450/405) was calculated for each organelle, and ratios were converted to pH values by using standard curves for F450/405 vs. pH. Proper calibration is critical for accurate measurement of pH values: standard curves generated in vitro yielded artifactually low organelle pH values. Calibration was unaffected by the use of culture medium buffered with various buffers or different cell types. By using this technique, we show that both acidic and neutral endocytically derived organelles exist in the axons of sympathetic neurons in different steady-state proportions than in the cell body. Furthermore, we demonstrate that these axonal organelles have a bimodal pH distribution, indicating a rapid acidification step in their maturation that reduces the average pH of a fraction of the organelles by 2 pH units while leaving few organelles of intermediate pH at steady state. Finally, we demonstrate a spatial gradient or organelle pH along axons, with the relative frequency of acidic organelles increasing with proximity to the cell body.
The motor protein kinesin is implicated in organelle movement toward the plus ends of microtubules, but little is known about its interaction with organelle membranes or about the physiological role of the phosphorylation of kinesin and its associated protein kinectin seen in neurons in vivo (Hollenbeck, P. J. (1993) J. Neurochem. 60, 2265-2275). Here we have demonstrated that the kinesin heavy chain (KHC), light chain, and kinectin isolated from chick brain or sympathetic neurons exist in several isoelectric forms. Metabolic labeling followed by phosphatase treatment showed that these are phosphoisoforms, and that phosphorylation is reversible in vitro. To assess the capability of phosphorylation to regulate kinesin's state and/or activity, we performed 32P and 35S pulse-chase experiments with neuronal cultures and determined that kinesin-associated phosphate turns over 3-4 times faster than the proteins themselves. When the phosphoisoform distributions for different kinesin pools were analyzed, it was found that membrane-associated KHC contained predominantly the most highly phosphorylated isoform, while soluble kinesin consisted of less phosphorylated KHC isoforms. Nerve growth factor-induced neurite outgrowth in PC12 cells was found to increase significantly kinesin's 32P specific activity while doubling the relative abundance of the most highly phosphorylated KHC isoform. These results demonstrate that the phosphorylation state of kinesin is closely coupled to its organelle binding and to the magnitude of organelle transport in the cell. We propose that the phosphorylation state of kinesin and associated proteins may regulate motility via association with organelle membranes and, specifically, that KHC phosphorylation induces membrane association.
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