Biomedical subjects

M J Cho

Publications and source records attributed to M J Cho.

At least 19 recordsLinked to original sources

PubMed2006

AtBAG6, a novel calmodulin-binding protein, induces programmed cell death in yeast and plants.

Calmodulin (CaM) influences many cellular processes by interacting with various proteins. Here, we isolated AtBAG6, an Arabidopsis CaM-binding protein that contains a central BCL-2-associated athanogene (BAG) domain. In yeast and plants, overexpression of AtBAG6 induced cell death phenotypes consistent with programmed cell death (PCD). Recombinant AtBAG6 had higher affinity for CaM in the absence of free Ca2 + than in its presence. An IQ motif (IQXXXRGXXXR, where X denotes any amino-acid) was required for Ca2 +-independent CaM complex formation and single amino-acid changes within this motif abrogated both AtBAG6-activated CaM-binding and cell death in yeast and plants. A 134-amino-acid stretch, encompassing both the IQ motif and BAG domain, was sufficient to induce cell death. Agents generating oxygen radicals, which are known to be involved in plant PCD, specifically induced the AtBAG6 transcript. Collectively, these results suggest that AtBAG6 is a stress-upregulated CaM-binding protein involved in plant PCD.

Amino Acid Motifs↗

PubMed2006

Thiobarbituric acid reactive substances and volatile compounds in chicken breast meat infused with plant extracts and subjected to electron beam irradiation.

The effect of irradiation on thiobarbituric acid reactive substances (TBARS) and volatile compounds in raw and cooked nonirradiated and irradiated chicken breast meat infused with green tea and grape seed extracts was investigated. Chicken breast meat was vacuum infused with green tea extract (3,000 ppm), grape seed extract (3,000 ppm), or their combination (at a total of 6,000 ppm), irradiated with an electron beam, and stored at 5 degrees C for 12 d. The targeted irradiation dosage was 3.0 kGy and the average absorbed dosage was 3.12 kGy. Values of TBARS and volatile compound contents of raw and cooked chicken meat were determined during the 12-d storage period. Thiobarbituric acid reactive substances values ranged from 15.5 to 71.4 mg of malondialdehyde/kg for nonirradiated raw chicken and 17.3 to 80.1 mg of malondialdehyde/kg for irradiated raw chicken. Values for cooked chicken ranged from 31.4 to 386.2 and 38.4 to 504.1 mg of malondialdehyde/kg for nonirradiated and irradiated chicken, respectively. Irradiation increased TBARS and hexanal values of controls and meat infused with plant extracts. Hexanal had the highest intensity of volatiles followed by pentanal and other volatiles. Cooking the samples significantly (P < 0.05) increased the amounts of TBARS and volatiles. Addition of plant extracts decreased the amount of TBARS as well as hexanal and pentanal values. Although irradiation increases lipid oxidation, infusion of chicken meat with plant extracts could reduce lipid oxidation caused by irradiation.

Aldehydes↗

PubMed2006

Expression profiling of radiation-induced genes in radiodermatitis of hairless mice.

BACKGROUND: Radiation induces many cellular events leading to radiodermatitis. OBJECTIVES: The aim of this study was to establish a radiodermatitis model using experimental animals, and to examine the expression profile of radiation-induced genes. METHODS: Hairless mice were irradiated on the dorsal skin; then total RNAs were isolated and microarray hybridizations were performed. RESULTS: Irradiation with a total of 40 Gy (10 Gy day-1 for four consecutive days) provokes radiodermatitis in the hairless mouse. After microarray analysis, 130 genes that showed upregulation by radiation were selected and organized into four different clusters, depending on the time-kinetic pattern. Classification of these genes into several functional categories revealed that various biological processes were globally affected by radiation. These include transcription regulation, signal transduction, cell communication, cell death regulation and metabolism. CONCLUSIONS: These results demonstrate the complexity of the transcriptional profile of the radiation response, providing important clues on which to base further investigations of the molecular events underlying radiodermatitis.

Animals↗

PubMed2005

Antibodies as drug carriers III: design of oligonucleotides with enhanced binding affinity for immunoglobulin G.

PURPOSE: To understand the structural requirements in designing epitope-bearing oligonucleotides with high antibody-binding affinity. METHODS: Binding affinity (KA) and stoichiometry (n) of dinitrophenyl (DNP)-derivatized model 27-mer oligonucleotides (ODNs), GGG(AAA)7GGG, to monoclonal anti-trinitrophenyl (TNP) antibodies were determined using isothermal titration calorimetry (ITC). Structural variations were made in the ODNs to assess the effects of antigenic valence, epitope density, inter-epitope linker length, and linker flexibility. Binding isotherms were fitted with a single binding-site model to obtain K(A) and n, from which changes in Gibbs free energy (deltaG(0)), entropy (deltaS(0)), and enthalpy (deltaH(0)) were derived. RESULTS: As expected, ligands displaying increased epitope density showed increases in K(A): for example, K(A) for (DNP)2-Cys is 3.3-fold greater than that for DNP-Lys. Introduction of multiple DNP groups via long and flexible linkers to one end of the 27-mer ODN resulted in a bivalent behavior with n value of 1. A bivalent ligand, derivatized at both ends with a long and flexible linker, failed to form an immune complex when hybridized to its antisense strand, presumably due to intercalation of the DNP moiety to the double strand. ODNs derivatized with flexible linkers exhibited a higher K(A) than those with a rigid linker. Ligands with flexible inter-epitope linkers measuring distances of 110, 60, and 40 angstroms yielded 13-, 30-, and 13-fold increases in K(A), respectively. The combination of these factors; namely, bivalence, flexible inter-epitope linkers, and optimal inter-epitope distance, resulted in an overall 66-fold increase in K(A). Thermodynamic analysis of binding indicates that the formation of high-affinity ODN-IgG complexes was a spontaneous and exothermic event, characterized by large negative deltaS degrees, deltaH degrees, and deltaG degrees values. CONCLUSIONS: All four strategies tested during this investigation, namely bivalence, epitope density, inter-epitope linker flexibility, and optimal inter-epitope distance, proved to be useful in improving the binding affinity of DNP-labeled ODNs to anti-TNP IgG. The final ODN design incorporating these strategies will be used in testing the systemic pharmacokinetic advantage gained from complexing such ODNs to IgG.

Binding Sites, Antibody↗

PubMed2004

Inactivation of pathogenic bacteria by cucumber volatiles (E,Z)-2,6-nonadienal and (E)-2-nonenal.

The effects of (E,Z)-2,6-nonadienal (NDE) and (E)-2-nonenal (NE) on Bacillus cereus, Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella Typhimurium were investigated. A suspension of each organism of 6 to 9 log CFU/ml was incubated for 1 h at 37 degrees C in brain heart infusion solution that contained 0 to 500 or 1,000 ppm of NDE or NE. Depending on concentration, exposure to either NDE or NE caused a reduction in CFU of each organism. Treatment with 250 and 500 ppm NDE completely eliminated viable B. cereus and Salmonella Typhimurium cells, respectively. L. monocytogenes was the most resistant to NDE, showing only about a 2-log reduction from exposure to 500 ppm for 1 h. Conversely, this concentration of NDE caused a 5.8-log reduction in E. coli O157:H7 cells. NE was also effective in inactivating organisms listed above. A higher concentration of NE, 1,000 ppm, was required to kill E. coli O157:H7, L. monocytogenes, or Salmonella Typhimurium compared with NDE. In conclusion, both NDE and NE demonstrated an apparent bactericidal activity against these pathogens.

Aldehydes↗

PubMed2003

AlphaIIbbeta3-mediated outside-in signaling induced by the agonist peptide LSARLAF utilizes ADP and thromboxane A2 receptors to cause alpha-granule secretion by platelets.

The peptide LSARLAF (LSA) causes alphaIIbbeta3-dependent platelet activation that results in alpha-granule secretion and aggregation. LSARLAF-induced, alphaIIbbeta3-mediated outside-in signaling causing alpha-granule secretion and platelet aggregation was studied using washed mouse platelets. ADP receptor antagonists, enzyme inhibitors, normal platelets and platelets from mice that lack either Galphaq or thromboxane (Tx) A2 receptors were used for this investigation. The results demonstrate that LSA-induced alphaIIbbeta3-mediated signaling producing aggregation of washed platelets is mediated through the release of ADP and thromboxane, which cause alpha-granule release by mediating their effects though Galphaq and/or Gi depending on the level of LSA used to activate the platelets. Specifically, alphaIIbbeta3 elicited aggregation of washed platelets in response to a low level of LSA requires signaling through the ADP receptor P2Y1 and Galphaq, and the ADP receptor P2Y12 and Gi as well as TxA2 receptors. However, this aggregation is independent of Galphaq and TxA2 signaling in response to high LSA concentrations, but is dependent on ADP signaling through its receptor P2Y12, and therefore presumably Gi, regardless of the level of LSA used to activate the platelets. PKC function is required for ADP secretion and the subsequent signaling through P2Y12 regardless of the level of LSA used to activate the platelets. The end point of the LSA-induced alphaIIbbeta3-mediated signaling characterized in this study is alpha-granule secretion, which provides the fibrinogen required for aggregation of washed platelets.

Adenosine Diphosphate↗

PubMed2001

Cold accumulation of SCOF-1 transcripts is associated with transcriptional activation and mRNA stability.

Cold acclimation enhances the transcription of several cold regulated (COR) genes. However, little is known about whether the elevation of the transcriptional level of the COR genes is due to transcriptional activation, or mRNA stability by a low temperature. Recently, we cloned a novel cold-inducible zinc finger protein gene from soybean, SCOF-1, which may function as a positive regulator of the COR gene expression . Here we report that the elevation of the SCOF-1 transcript level by cold stress is associated with both transcriptional activation and post-transcriptional mRNA stability under a low temperature. A nuclear run-on assay reveals that cold acclimation elevates the SCOF-1 transcript about three-fold compared to that of non-acclimated soybean nuclei. Furthermore, SCOF-1 transcripts increased substantially by a low temperature in transgenic tobacco plants that constitutively expressed SCOF-1 under the control of a constitutive cauliflower mosaic virus (CaMV) 35S promoter. When a transcription inhibitor, cordycepin, was treated with the deacclimating soybean cell, the decay level of the SCOF-1 transcripts was delayed significantly. This suggests that it may affect de novo protein synthesis, which degrades the SCOF-1 mRNA at room temperature. In addition, a secondary structure may be involved in the mRNA stability of SCOF-1 under a low temperature.

Acclimatization↗

PubMed2001

Quantitation of intracellular concentration of a delivered morpholino oligomer by capillary electrophoresis-laser- induced fluorescence: correlation with upregulation of luciferase gene expression.

Antisense oligonucleotides have shown great promise over the past several years as viable drugs to combat various forms of cancer and viral diseases. However, quantitative detection to monitor cellular association is difficult using conventional methods such as radiolabeling of the oligonucleotide or fluorescence confocal microscopy. In this paper quantitation of intracellular concentration of the morpholino oligonucleotide is investigated using capillary electrophoresis coupled with laser-induced fluorescence detection (CE-LIF). HeLa cells, which produce luciferase as the antisense oligomer enters the cell, were scrape-loaded with varying concentrations of the morpholino antisense. The intracellular antisense concentration measured by CE-LIF was found to correlate with those obtained with the cellular functional assay based on upregulation of luciferase. Intracellular concentrations of the antisense were found to be in the range of 6 to 29 nmol/g total cell protein, depending on the amounts that were scrape-loaded. To our best knowledge, this is the first reported quantitative correlation between delivered antisense concentration in a cell extract and the subsequent antisense upregulation of gene expression.

Cell Membrane↗

PubMed2001

A strategy for the identification of proteins targeted by thioredoxin.

Thioredoxins are 12-kDa proteins functional in the regulation of cellular processes throughout the animal, plant, and microbial kingdoms. Growing evidence with seeds suggests that an h-type of thioredoxin, reduced by NADPH via NADP-thioredoxin reductase, reduces disulfide bonds of target proteins and thereby acts as a wakeup call in germination. A better understanding of the role of thioredoxin in seeds as well as other systems could be achieved if more were known about the target proteins. To this end, we have devised a strategy for the comprehensive identification of proteins targeted by thioredoxin. Tissue extracts incubated with reduced thioredoxin are treated with a fluorescent probe (monobromobimane) to label sulfhydryl groups. The newly labeled proteins are isolated by conventional two-dimensional electrophoresis: (i) nonreducing/reducing or (ii) isoelectric focusing/reducing SDS/PAGE. The isolated proteins are identified by amino acid sequencing. Each electrophoresis system offers an advantage: the first method reveals the specificity of thioredoxin in the reduction of intramolecular vs. intermolecular disulfide bonds, whereas the second method improves the separation of the labeled proteins. By application of both methods to peanut seed extracts, we isolated at least 20 thioredoxin targets and identified 5-three allergens (Ara h2, Ara h3, and Ara h6) and two proteins not known to occur in peanut (desiccation-related and seed maturation protein). These findings open the door to the identification of proteins targeted by thioredoxin in a wide range of systems, thereby enhancing our understanding of its function and extending its technological and medical applications.

Amino Acid Sequence↗

PubMed2001

Antibodies as drug carriers. II. For proteins.

PURPOSE: To evaluate the potential use of antibodies as a carrier for monovalent protein haptens. METHODS: A single -SH functionality present in the human IgG light chain was fluoresceinated. This conjugate, FL-LC, was treated with pepsin to obtain FL conjugate of half light chain, FL-(LC)1/2, of MW 11 kDa. These two were radiolabeled using [3H]-propionic acid N-hydroxysuccinimide ester, and administered via tail vein to FL-immunized or mock-immunized mice. The blood radioactivity was measured over a 72-h period. Attempts were made to measure the affinity constant for the interaction between the conjugates and anti-FL antibodies by fluorescence quenching, surface plasmon resonance spectroscopy, and competitive ELISA. RESULTS: All of the three methods used produced supportive, if not conclusive, evidence of decreased binding affinity with increased conjugate size. Subsequent to tail-vein injection to FL-immunized mice, FL-LC showed approximately 4-fold smaller volume of distribution than mock-immunized mice: 0.041 +/- 0.005 vs. 0.16 +/- 0.02 mL/g. Corresponding values for FL-(LC)1/2, were significantly larger: 0.070 +/-0.013 and 0.30 +/- 0.02 mL/g, respectively. Compared with a small FL conjugate of ethanolamine, FL-EA, we studied earlier, the dose-normalized concentrations of the protein conjugates started at a higher level but declined more rapidly with time. In mock-immunized mice, the radioactivity disappeared very rapidly after administration, followed by an extremely slow decline with half-life close to 60 h. Evidence is provided to support that the radiolabel dissociated in the kidney, however, binding to anti-FL antibodies greatly stabilized the conjugate. CONCLUSIONS: Based on an entropic principle alone the affinity of monovalent hapten-antibody interaction is expected to diminish with increase in hapten size. As such, the size of a hapten should be an important determinant of its pharmacokinetics in animals harboring antibodies that recognize the hapten. Relative to what was observed with small MW FL-EA, the protein conjugates showed substantially sustained circulation as a result of antibody binding, but this effect was diminished at later time points. Both affinity and pharmacokinetic data are consistent with the hypothesis of reduced affinity with increasing MW for monovalent hapten conjugates, but neither offered overwhelming proof.

Animals↗

PubMed2001

Anti-drug antibodies as drug carriers. I. For small molecules.

PURPOSE: To better understand the pharmacokinetics of drugs compounds that bind endogenous antibodies METHODS: Three groups of mice with differing anti-fluorescein (FL) titers were established by empirically developed immunization protocols. These with two control groups were given intravenously [3H]-ethanolamine conjugate of FL (FL-EA). The latter was synthesized using isothiocyanate chemistry. Radioactivity in the circulation, and occasionally in peritoneal ascites, was monitored for 7 days. A group of mice was immunized with eosin Y and given FL-EA. Conversely, eosin Y conjugate of radiolabeled EA (EY-EA) was given to mice immunized with FL. These two groups represented animals of low affinity to probe haptens. The affinity was assessed by a precipitation procedure, while titer was determined by a standard ELISA. Dose of FL-EA varied over a 100-fold. RESULTS: On average, the three immunized groups showed a 1:13:85 ratio of anti-FL titer, with remarkably consistent levels within each group. Elimination rates of FL-EA from the serum of very high-titer mice and high-titer mice were similar, however, were substantially lower than that found in low-titer mice. The latter was in turn lower than that found in non- or mock-immunized mice. Serum of mice immunized with FL showed approximately 200-fold lower affinity towards EY-EA than FL-EA. In these mice and in mice immunized with eosin Y and given FL-EA, the elimination of the probe haptens was again fast, reminiscent of low-titer mice. Mice of either low titer or low affinity showed more rapid redistribution of the conjugate between serum and peritoneal fluid. In a group of mice with comparable anti-FL titer, elimination from serum was independent of dose over a 100-fold difference. The bi-phasic concentration-time profile observed was accommodated by a physiologically meaningful pharmacokinetic model incorporating two compartments in which antibody binding can occur. CONCLUSIONS: Monovalent antigenic substance cannot trigger immune clearance. As such, endogenous antibodies that recognize the molecule can serve as a carrier to result in a substantial decrease in clearance.

Animals↗

PubMed2001

A novel cold-inducible zinc finger protein from soybean, SCOF-1, enhances cold tolerance in transgenic plants.

Cold stress on plants induces changes in the transcription of cold response genes. A cDNA clone encoding C2H2-type zinc finger protein, SCOF-1, was isolated from soybean. The transcription of SCOF-1 is specifically induced by low temperature and abscisic acid (ABA) but not by dehydration or high salinity. Constitutive overexpression of SCOF-1 induced cold-regulated (COR) gene expression and enhanced cold tolerance of non-acclimated transgenic Arabidopsis and tobacco plants. SCOF-1 localized to the nucleus but did not bind directly to either C-repeat/dehydration (CRT/DRE) or ABA responsive element (ABRE), cis-acting DNA regulatory elements present in COR gene promoters. However, SCOF-1 greatly enhanced the DNA binding activity of SGBF-1, a soybean G-box binding bZIP transcription factor, to ABRE in vitro. SCOF-1 also interacted with SGBF-1 in a yeast two-hybrid system. The SGBF-1 transactivated the beta-glucuronidase reporter gene driven by the ABRE element in Arabidopsis leaf protoplasts. Furthermore, the SCOF-1 enhanced ABRE-dependent gene expression mediated by SGBF-1. These results suggest that SCOF-1 may function as a positive regulator of COR gene expression mediated by ABRE via protein-protein interaction, which in turn enhances cold tolerance of plants.

Abscisic Acid↗

PubMed2001

Redox changes accompanying the degradation of seed storage proteins in germinating rice.

The mobilization of storage proteins (glutelins) in germinating rice seeds was accompanied by an ordered sequential combination of proteolysis and reduction of disulfide groups. Mobilization was followed by application of non-reducing/reducing two dimensional-PAGE after monobromobimane labeling of the sulfhydryl groups of the proteins in intact seeds.

Electrophoresis, Gel, Two-Dimensional↗

PubMed2001

Crystallization and preliminary crystallographic studies of an antimicrobial protein from Pharbitis nil.

An antimicrobial protein from seeds of Pharbitis nil (Pn-AMP) which shows an antifungal activity towards several agriculturally important plant pathogens has been crystallized in the presence of equimolar N-acetylglucosamine with sodium citrate as precipitant. The crystal belongs to the hexagonal space group P6(1)22 (or P6(5)22), with unit-cell parameters a = b = 29.33 (5), c = 133.44 (12) A. Native data were collected using a crystal at 100 K to a resolution of 1.78 A.

Amino Acid Sequence↗

PubMed2001

CNS cholinergic innervation to the hippocampus in the rat using pseudorabies virus as a neurotracer.

The hippocampus is a central area of the memory-related neural system. Combined immunohistochemistry against choline acetyl transferase and retrograde transneuronal labelling of the pseudorabies virus were used to identify cholinergic neurons in the central nervous system projecting to the hippocampal formation of the rat. Five to ten microL of Bartha strain of pseudorabies virus were injected into the dentate gyrus, CA1 and CA3 of the hippocampus of 20 Sprague Dawley rats using stereotaxic instrument. Forty eight to 96 hr after the injection, the brains were removed and the tissue sections were processed for double immunofluorescence procedure using polyclonal antibodies against pseudorabies virus or choline acetyl transferase. The double labelled neurons were distributed at several different nuclei and the labelling patterns of three different areas of the hippocampus were similar. These data suggests that the cholinergic innervation to the hippocampus were distributed in a transsynaptic manner throughout the whole brain area.

Animals↗

PubMed2001

Serum amylase and lipase elevation is associated with intracranial events.

Serum amylase and lipase elevation has been observed in trauma patients and patients with traumatic intracranial bleeding. However, the causes of this elevation have not been clearly elucidated. A further question remains as to whether other intracranial events are associated with such enzyme elevation as well. We retrospectively reviewed 75 patients consecutively admitted to Cook County Hospital Neurosurgical Intensive Care Unit over a 3-month period for trauma, infection, tumor, or other space-occupying lesions with an unstable condition or neurological deficit. Eleven patients (15%) had elevated amylase and lipase levels. The patients were divided into two groups: Group I (n = 64) had normal and Group II (n = 11) had raised amylase and lipase levels [amylase 402 +/- 444 U/L with normal < or = 125 U/L and lipase 474 +/- 313 U/L with normal < or = 55 U/L]. All Group II patients suffered an intracranial event. Twenty-four Group I (38%) and 10 Group II (91%) patients required craniotomy (P < 0.01). No patients had clinical or radiographic evidence of pancreatitis. In summary, intracranial events are associated with serum amylase and lipase elevation probably through centrally activated pathways. Because of the lack of diagnostic value, routine pancreatic enzyme monitoring should not be performed in this patient population.

Aged↗

PubMed2000

Rice 1Cys-peroxiredoxin over-expressed in transgenic tobacco does not maintain dormancy but enhances antioxidant activity.

Possible functions that have been proposed for the plant 1Cys-peroxiredoxin, include activity as a dormancy regulator and as an antioxidant. The transcript level of rice 1Cys-peroxiredoxin (R1C-Prx) rapidly decreased after imbibition of rice seeds, but the protein was detected for 15 days after imbibition. To investigate the function of this protein, we generated transgenic tobacco plants constitutively expressing the R1C-Prx gene. The transgenic R1C-Prx plants showed a germination frequency similar to control plants. However, the transgenic lines exhibited higher resistance against oxidative stress, suggesting that antioxidant activity may be its primary function.

Animals↗

PubMed2000

Differential regulation of Ca2+/calmodulin-dependent enzymes by plant calmodulin isoforms and free Ca2+ concentration.

Multiple calmodulin (CaM) isoforms are expressed in plants, but their biochemical characteristics are not well resolved. Here we show the differential regulation exhibited by two soya bean CaM isoforms (SCaM-1 and SCaM-4) for the activation of five CaM-dependent enzymes, and the Ca(2+) dependence of their target enzyme activation. SCaM-1 activated myosin light-chain kinase as effectively as brain CaM (K(act) 1.8 and 1.7 nM respectively), but SCaM-4 produced no activation of this enzyme. Both CaM isoforms supported near maximal activation of CaM-dependent protein kinase II (CaM KII), but SCaM-4 exhibited approx.12-fold higher K(act) than SCaM-1 for CaM KII phosphorylation of caldesmon. The SCaM isoforms showed differential activation of plant and animal Ca(2+)-ATPases. The plant Ca(2+)-ATPase was activated maximally by both isoforms, while the erythrocyte Ca(2+)-ATPase was activated only by SCaM-1. Plant glutamate decarboxylase was activated fully by SCaM-1, but SCaM-4 exhibited an approx. 4-fold increase in K(act) and an approx. 25% reduction in V(max). Importantly, SCaM isoforms showed a distinct Ca(2+) concentration requirement for target enzyme activation. SCaM-4 required 4-fold higher [Ca(2+)] for half-maximal activation of CaM KII, and 1.5-fold higher [Ca(2+)] for activation of cyclic nucleotide phosphodiesterase than SCaM-1. Thus these plant CaM isoforms provide a mechanism by which a different subset of target enzymes could be activated or inhibited by the differential expression of these CaM isoforms or by differences in Ca(2+) transients.

Amino Acid Sequence↗