Pyonephrosis and urinary retention secondary to a large Gartner's duct cyst associated with single ectopic ureter in a pregnant woman.
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Adult↗
Biomedical subjects
T C Cheng
Publications and source records attributed to T C Cheng.
Stereochemical specificity of organophosphorus acid anhydrolase toward p-nitrophenyl analogs of soman and sarin.
Organophosphorus acid anhydrolase (OPAA) catalyzes the hydrolysis of p-nitrophenyl analogs of the organophosphonate nerve agents, sarin and soman. The enzyme is stereoselective toward the chiral phosphorus center by displaying a preference for the R(P)-configuration of these analogs. OPAA also exhibits an additional preference for the stereochemical configuration at the chiral carbon center of the soman analog. The preferred configuration of the chiral carbon center is dependent upon the configuration at the phosphorus center. The enzyme displays a two- to four-fold preference for the R(P)-enantiomer of the sarin analog. The k(cat)/K(m) of the R(P)-enantiomer is 250 M(-1) s(-1), while that of the S(P)-enantiomer is 110 M(-1) s(-1). The order of preference for the stereoisomers of the soman analog is R(P)S(C) > R(P)R(C) > S(P)R(C) > S(P)S(C). The k(cat)/K(m) values are 36,300 M(-1)s(-1), 1250 M(-1) s(-1), 80 M(-1) s(-1) and 5 M(-1) s(-1), respectively. The R(P)S(C)-isomer of the soman analog is therefore preferred by a factor of 7000 over the S(P)S(C)-isomer.
Alteromonas↗
Cryopreservation of heart cells from the eastern oyster.
Conditions were developed to cryopreserve cells from pronase-dissociated atria and ventricles of eastern oysters (Crassostrea virginica). The effect of three concentrations (5, 10, 15%) of the cryoprotectants (dimethyl sulfoxide, glycerol, and propylene glycol), three thawing temperatures (25, 45, 75 degrees C), and three cooling rates (slow, medium, fast) were compared. Cells were frozen at -80 degrees C and plunged in liquid nitrogen. Thawed cells were seeded in 96-well plates and primary cultures were evaluated after 3 d by measuring the metabolic activity using a tetrazolium compound, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, and by comparing the relative spreading of cells between treatments. The best conditions for freezing and thawing of cells for each cryoprotectant were selected and a final study was performed to compare cryoprotectants. For this final study, we measured the number of cells and their viability 3 d after thawing, in addition to determining cell metabolic activity and cell spreading. Primary cultures of cells fozen without cryoprotectant and of nonfrozen cells were used as controls in all studies. Atrial cells were best cryopreserved with glycerol at a concentration of 10%, a medium cooling rate, and thawing at 45 degrees C. After thawing, atrial cells showed 53+/-5% of the metabolic activity, 84+/-5% of the number, and 92+/-2% of the viability of nonfrozen cells. For ventricular cells, 10% glycerol, a medium cooling rate, and thawing at 25 degrees C yielded the best results. The thawed ventricular cells showed 83+/-5% of the metabolic activity, 91+/-5% of the number, and 96+/-2% of the viability of nonfrozen cells.
Animals↗
TCL1 oncogene expression in B cell subsets from lymphoid hyperplasia and distinct classes of B cell lymphoma.
Activation of the TCL1 oncogene has been implicated in T cell leukemias/lymphomas and recently was associated with AIDS diffuse large B cell lymphomas (AIDS-DLBCL). Also, in nonmalignant lymphoid tissues, antibody staining has shown that mantle zone B cells expressed abundant Tcl1 protein, whereas germinal center (GC; centrocytes and centroblasts) B cells showed markedly reduced expression. Here, we analyze isolated B cell subsets from hyperplastic tonsil to determine a more precise pattern of Tcl1 expression with development. We also examine multiple B cell lines and B lymphoma patient samples to determine whether different tumor classes retain or alter the developmental pattern of expression. We show that TCL1 expression is not affected by Epstein-Barr virus (EBV) infection and is high in naïve B cells, reduced in GC B cells, and absent in memory B cells and plasma cells. Human herpesvirus-8 infected primary effusion lymphomas (PEL) and multiple myelomas are uniformly TCL1 negative, whereas all other transformed B cell lines tested express moderate to abundant TCL1. This observation supports the hypothesis that PEL, like myeloma, usually arise from post-GC stages of B cell development. Tcl1 protein is also detected in most naïve/GC-derived B lymphoma patient samples (23 of 27 [85%] positive), whereas most post-GC-derived B lymphomas lack expression (10 of 41 [24%] positive). These data indicate that the pattern of Tcl1 expression is distinct between naïve/GC and post-GC-derived B lymphomas (P < 0.001) and that the developmental pattern of expression is largely retained. However, post-GC-derived AIDS-DLBCL express TCL1 at a frequency equivalent to naïve/GC-derived B lymphomas in immune-competent individuals (7 of 9 [78%] positive), suggesting that TCL1 down-regulation is adversely affected by severe immune system dysfunction. These findings demonstrate that TCL1 expression in B cell lymphoma usually reflects the stage of B cell development from which they derive, except in AIDS-related lymphomas.
B-Lymphocyte Subsets↗
Effect of methylglyoxal on mouse embryogenesis.
BACKGROUND: Methylglyoxal (MG), a highly active and mutagenetic compound, has been found widely in a variety of foods and beverages. We investigated the effect of MG on mouse embryo development in-vitro. METHODS: Two-cell mouse embryos were divided into six groups according to the MG concentration in the culture medium: Group 1 (control group). 0 mM; Group 2, 10(-4) mM; Group 3, 10(-3) mM; Group 4, 10(-2) mM; Group 5, 10(-1) mM; Group 6, 1 mM. Embryo development and cleavage were compared every day for 5 days. RESULTS: The percentages of embryos reaching blastocyst/hatching stages were as follows: Group 1, 66.8%/34.2%; Group 2, 67.9%/38.7%; Group 3, 56.2%/31.5%; Group 4, 39.4%/14.1%; Group 5, 11.4%/10.2%; Group 6, 0%/0%. Higher MG concentrations (> or = 10(-2) mM) were associated with morphological aberrations and blocked development of embryos. CONCLUSION: The cutoff value of MG concentration on the mouse embryo development in-vitro is 10(-2) mM. An increased risk of embryotoxicity occurs with MG concentrations > or = 10(-2) mM in vitro. There were no significant effects on the growth rate at MG concentrations of 10(-3) and 10(-4) mM.
Animals↗
Substrate and stereochemical specificity of the organophosphorus acid anhydrolase from Alteromonas sp. JD6.5 toward p-nitrophenyl phosphotriesters.
The enzyme OPAA hydrolyzes p-nitrophenyl phosphotriesters bearing substituents at the phosphorus center ranging in size from methyl to phenyl. The enzyme exhibits stereoselectivity toward the hydrolysis of chiral substrates with a preference for the Sp enantiomer.
Alteromonas↗
Long circulating liposomes encapsulating organophosphorus acid anhydrolase in diisopropylfluorophosphate antagonism.
These studies are focused on antagonizing organophosphorous (OP) intoxications by a new conceptual approach using recombinant enzymes encapsulated within sterically stabilized liposomes to enhance diisopropylfluorophosphate (DFP) degradation. The OP hydrolyzing enzyme, organophosphorous acid anhydrolase (OPAA), encapsulated within the liposomes, was employed either alone or in combination with pralidoxime (2-PAM) and/or atropine. The recombinant OPAA enzyme, from the ALTEROMONAS: strain JD6, has high substrate specificity toward a wide range of OP compounds, e.g., DFP, soman, and sarin. The rate of DFP hydrolysis by liposomes containing OPAA (SL)* was measured by determining the changes in fluoride-ion concentration using a fluoride ion-selective electrode. This enzyme carrier system serves as a biodegradable protective environment for the OP-metabolizing enzyme (OPAA), resulting in an enhanced antidotal protection against the lethal effects of DFP. Free OPAA alone showed some antidotal protection; however, the protection with 2-PAM and/or atropine was greatly enhanced when combined with (SL)*.
Animals↗
Alteromonas prolidase for organophosphorus G-agent decontamination.
Enzymes catalyzing the hydrolysis of highly toxic organophosphorus compounds (OPs) are classified as organophosphorus acid anhydrolases (OPAA; EC 3.1.8.2). Recently, the genes encoding OPAA from two species of Alteromonas were cloned and sequenced. Sequence and biochemical analyses of the cloned genes and enzymes have established Alteromonas OPAAs to be prolidases (E.C. 3.4.13.9), a type of dipeptidase hydrolyzing dipeptides with a prolyl residue in the carboxyl-terminal position (X-Pro). Alteromonas prolidases hydrolyze a broad range of G-type chemical warfare (CW) nerve agents. Efforts to over-produce a prolidase from A. sp.JD6.5 with the goal of developing strategies for long-term storage and decontamination have been successfully achieved. Large-scale production of this G-agent degrading enzyme is now feasible with the availability of an over-producing recombinant cell line. Use of this enzyme for development of a safe and non-corrosive decontamination system is discussed.
Aryldialkylphosphatase↗
Birth after the injection of sperm and the cytoplasm of tripronucleate zygotes into metaphase II oocytes in patients with repeated implantation failure after assisted fertilization procedures.
OBJECTIVE: To assess the technique of injecting a single sperm and cytoplasm obtained from tripronucleate zygotes into metaphase II oocytes for the treatment of patients with repeated implantation failure after intracytoplasmic sperm injection or IVF. DESIGN: Clinical study. SETTING: Private infertility clinic. PATIENT(S): Patients with repeated implantation failure after intracytoplasmic sperm injection or IVF. INTERVENTION(S): The metaphase II oocytes of recipients were injected with their husbands' spermatozoa and cytoplasm aspirated from the tripronucleate zygotes of donors. MAIN OUTCOME MEASURE(S): Fertilization after cytoplasm and sperm injection, embryo development, and successful pregnancy. RESULT(S): In total, 62 metaphase II oocytes from nine recipients were injected. Of the 62 injected oocytes, 3 (5%) degenerated and 43 (69%) had two pronuclei 18 hours after injection. Thirty-nine oocytes with two pronuclei cleaved to the two-cell to six-cell stage after another 24 hours of culture. All cleaved embryos were transferred into the uteruses of recipients. Four clinical pregnancies occurred in four recipients. No abnormal chromosomes were observed after amniocentesis and karyotyping in all pregnancies. Five healthy infants were born. CONCLUSION(S): Injection of the cytoplasm of tripronucleate zygotes may enhance the clinical pregnancy rate in patients with repeated implantation failure after intracytoplasmic sperm injection or IVF.
Adult↗
Purification and characterization of a cobalt-activated carboxypeptidase from the hyperthermophilic archaeon Pyrococcus furiosus.
A novel metallocarboxypeptidase (PfuCP) has been purified to homogeneity from the hyperthermophilic archaeon, Pyrococcus furiosus, with its intended use in C-terminal ladder sequencing of proteins and peptides at elevated temperatures. PfuCP was purified in its inactive state by the addition of ethylenediaminetetraacetic acid (EDTA) and dithiothreitol (DTT) to purification buffers, and the activity was restored by the addition of divalent cobalt (K, = 24 +/- 4 microM at 80 degrees C). The serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF) had no effect on the activity. The molecular mass of monomeric PfuCP is 59 kDa as determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and 58 kDa by SDS-PAGE analysis. In solution, PfuCP exists as a homodimer of approximately 128 kDa as determined by gel filtration chromatography. The activity of PfuCP exhibits a temperature optimum exceeding 90 degrees C under ambient pressure, and a narrow pH optimum of 6.2-6.6. Addition of Co2+ to the apoPfuCP at room temperature does not alter its far-UV circular dichroism (CD) or its intrinsic fluorescence spectrum. Even when the CoPfuCP is heated to 80 degrees C, its far-UV CD shows a minimal change in the global conformation and the intrinsic fluorescence of aromatic residues shows only a partial quenching. Changes in the intrinsic fluorescence appear essentially reversible with temperature. Finally, the far-UV CD and intrinsic fluorescence data suggest that the overall structure of the holoenzyme is extremely thermostable. However, the activities of both the apo and holo enzyme exhibit a similar second-order decay over time, with 50% activity remaining after approximately 40 min at 80 degrees C. The N-blocked synthetic dipeptide, N-carbobenzoxy-Ala-Arg (ZAR), was used in the purification assay. The kinetic parameters at 80 degrees C with 0.4 mM CoCl2 were: Km, 0.9 +/- 0.1 mM; Vmax, 2,300 +/- 70 U mg(-1); and turn over number, 600 +/- 20 s(-1). Activity against other ZAX substrates (X = V, L, I, M, W, Y, F, N, A, S, H, K) revealed a broad specificity for neutral, aromatic, polar, and basic C-terminal residues. This broad specificity was confirmed by the C-terminal ladder sequencing of several synthetic and natural peptides, including porcine N-acetyl-renin substrate, for which we have observed (by MALDI-TOF MS) stepwise hydrolysis by PfuCP of up to seven residues from the C-terminus: Ac-Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Leu-Val-Tyr-Ser.
Amino Acid Sequence↗
G-type nerve agent decontamination by Alteromonas prolidase.
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Amino Acid Sequence↗
Enzymatic hydrolysis of Russian-VX by organophosphorus hydrolase.
The Russian-VX (R-VX) is the principle V-type nerve agent in the chemical warfare (CW) arsenal of the Former Soviet Union. We here report the enzymatic hydrolysis of the P-S bond of Russian-VX by organophosphorus hydrolase (OPH) from Pseudomonas diminuta. While the Michaelis constant, K(m) for R-VX (474 microM), was similar to that for VX (434 microM), the Vmax for R-VX (2.1 mumoles/mg/min) was about four-fold higher compared to that for VX (0.56 mumoles/mg/min). A 50% inhibition in the rate of the enzymatic hydrolysis of R-VX was observed in the presence of 0.5% ethanol, isoamyl-alcohol, or isopropanol. The presence of acetonitrile, diethylene glycol, or methanol had marginal effects. These results comprise the first demonstration of enzymatic detoxification of R-VX.
Aryldialkylphosphatase↗
Inhibition of protein kinase C and proto-oncogene expression by crocetin in NIH/3T3 cells.
Crocetin, a carotenoid isolated from the seeds of Gardenia jasminoides, was found to be a potent inhibitor of tumor promotion induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) in mouse skin. When mouse fibroblast NIH/3T3 cells were treated with TPA alone, protein kinase C (PKC) translocated from the cytosolic fraction to the particulate fraction. Pretreatment with 60 and 120 microM crocetin for 15 min inhibited the TPA-induced PKC activity in the particulate fraction by 50% and 66%, respectively, but did not affect the level of PKC protein. Crocetin also reduced the level of TPA-stimulated phosphorylation of cellular proteins. Cells pretreated with crocetin (120 microM) had 55% less PKC [3H]phorbol dibutyrate-binding capacity. Suppression of TPA (100 ng/mL)-induced c-jun and c-fos gene expression was also observed in the mouse fibroblast cells pretreated with crocetin (30, 60, and 120 microM). Our results provided a basis for understanding the inhibitory effect of crocetin on TPA-mediated tumor promotion.
3T3 Cells↗
Clinical evaluation of the bone marrow imaging agent 99Tcm-phytate in the detection of bone metastases.
The aims of this study were to assess whether 99Tcm-phytate can detect metastatic skeletal lesions, and to compare it with 99Tcm-methylene diphosphonate (99Tcm-MDP) and 99Tcm-labelled human serum albumin nanocolloids (99Tcm-NC). Twenty-four patients with multiple bony metastases, investigated by 99Tcm-MDP whole-body scintigraphy, underwent 99Tcm-phytate bone marrow imaging. A separate bone marrow scintigram with 99Tcm-NC was performed in 20 of the patients. All of the metastatic lesions detected on the 99Tcm-phytate scintigrams exhibited photon-abundant foci only. Most of the 99Tcm-phytate scintigrams detected fewer metastatic lesions than the corresponding bone scintigrams. Visual comparison of the 99Tcm-NC images showed that 13 of 20 99Tcm-NC images were superior to the 99Tcm-phytate images in the detection of metastatic involvement of the skeleton. Thus 99Tcm-phytate should not be used as a bone marrow imaging agent for the detection of skeletal metastases.
Adult↗
Cloning and expression of a gene encoding a bacterial enzyme for decontamination of organophosphorus nerve agents and nucleotide sequence of the enzyme.
Organophosphorus acid (OPA) anhydrolase enzymes have been found in a wide variety of prokaryotic and eukaryotic organisms. Interest in these enzymes has been prompted by their ability to catalyze the hydrolysis of toxic organophosphorus cholinesterase-inhibiting compounds, including pesticides and chemical nerve agents. The natural substrates for these enzymes are unknown. The gene (opaA) which encodes an OPA anhydrolase (OPAA-2) was isolated from an Alteromonas sp. strain JD6.5 EcoRI-lambda ZAPII chromosomal library expressed in Escherichia coli and identified by immunodetection with anti-OPAA-2 serum. OPA anhydrolase activity expressed by the immunopositive recombinant clones was demonstrated by using diisopropylfluorophosphate (DFP) as a substrate. A comparison of the recombinant enzyme with native, purified OPAA-2 showed they had the same apparent molecular mass (60 kDa), antigenic properties, and enzyme activity against DFP and the chemical nerve agents sarin, soman, and O-cyclohexyl methylphosphonofluoridate. The gene expressing this activity was found in a 1.74-kb PstI-HindIII fragment of the original 6.1-kb EcoRI DNA insert. The nucleotide sequence of this PstI-HindIII fragment revealed an open reading frame of 1,551 nucleotides, coding for a protein of 517 amino acid residues. Amino acid sequence comparison of OPAA-2 with the protein database showed that OPAA-2 is similar to a 647-amino-acid sequence produced by an open reading frame which appears to be the E. coli pepQ gene. Further comparison of OPAA-2, the E. coli PepQ protein sequence, E. coli aminopeptidase P, and human prolidase showed regions of different degrees of similarity or functionally conserved amino acid substitutions. These findings, along with preliminary data confirming the presence of prolidase activity expressed by OPAA-2, suggest that the OPAA-2 enzyme may, in nature, be used in peptide metabolism.
Amino Acid Sequence↗
Use of adverse conditions to stimulate a cellular stress response by equine articular chondrocytes.
OBJECTIVE: To determine the response of equine articular cartilage cells to heat and calcium stresses. DESIGN: Analysis of newly synthesized, [35S]methionine-labeled proteins after treatment of isolated primary equine chondrocytes. PROCEDURE: Primary cultures of equine articular chondrocytes were incubated at temperatures ranging from 37 to 42 C for heat stress experiments or incubated in the presence or absence of the intracellular calcium pump inhibitor, thapsigargin, for calcium stress experiments. Patterns of new protein synthesis were determined by incubating with [35S]methionine followed by separation of proteins by use of one- or two-dimensional gel electrophoresis and visualization of labeled proteins by use of fluorography. RESULTS: Equine chondrocytes cultured at temperature of 42 C had increased synthesis of specific proteins, compared with the profile of protein synthesis in control chondrocytes cultured at 37 C. These changes were characteristic of the heat shock stress response described in a number of other mammalian cell-types. Equine chondrocytes cultured in the presence of thapsigargin also had increased synthesis of specific proteins. Two-dimensional gel electrophoresis of these newly synthesized proteins revealed the changes to be consistent with the induction of the glucose-regulated protein family of stress proteins. CONCLUSIONS: Changes in the pattern of new protein synthesis can be induced in differentiated equine articular chondrocytes by heat shock or calcium stress. These responses are characteristic of a widely described mammalian stress response that has been postulated to be involved in cellular protective mechanisms. The ability of equine chondrocytes to mount a robust stress response may be important in the processes of tissue damage and recovery in articular joints of horses.
Animals↗
Activation of the myogenin promoter during mouse embryogenesis in the absence of positive autoregulation.
Myogenin, a member of the MyoD family of helix-loop-helix proteins, can induce myogenesis in a wide range of cell types. In addition to activating muscle structural genes, members of the MyoD family can autoactivate their own and cross-activate one another's expression in transfected cells. This has led to the hypothesis that autoregulatory loops among these factors provide a mechanism for amplifying and maintaining the muscle-specific gene expression program in vivo. Here, we make use of myogenin-null mice to directly test this hypothesis. To investigate whether the myogenin protein autoregulates the myogenin gene during embryogenesis, we introduced a myogenin-lacZ transgene into mice harboring a null mutation at the myogenin locus. Despite a severe deficiency of skeletal muscle in myogenin-null neonates, the myogenin-lacZ transgene was expressed normally in myogenic cells throughout embryogenesis. These results show that myogenin is not required for regulation of the myogenin gene and argue against the existence of a myogenin autoregulatory loop in the embryo.
Animals↗