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Biomedical subjects

B Liu

Publications and source records attributed to B Liu.

At least 415 records · Page 23Linked to original sources
PubMed1998

[Flotation spectrophotometric determination of platinum on isochromatic dye ion pair with tetrabromofluorescein and rhodamine 6G].

The flotation spectrophotometric method for the determination of platinum with isochromatic dye ion-pair is described. Rhodamine 6G (R6G) was produced and entered into aqueous phase when pH = 5.5 buffer solution was added to the Pt (SnCl3)5 (R6G)3 by isopropyl ether flotation. And then R6G. TBF was floated by tetrabromofluorescein(TBF)with toluene. The floatation was dissolved in acetone and absorbance of the solution was measured at 530nm. The sensitivity was raised because of the six dyes (Pt : R6G : TBF = 1 : 3 : 3) assistant effect. The molar absorptivity of this method is 5.92 x 10(5)L x mol(-1) x cm(-1). Beer's law was obeyed in the range of 0-2 microg/10mL with a relative standard deviation of 3.8%. The method was applied satisfactorily to the determination of platinum contained in catalysts without separation.

English Abstract↗
PubMed1998

Humanized M195 monoclonal antibody conjugated to recombinant gelonin: an anti-CD33 immunotoxin with antileukemic activity.

The recently characterized immunotoxin HuM195-gelonin consists of a humanized anti-CD33 monoclonal antibody conjugated to the single-chain plant toxin gelonin. Binding of the immunotoxin to hematopoietic cells that express the CD33 differentiation antigen has been demonstrated and results in cytotoxicity due to ribosomal inactivation by gelonin. Blast cells from most patients with acute myelogenous leukemia express CD33, whereas normal stem cells necessary for maintenance of hematopoiesis do not. We asked whether an immunoconjugate using recombinant gelonin rather than plant gelonin is toxic to acute myelogenous leukemia (AML) cell lines and primary AML blasts obtained from patients and exposed to the immunotoxin in vitro. The CD33pos cell lines HL60, OCI/AML2, and OCI/ AML5 showed decreased proliferation when exposed to immunotoxin for 24-72 h. The CD33neg cell line OCI/AML3 was relatively resistant to HuM195, and all cell lines were resistant to equimolar concentrations of unconjugated antibody and gelonin. Primary blast cultures from seven patients with AML had CD33 detectable on 75.7-99.8% of cells by flow cytometry, and all showed dose-dependent decreases in clonogenic cell survival during 24-h incubation with the immunotoxin. Cells selected for low CD33 expression by cell sorting or by prolonged incubation with immunotoxin could reexpress CD33 at baseline levels and remained sensitive to immunotoxin. We conclude that humanized M195 conjugated to recombinant gelonin has antileukemic activity and should be considered for clinical testing in Phase I trials.

Antibodies, Monoclonal↗
PubMed1997

Reductions in the activation of ERK and JNK are associated with decreased IL-2 production in T cells from elderly humans stimulated by the TCR/CD3 complex and costimulatory signals.

T cells from elderly humans often display impaired IL-2 production, but the mechanisms are unknown. Because the activities of extracellular signal-regulated kinases (ERK) and c-Jun NH2-terminal kinases (JNK) are important for IL-2 production, the current study evaluated if aberrancies in the expression and activation of ERK2 or JNK might underlie decreased IL-2 production by human T cells during aging. The present results show that diminished ERK2 and JNK catalytic activities were commonly detected in T cells from elderly humans stimulated with anti-CD3 mAb OKT3 plus PMA. These reductions did not represent temporal shifts in activation or altered expression of ERK2 or JNK. In addition, the reductions of ERK2 activation in stimulated T cells from elderly individuals were accompanied by decreased Raf-1 kinase activation and could be observed without coexisting impairments in JNK activation. Stimulation of ERK2 activation in elderly T cells correlated with IL-2 production and decreased ERK2 activation was consistently associated with reduced IL-2 production. Although the age-related decreases in JNK activation were accompanied by reduced IL-2 production, substantial impairments of JNK activation were observed with diminished ERK2 activation. Moreover, anti-CD3/PMA-stimulated T cells from elderly individuals that displayed normal JNK activation and impaired ERK2 activation continued to demonstrate reduced IL-2 production. These findings show that impairments in the activation of ERK2 and JNK can accompany decreased IL-2 production by T cells from elderly humans and further suggest that aberrancies in TCR/CD3-dependent activation of the Raf-1/MEK/ERK2 cascade may be rate-limiting for the full induction of IL-2.

Aged↗
PubMed1997

Thioredoxin peroxidase is a novel inhibitor of apoptosis with a mechanism distinct from that of Bcl-2.

Thioredoxin peroxidase (TPx) is a member of a newly discovered family of proteins that are conserved from yeast to mammals and to which natural killer enhancing factor belongs. These proteins are antioxidants that function as peroxidases only when coupled to a sulfhydryl reducing system. The physiological function of TPx in cells is not yet known. Here we demonstrate that when the human TPx II, a member of this family, is stably overexpressed in Molt-4 leukemia cells, it protects from apoptosis induced by serum deprivation, ceramide, or etoposide. TPx II, like Bcl-2, is able to inhibit release of cytochrome c from mitochondria to cytosol, and it inhibits lipid peroxidation in cells. TPx II, unlike Bcl-2, could prevent hydrogen peroxide accumulation in cells, suggesting that it functions upstream of Bcl-2 in the protection from apoptosis and may be implicated as an endogenous regulator of apoptosis.

Antioxidants↗
PubMed1997

Specific inhibition of Stat3 signal transduction by PIAS3.

The signal transducer and activator of transcription-3 (Stat3) protein is activated by the interleukin 6 (IL-6) family of cytokines, epidermal growth factor, and leptin. A protein named PIAS3 (protein inhibitor of activated STAT) that binds to Stat3 was isolated and characterized. The association of PIAS3 with Stat3 in vivo was only observed in cells stimulated with ligands that cause the activation of Stat3. PIAS3 blocked the DNA-binding activity of Stat3 and inhibited Stat3-mediated gene activation. Although Stat1 is also phosphorylated in response to IL-6, PIAS3 did not interact with Stat1 or affect its DNA-binding or transcriptional activity. The results indicate that PIAS3 is a specific inhibitor of Stat3.

Amino Acid Sequence↗
PubMed1997

4-hydroxy-5,6-dihydropyrones. 2. Potent non-peptide inhibitors of HIV protease.

The 4-hydroxy-5,6-dihydropyrone template was utilized as a flexible scaffolding from which to build potent active site inhibitors of HIV protease. Dihydropyrone 1c (5,6-dihydro-4-hydroxy-6-phenyl-3-[(2-phenylethyl)thio]-2H-pyran-2-one) was modeled in the active site of HIV protease utilizing a similar binding mode found for the previously reported 4-hydroxybenzopyran-2-ones. Our model led us to pursue the synthesis of 6,6-disubstituted dihydropyrones with the aim of filling S1 and S2 and thereby increasing the potency of the parent dihydropyrone 1c which did not fill S2. Toward this end we attached various hydrophobic and hydrophilic side chains at the 6-position of the dihydropyrone to mimic the natural and unnatural amino acids known to be effective substrates at P2 and P2'. Parent dihydropyrone 1c (IC50 = 2100 nM) was elaborated into compounds with greater than a 100-fold increase in potency [18c, IC50 = 5 nM, 5-(3,6-dihydro-4-hydroxy-6-oxo-2-phenyl-5-[2-phenylethyl)thio] -2H-pyran-2-yl)pentanoic acid and 12c, IC50 = 51 nM, 5,6-dihydro-4-hydroxy-6-phenyl-6-(2-phenylethyl)-3- [(2-phenyl-ethyl)thio]-2H-pyran-2-one]. Optimization of the 3-position fragment to fill S1' and S2' afforded potent HIV protease inhibitor 49 [IC50 = 10 nM, 3-[(2-tert-butyl-5-methylphenyl)sulfanyl]-5,6-dihydro-4 -hydroxy-6-phenyl-6-(2-phenylethyl)-2H-pyran-2-one]. The resulting low molecular weight compounds (< 475) have one or no chiral centers and are readily synthesized.

Anti-HIV Agents↗
PubMed1997

Cross-reactivity of C219 anti-p170(mdr-1) antibody with p185(c-erbB2) in breast cancer cells: cautions on evaluating p170(mdr-1)

BACKGROUND: Increased expression of the multidrug resistance gene (MDR-1)-encoded P-glycoprotein (p170[mdr-1]) is a major cause of tumor cell multidrug resistance. p170(mdr-1) functions as a drug-efflux pump to reduce the cellular accumulation of specific drugs. MDA-MB-435 human breast cancer cells that have been transfected with oncogene c-erbB2 complementary DNA (435.eb cells) express high levels of the transmembrane glycoprotein p185(c-erbB2) and exhibit increased resistance to the chemotherapeutic agent paclitaxel via p170(mdr-1)-independent mechanisms. We have recently discovered that the widely used monoclonal antibody C219, which is specific for p170(mdr-1), may cross-react with p185(c-erbB2) in 435.eb cells. In this study, we have investigated the nature of this cross-reactivity. METHODS: Immunoprecipitation experiments involving the use of breast cancer cells that express different levels of p185(c-erbB2) were performed, and C219 was used for western blot analysis of immunoprecipitated proteins. Immunohistochemical analyses were performed on acetone-fixed slides of human breast cancer cells. Peptide sequence comparisons and enzyme-linked immunosorbent assays were performed to determine the molecular basis of C219 cross-reactivity with p185(c-erbB2). RESULTS: The cross-reactivity of C219 with p185(c-erbB2) was demonstrated by both western blot and immunohistochemical analyses. Peptide sequence comparisons revealed that C219 recognizes an epitope in p170(mdr-1) (C219 epitope) that shares sequence homology with p185(c-erbB2). Enzyme-linked immunosorbent assays demonstrated that C219 recognizes synthetic peptides derived from both the C219 epitope in p170(mdr-1) and the C219 epitope-homologous region in p185(c-erbB2). CONCLUSIONS: The anti-p170(mdr-1) monoclonal antibody C219 cross-reacts with p185(c-erbB2) through a peptide sequence in p185(c-erbB2) that is homologous to the C219 epitope in p170(mdr-1).

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

Prolonged activation of phospholipase D in Chinese hamster ovary cells expressing platelet-activating-factor receptor lacking cytoplasmic C-terminal tail.

The mechanism and role of phospholipase D (PLD) activation by platelet-activating factor (PAF) were examined with Chinese hamster ovary cells stably expressing wild-type PAF receptor (WT-H cells) and truncated PAF receptor lacking the C-terminal cytoplasmic tail (D-H cells). Treatment of D-H cells with PAF resulted in the rapid formation of Ins(1,4,5)P3, which was followed by a sustained phase for more than 10 min. In these cells, PAF-induced PLD activation lasted for more than 20 min. In contrast, PLD activation in WT-H cells was transient. PAF stimulation caused the biphasic formation of 1,2-diacylglycerol (DG) in both types of cell. The first phase was rapid and transient, coinciding with the Ins(1,4,5)P3 peak. The second sustained phase of DG formation was attenuated by butanol, which produces phosphatidylbutanol at the expense of phosphatidic acid (PA) by transphosphatidylation activity of PLD, and by propranolol, a selective inhibitor for PA phosphohydrolase catalysing the conversion of PA into DG. The DG level returned nearly to basal at 20 min after PAF stimulation in WT-H cells, whereas in D-H cells the elevated DG level was sustained for more than 20 min. The profile of translocation of protein kinase Calpha (PKCalpha) to membrane was similar to that of DG formation. In WT-H cells, PKCalpha was transiently associated with membranes and then returned to the cytosol. However, in D-H cells PKCalpha was rapidly translocated to and remained in membranes for more than 20 min. Butanol suppressed this sustained translocation of PKCalpha. Furthermore the mRNA levels of c-fos and c-jun by PAF in WT-H cells were much lower than those in D-H cells. Propranolol and butanol at concentrations that inhibited the formation of DG suppressed the PAF-induced mRNA expression of c-fos and c-jun. Taken together, the prolonged PLD activation in D-H cells confirmed a primary role for phospholipase C/PKC in PLD activation by PAF. Furthermore the results obtained here suggest that sustained PLD activation in turn leads to chronic activation and membrane translocation of PKCalpha, which might play an important role in the expression of c-fos and c-jun.

1-Butanol↗
PubMed1997

Phospholipase A2 is necessary for tumor necrosis factor alpha-induced ceramide generation in L929 cells.

The role of cytosolic phospholipase A2 (cPLA2) in the regulation of ceramide formation was examined in a cell line (L929) responsive to the cytotoxic action of tumor necrosis factor alpha (TNFalpha). In L929 cells, the addition of TNFalpha resulted in the release of arachidonate, which was followed by a prolonged accumulation of ceramide occurring over 5-12 h and reaching 250% over base line. The formation of ceramide was accompanied by the hydrolysis of sphingomyelin and the activation of three distinct sphingomyelinases (neutral Mg2+-dependent, neutral Mg2+-independent, and acidic enzymes). The variant cell line C12, which lacks cPLA2, is resistant to the cytotoxic action of TNFalpha. TNFalpha was able to activate nuclear factor kappaB in both the wild-type L929 cells and the C12 cells. However, TNFalpha was unable to cause the release of arachidonate or the accumulation of ceramide in C12 cells. C6-ceramide overcame the resistance to TNFalpha and caused cell death in C12 cells to a level similar to that in L929 cells. The introduction of the cPLA2 gene into C12 cells resulted in partial restoration of TNFalpha-induced arachidonate release, ceramide accumulation, and cytotoxicity. This study suggests that cPLA2 is a necessary component in the pathways leading to ceramide accumulation and cell death.

Animals↗
PubMed1997

Characterization of immobilization of an enzyme in a modified Y zeolite matrix and its application to an amperometric glucose biosensor.

A new approach to construct an amperometric biosensor is described. Without using bovine serum albumin-glutaraldehyde, glucose oxidase (GOx) was immobilized on a dealuminized Y zeolite (DAY)-modified platinum electrode to construct a glucose sensor. The large specific surface area of the zeolite substrate resulted in high enzyme loading. The immobilized GOx in this manner was stable and could maintain its high activity for at least 3 months. The interactions between the zeolite and the enzyme were investigated by means of Fourier transform infrared spectra, and the pore distribution and the surface acid property of DAY were preliminarily studied. The results showed that the hydrophilic property and the existing mesopores of DAY played important roles in the enzyme immobilization. This resulting biosensor exhibited good reproducibility and selectivity, owing to the uniform pore structure and unique ion-exchange property of the zeolite. The biosensor responded rapidly to glucose in the linear range from 2.0 x 10(-6) to 3.0 x 10(-3) M, with a detection limit of 0.5 microM.

Biosensing Techniques↗
PubMed1997

Inhibition of the neutral magnesium-dependent sphingomyelinase by glutathione.

Sphingomyelin hydrolysis through the activation of sphingomyelinases has become a potentially important signaling pathway with the product ceramide implicated in the regulation of cell growth, differentiation, apoptosis, and inflammatory responses. However, little is known about the regulation of sphingomyelinases. In this study, we show that the magnesium-dependent, neutral pH-optimum and membrane-associated sphingomyelinase (N-SMase) is inhibited, in a dose-dependent manner, by glutathione (GSH) at physiological concentrations with a greater than 95% inhibition observed at 5 mM GSH. The inhibitory effect of GSH was reproduced by gamma-glutamyl-cysteine, but not the cysteinyl-glycine fragment of GSH. The S-modified GSH analogs were as effective as GSH in inhibiting the N-SMase. On the other hand, neither dithiothreitol nor beta-mercaptoethanol had any effect on the N-SMase, suggesting that the sulfhydryl in GSH is not required for inhibition of N-SMase. GSH had no effect on the acid pH-optimum SMase, whereas dithiothreitol inhibited the acid SMase. These results suggest that in cells the N-SMase is inactive in the presence of physiological concentrations of GSH (1-20 mM). Finally, treatment of cultured Molt-4 cells with the GSH synthesis inhibitor, L-buthionine-(SR)-sulfoximine, resulted in a time-dependent depletion of GSH, accompanied by an increased hydrolysis of sphingomyelin and production of ceramide. Since GSH depletion is observed in a variety of cells in the process of cellular injury and apoptosis, these studies suggest that depletion of GSH may be an important mechanism in activation of N-SMase. This mechanism may therefore bring together the fields of oxidative stress and signaling through products of sphingomyelin hydrolysis.

Buthionine Sulfoximine↗
PubMed1997

Selective involvement of ceramide in cytokine-induced apoptosis. Ceramide inhibits phorbol ester activation of nuclear factor kappaB.

Among its diverse biologic effects, the cytokine tumor necrosis factor alpha causes the rapid nuclear translocation of the transcription factor, nuclear factor kappaB (NF-kappaB). The p55 tumor necrosis factor (TNF) receptor shares with the related APO-1/Fas antigen the ability to initiate apoptosis. We investigated the role of the sphingolipid mediator ceramide in the cytokine-induced signaling mechanisms leading to NF-kappaB activation and cell death. Several lines of evidence presented here suggest that ceramide generated in response to TNFalpha or Fas activation is not involved in NF-kappaB activation. (i) Cell-permeable ceramides and exogenous sphingomyelinase failed to induce either nuclear translocation of NF-kappaB or degradation of its cytosolic inhibitor, I-kappaB, in Jurkat T cells. (ii) Ceramide treatment of cells inhibited phorbol ester-induced activation of NF-kappaB. (iii) TNFalpha potently activated NF-kappaB in a cell line deficient in acid sphingomyelinase. (iv) TNFalpha activated NF-kappaB within minutes without altering ceramide levels. (v) Treatment of Jurkat cells with cross-linking antibodies to APO-1/Fas induced large scale increases in ceramide and apoptosis without affecting NF-kappaB. (vi) Ceramide generation in response to Fas activation was inhibited by N-acetyltyrosinylvalinylalanylaspartyl chloromethyl ketone, a peptide inhibitor of interleukin-1beta-converting enzyme-like proteases, whereas TNFalpha-induced NF-kappaB activation was unaffected by the inhibitor. These results show that ceramide accumulation belongs selectively to the apoptotic pathway(s) induced by cytokines, and, if anything, ceramide may participate in negative feedback regulation of NF-kappaB.

Apoptosis↗
PubMed1997

Expression of neutral sphingomyelinase identifies a distinct pool of sphingomyelin involved in apoptosis.

The activation of sphingomyelinase and generation of ceramide have been implicated as important regulatory pathways in cell growth and apoptosis. Bacterial sphingomyelinase has been used in many cell systems to mimic the activation of endogenous sphingomyelinase. These studies, however, have been complicated by the inability of exogenously applied bacterial sphingomyelinase to perform many of the effects of short chain cell permeable ceramides, indicating that there may be a distinct signal transducing pool of sphingomyelin not accessible to exogenous sphingomyelinase or that endogenous ceramide is not sufficient to induce these changes. We cloned the Bacillus cereus sphingomyelinase gene by polymerase chain reaction and subcloned it into a mammalian expression vector under the control of an inducible promoter. Upon stable transfection and induction of B. cereus sphingomyelinase, there were increases in neutral sphingomyelinase activity, cellular ceramide levels, cleavage of the death substrate poly(ADP-ribosyl)polymerase, and cell death. In contrast, exogenously applied B. cereus sphingomyelinase, despite causing higher elevations in ceramide levels, was unable to induce poly(ADP-ribosyl)polymerase cleavage or cell death. These results support the existence of a signal transducing pool of sphingomyelin that is distinct from the pool accessible to exogenous sphingomyelinase.

Animals↗
PubMed1997

Sphingomyelinases in cell regulation.

Sphingomyelin hydrolysis and ceramide generation have emerged as key events in cellular regulation. Sphingomyelinases (SMases) catalyse the breakdown of sphingomyelin to form ceramide and phosphorylcholine. Ceramide formed through activation of SMases may function as a second messenger in mediating cell growth, differentiation, stress responses, and programmed cell death (apoptosis). So far, five types of SMases have been described and they include the acidic, the acidic zinc-dependent, the neutral magnesium-dependent, the neutral magnesium-independent, and the alkaline SMase. These SMases differ in tissue distribution, cofactor dependence, mechanism for regulation, and involvement in diverse cellular processes. At least two of these sphingomyelinases may regulate the intracellular levels of ceramide and subsequent ceramide-mediated responses. This review will focus on the identification, regulation and roles of SMases in cell function.

Journal Article↗
PubMed1997

A two-backbone polymer model for interphase chromosome geometry.

A polymer model for the overall geometric structure of a human chromosome during the G0/G1 portion of cell-cycle interphase is constructed, based on fluorescence in situ hybridization data on distances between defined genomic sequences. The model consists of flexible giant loops, averaging about 6 million base pairs, with two random-walk backbones; it involves essentially three parameters. Numerical results based on properly selected values of parameters fit the data well.

Chromosomes, Human↗
PubMed1997

Mutations in the heavy chain of cytoplasmic dynein suppress the nudF nuclear migration mutation of Aspergillus nidulans.

To identify proteins that interact directly or indirectly with the NUDF protein, which is required for nuclear migration in Aspergillus nidulans, we initiated a screen for extragenic suppressors of the heat-sensitive nudF6 mutation. Suppressor mutations in at least five genes, designated snfA-snfE, caused improved growth and nuclear migration at high temperatures compared to the nudF6 parent. Two snfC mutations mapped near the nudA gene, which encodes the cytoplasmic dyncin heavy chain, and could be repaired by transformation with wild-type nudA DNA, demonstrating that they are mutations in nudA. The snfC mutations are bypass suppressors of nudF and genetic evidence indicated that NUDA and NUDF act in the same nuclear migration pathway. Taken together, our data suggests that NUDF affects nuclear migration by acting on the dynein motor system.

Aspergillus nidulans↗