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Biomedical subjects

P Lu

Publications and source records attributed to P Lu.

At least 91 records · Page 5Linked to original sources
PubMed1999

Two approaches for determining the urinary excretion patterns of nucleosides--HPLC and CE.

Cancer patients excrete in their urine increased amounts of modified purines and pyrimidines. These modified mucleosides are primary constituents of RNA and are synthesized at the macromolecular level. When RNA is catabolized, most of these modified nucleosides cannot be reutilized; consequently, they are excreted. Due to their increased urinary excretion in conjunction with altered RNA turnover in carcinogenesis, they have been proposed as tumour markers. We developed both reversed-phase high performance liquid chromatographic and capillary electrophoretic approaches to determine normal and modified nucleosides in urine. Data obtained with CE are in good accordance with those from HPLC. The reference excretion levels of urinary nucleosides from healthy volunteers were established, and elevated ones of modified nucleosides from 34 patients with different kinds of cancer were observed. The developed methods are suitable for the analysis of large series of samples for clinical studies.

Biomarkers, Tumor↗
PubMed1999

[Development of column switching systems in industrial gas chromatography].

According to the home-made requirement of column switching systems in 23 industrial gas chromatographs in 300,000 Tons of olefin plant, based on the research work of the expert system on gas chromatography and movement laws of compounds of interest in columns, the suitable stationary phases were defined, and the column switching systems were designed by using combined cutting techniques which consist of backflushing, fore-flushing and heart cutting, and by controlling column lengths, flows, flow directions and temperatures. The practical applications showed the column systems satisfy the on-line monitoring requirement of the production process and can be used continually for 1-3 years.

English Abstract↗
PubMed1999

[Gas chromatographic method for the analysis of polychlorinated biphenyls in pine needles].

Polychlorinated biphenyls (PCBs) are a class of 209 chemical compounds, in which 1-10 chlorine atoms are attached to a biphenyl molecule. PCBs are members of halogenated aromatic group of environmental pollutants that have been identified worldwide in diverse environmental matrices. PCBs in air, soils, sediment, water, transformer oils and other environmental matrices have been determined in the past years. In this work a method for routine analysis of PCBs in pine needles has been developed. First, extractions were carried out in Soxhlet apparatus with n-hexane as solvent. Then, a silica gel chromatographic column was applied to pretreat the pine needle samples. The reference standard used was clophen 50. The recovery was about 90%, so the feasibility and reliability were assured. After the analysis of PCBs in pine needle samples from four different regions, the distribution of PCBs in environment can be discussed and monitored. In this method, a Shimadzu GC-7A gas chromatograph equipped with a 63Ni electron capture detector was used for the analysis of PCBs. From the results, we can see the PCB pollution in different regions. So evaluation of air pollution level through foliage data is feasible. The sample preparation and analytical method mentioned in this paper is reliable and simple.

Chromatography, Gas↗
PubMed1999

[Purification and specific detection of two major Schistosoma gut-associated circulating antigens, CAA and CCA].

AIM: To investigate the specificity differences of the 2 major gut-associated diagnostic circulating molecules in Schistosoma japonicum infection, CAA and CCA, and to obtain sufficient purified material for setting up a standard series in quantitative determinations. METHODS: Isolation and purification of the two worm fractions from a trichloroacetic acid (TCA) soluble preparation of S. japonicum adult worm antigen (AWAj-TCA) by Mono-Q anion exchange chromatography were performed and the specific reactivity of the eluted fractions by antigen-capture ELISA (specific for CAA or CCA) with reference to affinity purified preparations of S. mansoni CAA and CCA was analysed. RESULTS: By using an ionic strength gradient, CCA was eluted in two major peaks, an unbound fraction CCA-1, and a major bound fraction CCA-2. Two additional minor peaks, CCA-3 and CCA-4, were eluted at higher ionic strengths. CAA was only detected in a bound fraction, partly overlapping with CCA-3. In the CCA-1 and CCA-2 fractions reactivity was only found in the antigen-capture ELISA using anti-CCA McAbs both for capture and detection. The CAA fraction was predominantly found to be positive in the antigen-capture ELISA using anti-CAA McAbs both for capture and detection. However, when using combinations of anti-CCA and anti-CAA McAbs for capture and detection by ELISA this fraction showed some reactivity. CONCLUSION: Two CCA fractions contain molecules which bear at least two CCA-epitopes; while the CAA fraction contains molecules which contain at least two CAA-epitopes, and one CCA-epitope.

Animals↗
PubMed1998

Real time detection of DNA.RNA hybridization in living cells.

Demonstrating hybridization between an antisense oligodeoxynucleotide and its mRNA target has proven to be extremely difficult in living cells. To address this fundamental problem in antisense research, we synthesized "molecular beacon" (MB) reporter oligodeoxynucleotides with matched fluorescent donor and acceptor chromophores on their 5' and 3' ends. In the absence of a complementary nucleic acid strand, the MB remains in a stem-loop conformation where fluorescence resonance energy transfer prevents signal emission. On hybridization with a complementary sequence, the stem-loop structure opens increasing the physical distance between the donor and acceptor moieties thereby reducing fluorescence resonance energy transfer and allowing a detectable signal to be emitted when the beacon is excited by light of the appropriate wavelength. Solution hybridization studies revealed that in the presence of a complementary strand targeted MB could yield up to a 60-fold increase in fluorescence intensity in comparison to control MB. By using a fluorescence microscope fitted with UV fluoride lenses, the detection limit of preformed MB/target sequence duplexes microinjected into cells was found to be >/=1 x 10(-1) ag of MB, or approximately 10 molecules of mRNA. On the basis of this exquisite sensitivity, real-time detection of MB/target mRNA hybridization in living cells was attempted by microinjecting MB targeted to the vav protooncogene, or control MB, into K562 human leukemia cells. Within 15 min, confocal microscopy revealed fluorescence in cells injected with targeted, but not control, MB. These studies suggest that real-time visualization and localization of oligonucleotide/mRNA interactions is now possible. MB could find utility in studying RNA processing, trafficking, and folding in living cells. We hypothesize that MB may also prove useful for finding targetable mRNA sequence under physiologic conditions.

Animals↗
PubMed1998

Linear and cyclic peptides as substrates and modulators of P-glycoprotein: peptide binding and effects on drug transport and accumulation.

One cause of multidrug resistance (MDR) in human cancers is the overexpression of the P-glycoprotein multidrug transporter, a member of the ABC superfamily of membrane proteins. Natural products and chemotherapeutic drugs are pumped out of the cell by P-glycoprotein in an ATP-dependent fashion. There is growing evidence that many hydrophobic peptides are also P-glycoprotein substrates. With the use of a fluorescence-quenching assay, we have shown that some linear and cyclic hydrophobic peptides interact with P-glycoprotein, whereas others do not. The measured values of the quenching constant, Kq, for interaction of peptides with P-glycoprotein ranged from 200 nM for cyclosporine A to 138 microM for the tripeptide N-acetyl-leucyl-leucyl-norleucinal. Peptides that interacted with P-glycoprotein in the fluorescence assay also blocked colchicine transport into plasma membrane vesicles from MDR cells. The values of Dm, the peptide concentration causing 50% inhibition of drug uptake, were highly correlated with the values of Kq, over three orders of magnitude. The P-glycoprotein ATPase stimulation/inhibition profile of the peptides was not helpful in making a quantitative assessment of the ability of a peptide to interact with P-glycoprotein or to block drug transport. Some hydrophobic peptides were able to restore accumulation in MDR cells of the chemotherapeutic drug daunorubicin and the fluorescent dye rhodamine 123 to the levels observed in the drug-sensitive parent. Peptides that interacted with P-glycoprotein also displayed a relatively low overall toxicity to intact MDR cells, and inhibited drug transport at concentrations below the toxic range. Hydrophobic peptides should be given serious consideration for development as clinical chemosensitizing agents.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1998

Cloning and disruption of the beta-isopropylmalate dehydrogenase gene (LEU2) of Pichia stipitis with URA3 and recovery of the double auxotroph.

Transformation of Pichia stipitis is required to advance genetic studies and development of xylose metabolism in this yeast. To this end, we used P. stipitis URA3 (PsURA3) to disrupt P. stipitis LEU2 in a P. stipitis ura3 mutant. A highly fermentative P. stipitis mutant (FPL-DX26) was selected for resistance to 5'-fluoroorotic acid to obtain P. stipitis FPL-UC7 (ura3-3). A URA3:lacZ "pop-out" cassette was constructed containing PsURA3 flanked by direct repeats from segments of the lacZ reading frame. The P. stipitis LEU2 gene (PsLEU2) was cloned from a P. stipitis CBS 6054 genomic library through homology to Saccharomyces cerevisiae LEU2, and a disruption cassette was constructed by replacing the PsLEU2 reading sequence with the PsURA3:lacZ cassette. FPL-UC7 (ura3-3) was transformed with the disruption cassette, and a site-specific integrant was identified by selecting for the Leu- Ura+ phenotype. The ura3 marker was recovered from this strain by plating cells onto 5'-fluoroorotate and screening for spontaneous URA3 deletion mutants. Excision of the flanked PsURA3 gene resulted in the Leu- Ura- phenotype. The double auxotrophs are stable and can be transformed at a high frequency by PsLEU2 or PsURA3 carried on autonomous-replication-sequence-based plasmids.

3-Isopropylmalate Dehydrogenase↗
PubMed1998

Arabidopsis SKP1, a homologue of a cell cycle regulator gene, is predominantly expressed in meristematic cells.

The yeast SKP1 gene and its human homolog p19skp1 encode a kinetochore protein required for cell cycle progression at both the DNA synthesis and mitosis phases of the cell cycle. In orchids we identified a cDNA (O 108) that is expressed in early stages of ovule development and is homologous to the yeast SKP1. Based on the orchid O 108 cDNA clone, we identified and characterized an Arabidopsis thaliana (L.) Heynh. cDNA designated ATskp1 that also has high sequence similarity to yeast SKP1. The Arabidopsis ATskp1 is a single-copy gene that mapped to chromosome 1. The expression of the ATskp1 gene was highly correlated with meristem activity in that its mRNA accumulated in all of the plant meristems including the vegetative shoot meristem, inflorescence and floral meristems, root meristem, and in the leaf and floral organ primordia. In addition, ATskp1 was also highly expressed in the dividing cells of the developing embryo, and in other cells that become multinucleate or undergo endoreplication events such as the endosperm free nuclei, the tapetum and the endothelium. Based on its spatial pattern of expression, ATskp1 is a marker for cells undergoing division and may be required for meristem activity.

Arabidopsis↗
PubMed1998

PNZIP is a novel mesophyll-specific cDNA that is regulated by phytochrome and the circadian rhythm and encodes a protein with a leucine zipper motif.

We isolated and characterized a novel light-regulated cDNA from the short-day plant Pharbitis nil that encodes a protein with a leucine (Leu) zipper motif, designated PNZIP (Pharbitis nil Leu zipper). The PNZIP cDNA is not similar to any other gene with a known function in the database, but it shares high sequence homology with an Arabidopsis expressed sequence tag and to two other sequences of unknown function from the cyanobacterium Synechocystis spp. and the red alga Porphyra purpurea, which together define a new family of evolutionarily conserved Leu zipper proteins. PNZIP is a single-copy gene that is expressed specifically in leaf photosynthetically active mesophyll cells but not in other nonphotosynthetic tissues such as the epidermis, trichomes, and vascular tissues. When plants were exposed to continuous darkness, PNZIP exhibited a rhythmic pattern of mRNA accumulation with a circadian periodicity of approximately 24 h, suggesting that its expression is under the control of an endogenous clock. However, the expression of PNZIP was unusual in that darkness rather than light promoted its mRNA accumulation. Accumulation of PNZIP mRNA during the dark is also regulated by phytochrome, since a brief exposure to red light in the middle of the night reduced its mRNA levels. Moreover, a far-red-light treatment at the end of day also reduced PNZIP mRNA accumulation during the dark, and that effect could be inhibited by a subsequent exposure to red light, showing the photoreversible response attributable to control through the phytochrome system.

Amino Acid Sequence↗
PubMed1998

Cloning and characterization of two pyruvate decarboxylase genes from Pichia stipitis CBS 6054.

In Pichia stipitis, fermentative and pyruvate decarboxylase (PDC) activities increase with diminished oxygen rather than in response to fermentable sugars. To better characterize PDC expression and regulation, two genes for PDC (PsPDC1 and PsPDC2) were cloned and sequenced from P. stipitis CBS 6054. Aside from Saccharomyces cerevisiae, from which three PDC genes have been characterized, P. stipitis is the only organism from which multiple genes for PDC have been identified and characterized. PsPDC1 and PsPDC2 have diverged almost as far from one another as they have from the next most closely related known yeast gene. PsPDC1 contains an open reading frame of 1,791 nucleotides encoding 597 amino acids. PsPDC2 contains a reading frame of 1,710 nucleotides encoding 570 amino acids. An 81-nucleotide segment in the middle of the beta domain of PsPDC1 codes for a unique segment of 27 amino acids, which may play a role in allosteric regulation. The 5' regions of both P. stipitis genes include two putative TATA elements that make them similar to the PDC genes from S. cerevisiae, Kluyveromyces marxianus, and Hanseniaspora uvarum.

Amino Acid Sequence↗
PubMed1998

[Hepatitis B virus infection and aflatoxin exposure in the development of primary liver cancer].

OBJECTIVE: To investigate the role of hepatitis B Virus (HBV) infection and aflatoxin (AF) exposure in the development of primary liver cancer (PLC). METHODS: A 10-year prospective case-control study was carried out in 737 HBsAg carriers and 699 HBsAg negative cases, and the aflatoxin B1 albumin adducts (AFB1-Alb) were detected in the serum of the cohort including 30 HBsAg postive cases and 150 control individuals according to the case-control study model (ratio 1:5) at random in the high prevalance area of PLC. RESULTS: The average year-incidence rate was significantly higher in the HBsAg postive group (824.13/100,000) than in the control group (70.97/100,000, RR = 11.61). There was no significant difference in the incidence rate of other tumors between the two groups (P > 0.05). The serum positive rate of AFB1-Alb was significantly higher in the PLC group (76.67%) than in the control group (48.67%, OR = 3.47), and the serum concentration of AFB1-Alb was also significantly higher in the PLC group than in the control group (P < 0.01). CONCLUSION: HBV infection and AF exposure are important etiological factors in the development of PLC and both result in carcinogenic synergy.

Adult↗
PubMed1998

[Comparative observation on the efficacy of several test/probe systems for detecting schistosome circulating antigen].

AIM: To compare the efficacy of 5 test/probe systems for the detecting of schistosomal circulation antigen (SCA) in sera from infected rabbits. METHODS: Nine rabbits were infected with monosexual cercariae (either male or female), 7 rabbits were infected with bisexual cercariae as controls. Blood samples were collected periodically post infection (p.i). 15 rabbits were infected each with 250 bisexual cercaria and divided into 3 groups, 2 groups were treated with allyl thiourea (295-590 mg/kg) from 19 d or 46 d respectively, to inhibit their egg formation. One untreated group was used as control. Blood samples were collected weekly until 8 wk post infection. SCA detecting methods include (1) dot-ELISA/McAb anti-surface membrane antigen of adult worms (8SE4), (2) dot-ELISA/McAb anti-CCA (3D10), (3) Sandwich dot-ELISA/McAb anti-egg antigen (MG2), (4) Sandwich-ELISA/McAb anti-egg antigen (2H10), (5) Sandwich-ELISA/McAb anti-CAA (1B10). RESULTS: Using method (1) all of the 18 rabbits infected with monosexual cercariae were negative. Using method (2) only 1 rabbit harboured 133 male worms showed positive. Using method (5) no SCA were detected in sera from female cercariae-infected rabbits but 3 out of 9 male cercariae-infected rabbits showed positive reaction. SCA detecting results from rabbits treated with allyl thiourea: both method (1) and (3) showed negative in rabbits treated from 19 d (p.i.) but all gave positive reaction in 6-7 wk in rabbits treated from 46 d (p.i.). There were all negative detected by using method (4) and all positive at 6 wk (p.i.) detected by method (5) in rabbits of 3 groups whether treated or non-treated. CONCLUSION: Various test/probe systems have different efficacy in detecting SCA but (5) of the methods (1)(2) tests/probe systems could not detect SCA in rabbits with single female cercariae infection. From the egg formation inhibition study, the SCA detected by McAb 8SE4 dot-ELISA might be related to egg maturation. Whether the surface membrane antigen of adult worm has common antigenic component with egg antigen remains to be studied.

Animals↗
PubMed1997

Interaction of the cytoplasmic tail of CTLA-4 (CD152) with a clathrin-associated protein is negatively regulated by tyrosine phosphorylation.

CTLA-4 (CD152), high-avidity receptor for CD80 and CD86, is a powerful regulator of T cell activation. While CTLA-4 functions at the cell surface, it is primarily localized in intracellular vesicles and cycles to the cell surface. The CTLA-4 cytoplasmic domain contains sequences that direct its intracellular localization and regulate its signaling. Here we demonstrate that effector molecules involved in receptor trafficking and signaling interact with distinct, but overlapping, sequences in the CTLA-4 cytoplasmic domain. Using the yeast two-hybrid method, we demonstrate association of the mu2 subunit of AP-2, the clathrin-associated complex found in plasma membrane-associated coated pits, with the cytoplasmic tail of CTLA-4, but not CD28. The mu1 subunit of AP-1, found in Golgi-associated coated pits, associated with neither CTLA-4 nor CD28. Sequences required for interaction of mu2 and CTLA-4 were localized to residues, 161TTGVY in CTLA-4; this sequence is N-terminal to, but overlaps with, a previously identified SH2 binding motif, 165YVKM, involved in CTLA-4 signaling. Mu2 interacted preferentially with CTLA-4 when residue 165Y was nonphosphorylated, whereas a PI3 kinase SH2 domain interacted preferentially when 165Y was phosphorylated. In co-transfection experiments, both tyrosine residues in the cytoplasmic tail of CTLA-4 (165Y and 182Y) were phosphorylated by the T lymphocyte-associated tyrosine kinase, p56lck. Thus, phosphorylation of CTLA-4 residue 165Y may reciprocally regulate signaling and trafficking of CTLA-4 by determining which effector molecules bind to its cytoplasmic tail.

Abatacept↗
PubMed1997

Characterization of monoclonal antibodies for the rapid detection of foodborne campylobacters.

The specificity of 97 monoclonal antibodies (MAbs) to the Campylobacter jejuni Lior serogroup 6 reference strain was assessed using an indirect enzyme linked immunosorbent assay (ELISA). Four MAbs, M316, M337, M357 and M637, reacted with whole cells of the C. jejuni, C. coli and C. lari reference strains of the 20 most common Lior serogroups and 25 recent C. jejuni and C. coli isolates, and did not react with most of the 42 other Campylobacter and non-Campylobacter spp. tested. Immunoblot analysis revealed that MAbs M337 and M357 reacted with a protein component with molecular mass of approximately 62 kiloDaltons (kDa) while M316 and M637 reacted with protein components of approximately 92 and 31 kDa, respectively. The detection limit of M357 in an indirect ELISA was 10(5) colony forming units. These four highly specific MAbs may be useful reagents of an immunoassay for the rapid detection of thermophilic campylobacters in foods and clinical samples.

Antibodies, Monoclonal↗
PubMed1997

CD28 dependence of T cell differentiation to IL-4 production varies with the particular type 2 immune response.

T cell differentiation to effector cell function is required for the development of a type 2 immune response. The T cell surface molecule, CD28, is widely considered to be the principal costimulatory molecule involved in T cell differentiation to effector function, including IL-4 production, although this has been difficult to directly examine in vivo. We have studied in vivo differentiation to T cell effector function during two type 2 immune responses in CD28 knockout mice: the systemic immune response to goat anti-mouse IgD Ab and the mucosal immune response following oral inoculation with the nematode parasite, Heligmosomoides polygyrus. Our results show that in C57BL/6 CD28 knockout mice elevations in IL-4 gene expression and protein secretion are blocked during the immune response to goat anti-mouse IgD, and associated increases in serum IgG1 and IgE are also inhibited to untreated control levels. In marked contrast, T cell differentiation to IL-4 production is comparable in C57BL/6 CD28 -/- and CD28 +/+ H. polygyrus-inoculated mice, and elevations in both serum IgG1 and IgE levels occur. These results indicate that the specific kind of type 2 immune response determines whether T cell differentiation to IL-4 production is CD28 dependent.

Animals↗
PubMed1997

Effects of blocking B7-1 and B7-2 interactions during a type 2 in vivo immune response.

The costimulatory signal provided to T cells through CD28/CTLA-4 interactions is required for in vivo Th cell effector function associated with cytokine production. However, it is uncertain whether the two well-characterized ligands for these molecules, B7-1 and B7-2, differentially influence the consequent development of a type 1 or a type 2 primary response. We have examined the in vivo effects of blocking B7-1 and/or B7-2 ligand interactions on the type 2 mucosal immune response that follows oral infection of mice with the nematode parasite, Heligmosomoides polygyrus. Administration of the combination of anti-B7-1 and anti-B7-2 Abs inhibited H. polygyrus-induced increases in serum IgG1 and IgE levels, the expansion of mesenteric lymph node (MLN) germinal centers, in situ CD4+ T cell expansion, elevated blood eosinophils, and increased intestinal mucosal mast cells. Similarly, both Abs blocked MLN and Peyer's patch cytokine gene expression and elevations in MLN T cell-derived IL-4 protein secretion. However, in the same experiments, administration of either anti-B7-1 or anti-B7-2 Abs alone had little effect on any of these parameters. T cell and B cell activation was also blocked by the combination of anti-B7-2 and a B7-1-specific mutant Y100F CTLA-4Ig construct. These results suggest that to the extent that anti-B7-1 and anti-B7-2 mAbs block B7 interactions, either B7-1 or B7-2 ligand interactions can provide the required costimulatory signals that lead to T cell effector function during a type 2 in vivo immune response.

Animals↗
PubMed1997

Escherichia coli lac repressor-lac operator interaction and the influence of allosteric effectors.

The wild type E. coli lac operator is embedded in a 35 base-pair DNA sequence containing extensive 2-fold symmetry, suggesting a symmetric repressor operator complex. However, deviations from strict 2-fold symmetry occur at the central base-pair and at three additional base-pairs. Using an operator fragment binding analysis we have determined: (a) a relative contribution each pair provides to the lac repressor-lac operator DNA complex, (b) the operator DNA length necessary for maximum binding to lac repressor; and (c) the contribution of the several non-symmetric base in the wild-type operator to the binding affinity. Since lac repressor-lac operator DNA interaction is reduced upon binding of the gratuitous inducer, isopropyl-beta-D-galactoside (IPTG), the same DNA fragment binding analysis was performed with the low affinity form of lac repressor. In the presence of inducer, the affinity for the left half site of the wild-type lac operator is reduced without significant reduction on the right half of the operator. Conversely, the anti-inducer orthonitrophenylfucoside (ONPF) which stabilizes the lac repressor-lac operator complex increases the binding affinity, particularly to the right half of the operator.

Allosteric Regulation↗