Biomedical subjects

D Lu

Publications and source records attributed to D Lu.

At least 289 records · Page 16Linked to original sources

PubMed1993

Effect of sample numbers on the kinetic data analysis of MR contrast agents.

The Monte Carlo simulation method is used to determine the uncertainties of the estimated are and mean transit time (MTT) of a regional cerebral concentration-time curve of a bolus injected paramagnetic contrast agent. Many factors contribute to these uncertainties. This study concentrates on the error propagation from the sample noise at different sample time points along the concentration-time curve. The uncertainties of area and MTT were determined as a function of the sample noise and the number of sample time points. For example, the percentage standard deviations (uncertainties) of both area and MTT are less than 12% with five samples whose signal-to-noise ratios are greater than 50.

Bias↗

PubMed1993

Glycosphingolipid acyl chain orientational order in unsaturated phosphatidylcholine bilayers.

The glycosphingolipid, galactosyl ceramide (GalCer), was studied by 2H nuclear magnetic resonance (NMR) in fluid phospholipid bilayer membranes, with regard to arrangement of its acyl chain. For this purpose, species with perdeuterated 18-carbon fatty acid (18:0[d35]GalCer) or with perdeuterated 24-carbon fatty acid (24:0[d47] GalCer) were dispersed in bilayers of the 18-carbon phospholipid, 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC). For 18:0[d35] GalCer, smoothed profiles of the order parameter, SCD, were found to be very similar to one another over the range of glycolipid concentration, 5-40 mol%. In addition, they were very similar to orientational order parameter profiles well known from the literature on phospholipid and glycolipid acyl chains (which deals in general with membranes of homogeneous chain length in the range 14-18 carbons). Corresponding order parameter profiles for the long-chain species, 24:0[d47] GalCer, were also similar to one another for glycolipid concentrations between 5 and 40 mol%. Their shapes, however, were distinctly different from those of the shorter chain analogues. SCD profiles for the two species were quantitatively similar to a membrane depth of C15. SCD values at C16 and C17 were approximately 20 and 30%, respectively, higher for the long-chain glycosphingolipid than for its short-chain analogue in SOPC. Nitroxide spin labels attached rigidly to C16 of the long-chain glycolipid in SOPC gave electron paramagnetic resonance (EPR) order parameters that were twice as high as for a spin label at C16 on the shorter chain glycolipid. Comparison was made between spectra of 24:0[d47] GalCer in SOPC and fully hydrated bilayers of the pure 24:0[d47] GalCer, a system that is considered to be partially interdigitated in fluid and gel phases. The resultant 2H NMR order parameter profiles displayed similar features, indicating that related organizational properties exist in these fluid systems. Effective chain length of 24:0[d47] GalCer within the SOPC membrane was calculated using the method of Schindler and Seelig (1975. Biochemistry, 14:2283-2287). The result suggested that the long-chain fatty acid should protrude roughly one third of the host matrix chain length across the bilayer midplane. However, a treatment of the same order parameters making very few assumptions about chain conformation indicated a high degree of orientational flexibility for the "extra" length of the long chain fatty acid. It seems likely that a realistic treatment of the long-chain fatty acidin a shorter chain fluid host matrix considers interdigitation as a subset of the conformational possibilities, many of which are rapidly interconverting on the NMR timescale of 10-4_10-5 s and longer lived on the EPR timescale of 10-8_10-9 s.

Biophysical Phenomena↗

PubMed1993

Angiotensin II type 1 receptor mRNA levels in the brains of normotensive and spontaneously hypertensive rats.

The type 1 angiotensin II (AII) receptor (AT1-R) has been implicated in the physiological actions mediated by AII in the brain. In view of the reported hyperactivity of the brain AII system in the spontaneously hypertensive rat (SHR), we compared the expression of AT1-R mRNAs in the brains of normotensive [Wistar Kyoto (WKY)] and SHR animals. Northern blot analysis showed about three- and approximately 20-fold increases in the levels of AT1-R mRNAs from the hypothalamus and brainstem areas, respectively, of the SHR compared with the WKY rat brain. This was attributable to greater levels of both AT1A- and AT1B-R mRNA subtypes in these areas from the SHR. These observations suggest that increased AII receptor levels in SHR brain may, in part, be a result of increased expression of the AT1-R gene.

Animals↗

PubMed1993

Molecular adaptation of vascular endothelial cells to oxidative stress.

Cellular organisms respond at the cellular and molecular level when confronted with sudden changes in environment, and molecular adaptation represents the ability of the cells to acclimate themselves to their new environment. In this study we examined the response of bovine vascular endothelial cells (VEC) to the oxidative stress by exposing the cultured cells to two different concentrations of H2O2, 0.04 or 0.08 mM, for 18-24 h. H2O2-exposed VEC displayed good viability (85-90% for 0.04 mM H2O2; 75-80% for 0.08 mM H2O2) and exhibited normal morphology. H2O2 treatment of the VEC was associated with the expression of a number of new proteins, as demonstrated by two-dimensional gel electrophoresis of total cell lysate. Cells exposed to 0.04 mM H2O2 expressed 25 new proteins, whereas 19 newly expressed proteins were detected when the cells were exposed to 0.08 mM H2O2. Western blot analysis of H2O2-treated VEC using specific antibodies to heat-shock proteins (HSP) identified one of these proteins as a member of the HSP 70 family. In addition, H2O2 induced an increase in antioxidative enzyme activities in the VEC, including superoxide dismutase, catalase, and glutathione peroxidase. Moreover, these changes were a truly adaptive phenomenon because challenging the VEC with brief exposure to toxic levels of H2O2 (1 mM for 30 min) showed increased viability (by Trypan blue exclusion test) and decreased injury (by lactate dehydrogenase supernatant-to-cellular ratio determination) in adapted cells (preexposed to 0.04 or 0.08 mM H2O2) compared with control cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Adaptation, Physiological↗

PubMed1993

Angiotensin II type 1 receptor-mediated stimulation of c-fos gene expression in astroglial cultures.

Angiotensin II (ANG II) stimulates plasminogen activator inhibitor 1 (PAI-1) gene expression in astroglial cells prepared from rat brains. In this study, we investigated whether c-fos gene expression may be involved in this cellular action of ANG II. Incubation of astroglial cultures with ANG II caused a time- and dose-dependent transient stimulation of the steady-state levels of c-fos mRNA, with a maximal stimulation of 50-fold observed with 100 nM ANG II within 30-45 min. This stimulation was completely abolished by the presence of the type 1 ANG II (AT1) receptor antagonist losartan but not by the type 2 ANG II receptor blocker PD-123177. Depolarization of brain cell cultures with 50 mM K+ also caused a 100-fold increase in c-fos mRNA levels, an effect partially blocked by losartan. These observations show that AT1 receptor activation stimulates expression of the c-fos gene, which may act as a third messenger in the regulation of cellular actions of ANG II, including PAI-1 gene expression in astroglial cells.

Angiotensin II↗

PubMed1993

Inhibition of central angiotensin responses by angiotensin type-1 receptor antibody.

Angiotensin type-1 receptor subtypes (AT1) are implicated in the physiological actions of angiotensin II in the brain. In the present study we used an AT1 receptor antibody and a polymerase chain reaction--synthesized AT1 receptor complementary DNA to show that the hypothalamus expresses significantly higher levels of AT1 receptor messenger RNA and protein compared with the brain stem. Intracerebroventricular injections of AT1-specific antibody blocks the dipsogenic and blood pressure responses induced by centrally injected angiotensin II. These results demonstrate the expression of AT1 receptor gene in the brain and that the AT1 receptor antibody is able to inhibit the physiological responses of angiotensin II mediated by the brain.

Angiotensin II↗

PubMed1993

Increased angiotensin II type-1 receptor gene expression in neuronal cultures from spontaneously hypertensive rats.

In this study we compared the expression of angiotensin II type 1 (AT1) receptor messenger RNA (mRNA) and AT1 receptors in neurons cultured from Wistar-Kyoto (WKY) and spontaneously hypertensive (SH) rat brains. Neuronal cultures from the hypothalamus and brain-stem of 1-day-old SH rats exhibited approximately 4-fold higher steady-state levels of AT1 receptor mRNA than the corresponding WKY cultures. This was attributable to greater levels of both AT1A and AT1B receptor mRNA subtypes in SH rat neuronal cultures compared with WKY rat neurons. SH rat neuronal cultures also exhibited increased numbers (approximately 2.3-fold) of binding sites for [3H]DuP753, an AT1 receptor selective ligand, and enhanced (approximately 3.4-fold) stimulation of inositol phospholipid hydrolysis by angiotensin II compared with WKY neurons. By contrast, cultured astroglia from SH and WKY rat brain exhibited no significant differences in either the levels of AT1 receptor mRNA or the specific binding of [3H]DuP753. These data suggest that in SH rat neurons, AT1 receptor transcription and translation is increased, compared with neurons from WKY rats.

Amino Acid Sequence↗

PubMed1993

Interleukin-1 alpha preconditioning reduces myocardial ischemia reperfusion injury.

BACKGROUND: Interleukin-1 (IL-1) has been shown to induce superoxide dismutase (SOD) activity and to express heat shock protein (HSP). Since the reperfusion of ischemic heart is associated with the reduction of antioxidative enzymes including SOD and expression of HSP, it was hypothesized that IL-1 could be beneficial against ischemic reperfusion injury. METHODS AND RESULTS: Rats were injected with recombinant IL-1 alpha (30 micrograms/kg IP); after 48 hours, they were anesthetized and hearts were removed, isolated, and perfused by the Langendorff technique. Myocardial functions were studied by measuring left ventricular developed pressure (LVDP) and its maximum first derivative (LV dP/dt), and cellular injury was studied by estimating creatine kinase (CK) release. Induction of the expression of HSP27 mRNA and HSP27 protein was examined by Western blot analysis and Northern blot analysis, respectively. Antioxidant enzymes were assayed by enzymatic analysis. Our results indicated reduction of ischemic reperfusion injury by IL-1 alpha, as evidenced by better recovery in postischemic ventricular functions (LVDP [mm Hg]: control, 63 +/- 14; IL-1, 102 +/- 11; P < .05), increased coronary flow (mL/min) (control, 2.93 +/- 0.58; IL-1, 5.17 +/- 0.43; P < .03), and reduced creatine kinase release (IU/L) (control, 110 +/- 5.78; IL-1, 81.76 +/- 7.71; P < .01). IL-1 alpha induced the expression of HSP27 mRNA within 2 hours as examined by Northern blot analysis and the expression of HSP27 after 48 hours. In addition, hearts pretreated with IL-1 alpha for 48 hours and then subjected to 30-minute ischemia and 60-minute reperfusion enhanced the activities (nmol/min/mg protein) of Cu/Zn SOD (control, 1.55 +/- 0.22; IL-1 alpha, 2.92 +/- 0.04; P < .004), Mn-SOD (control, 4.54 +/- 0.19; IL-1 alpha, 6.33 +/- 0.09, P < .001), catalase (control, 15.53 +/- 0.37; IL-1 alpha, 21.67 +/- 0.72; P < .002), glutathione peroxidase (control, 17.49 +/- 0.35; IL-1 alpha, 25.87 +/- 0.58; P < .001), and glucose-6-phosphate dehydrogenase (control, 22.71 +/- 0.44; IL-1 alpha, 29.53 +/- 0.48; P < .001). CONCLUSIONS: The results of this study indicate that low doses of IL-1 alpha can be used as a therapeutic agent to precondition a heart from ischemia reperfusion injury.

Animals↗

PubMed1993

[Production of transgenic rabbits by micro injection].

The feasibility of using whole animal instead of bioreactor in genetic engineering has been investigated with transgenic domestic rabbits. The gene chosen is the surface gene (S gene) of hepatitis B virus. Two plasmids were specifically constructed for this purpose, pHBV3.0 contains the promoter pre S gene and a part of c gene of the virus; while MT-SA, the S gene and mouse MT promotor. These plasmids were made linear by suitable restriction endonuclease before they were transferred into maleprounclei by means of microinjection. From 757 microinjected and transplanted fertilized eggs 101 rabbits were obtained. 57% of these animals were found with integrated microinjected genes. 28 of the transgenic animals were tested for the presence of the surface antigen of the virus in the serum by ELISA method. 8 animals were found positive, approximately 30% of the tested transgenic animals. The second generation transgenics were obtained either by first generation ransgenics crossed with non-trans-genics or transgenics. Among them 73% contained the transgene and 15% had the surface antigen in the serum. Some experiments were also carried out with human growth hormone gene.

Animals↗

PubMed1993

Inhibition of phosphorylation of p160 BCR within p210 BCR-ABL complexes during early stages of phorbol ester-induced differentiation of K562 cells.

The kinase activity of the BCR-ABL gene product is known to be down-regulated in K562 cells treated with low concentrations of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). The reduction of BCR-ABL kinase activity is followed by the loss of cell proliferation and progression to a more differentiated state. We have previously demonstrated that K562 cells possess protein complexes that contain p210 BCR-ABL and p160 BCR (M. L. Campbell, W. J. Li, and R. B. Arlinghaus, Oncogene, 5: 773-776, 1990). We performed experiments to determine whether BCR-ABL/BCR complexes were disrupted prior to alterations in cell growth and differentiation effects in TPA-treated K562 cells. Our results indicate that BCR-ABL/BCR complexes disappeared at precisely the same time after TPA treatment as the loss of autophosphorylation activity exhibited by total p210 BCR-ABL, which occurred 16-19 h after TPA treatment. The loss of kinase activity preceded the loss of p210 BCR by more than 24 h. A degraded form of p210 BCR-ABL (about 175 kilodaltons) accounted for the residual autophosphorylation activity seen during the later phases of kinase inactivation following TPA treatment, and this form was preferentially sequestered within BCR-ABL/BCR complexes. This altered BCR-ABL protein, although able to autophosphorylate, had reduced ability to phosphorylate p160 BCR. We conclude that 15 nM TPA treatment of K562 cells initiates effects that simultaneously interfere with the phosphorylation of p160 BCR in BCR-ABL complexes and inactivates the autophosphorylation activity of the full length BCR-ABL protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Differentiation↗

PubMed1993

BCR-ABL tyrosine kinase is autophosphorylated or transphosphorylates P160 BCR on tyrosine predominantly within the first BCR exon.

The role of BCR gene sequences in Philadelphia (Ph) chromosome-positive leukemia is not well understood. Our previous studies demonstrated that P210 BCR-ABL co-precipitates with P160 BCR following immunoprecipitation with antibodies to the C-terminal domain of P160 BCR, sequences lacking in P210 BCR-ABL. We now report that tryptic peptides shared by both P160 BCR and P210 BCR-ABL are phosphorylated on tyrosine in vitro either when using immune complexes containing P160 BCR complexed to BCR-ABL or when P160 BCR is phosphorylated in trans by P210 BCR-ABL immune complexes from cells lacking functional P160 BCR. P185 BCR-ABL produced in a cell line derived from a Ph chromosome-positive acute lymphocytic leukemia patient also co-immunoprecipitated with P160 BCR. As with P210 BCR-ABL, P160 BCR tyrosine phosphopeptides were shared with P185 BCR-ABL, indicating that the major sites of tyrosine phosphorylation in vitro are contained within the first exon of P160 BCR. Similarly, BCR-ABL autophosphorylation was found to occur predominantly at tyrosines within BCR exon 1 sequences. These results raise the possibility that the activated ABL protein kinase of BCR-ABL proteins modulates the putative signal transduction activities of P160 BCR by tyrosine phosphorylation of exon 1 sequences.

Animals↗

PubMed1992

A polymerase chain reaction assay for the detection and quantitation of cytokine gene expression in small numbers of cells.

A reverse transcriptase-polymerase chain reaction (RT-PCR) assay is described that allows the rapid detection and quantitation of mRNA encoding the cytokines interleukin-2 (IL-2), IL-4 and interferon-gamma (IFN-gamma). Analysis of cytokine production by defined CD4+ T cell clones and the thymoma cell line EL4, demonstrates that the oligonucleotide primers used in this assay are specific for the genes encoding the individual cytokines, generating PCR products of different sizes. This allows the simultaneous and unambiguous detection of all three cytokine mRNAs in the same cDNA sample. The assay is sensitive enough to reproducibly detect cytokine mRNA expressed in as few as ten cells and requires 10,000-fold less cells for the detection of IL-2 production than that required for its detection using a conventional bioassay. Reverse transcribed mRNA is quantitated in the PCR assay by amplifying in the presence of a known amount of competitive genomic DNA (gDNA) template containing a small intron using the same primers. The PCR products obtained form the target cDNA and gDNA templates, which are distinguished by size, are processed by Southern analysis and quantitated by scanning densitometry of autoradiographs. As little as two-fold differences in cytokine mRNA can be reliably detected using this assay. We have demonstrated the successful application of this assay to the quantitation of pg amounts of IL-2 mRNA that is constitutively produced at low levels by fetal thymocytes in vivo during T cell ontogeny. The sensitivity, specificity, reliability and speed of this assay will facilitate the analysis of cytokine production in in vivo-derived or, in vitro propagated cells which are not available in sufficient numbers for analysis using more conventional molecular and biochemical assays.

Animals↗

PubMed1992

Monoclonal antibodies specific for mercuric ions.

Monoclonal antibodies (mAbs) that react with soluble mercuric ions have been produced by injection of BALB/c mice with a hapten-carrier complex designed to maximize exposure of the metal to the immune system. Three hybridomas producing antibodies that reacted with bovine serum albumin (BSA)-glutathione-HgCl, but not with BSA-glutathione, were isolated from the spleen of a mouse given multiple injections with glutathione-HgCl conjugated to keyhole limpet hemocyanin. Stable subclones were established from two of these antibodies, designated mAb 4A10 and mAb 1F10. The binding of both antibodies to immobilized BSA-glutathione-HgCl was inhibited by soluble HgCl2, and dissociation constants for mercuric chloride binding were 2.3 and 3.7 nM for mAbs 4A10 and 1F10, respectively. Both antibodies bound mercuric acetate with similar affinities, demonstrating that the antibodies were capable of binding to mercuric ions in the presence of a different counterion than the one used in the immunogen. Reactions were not observed with other metal cations by either antibody. These data demonstrate the successful induction of antibodies that react very specifically with mercuric ions in solution regardless of the presence of a carrier.

Amino Acid Sequence↗

PubMed1992

Glycosphingolipid phase behaviour in unsaturated phosphatidylcholine bilayers: a 2H-NMR study.

2H-NMR was employed to consider the arrangement of a glycosphingolipid, N-(lignoceroyl-d47)galactosylceramide, in bilayers of the mono-unsaturated phospholipid, 1-stearoyl-2-oleoylphosphatidylcholine. The deuterated glycolipid prepared by partial synthesis was incorporated at concentrations ranging from 5 mol% to 53 mol% into unsonicated liposomes, and its spectra were recorded from +76 degrees C to -10 degrees C. First spectral moments were plotted as a function of temperature for each sample composition and, along with inspection of the spectra, were employed to infer a phase diagram describing glycolipid behaviour in the unsaturated phospholipid host matrix. It was possible to refine the result using 2H-NMR difference spectroscopy. The phase diagram obtained was indicative of peritectic behaviour. At glycolipid concentrations exceeding about 20 mol% there was considerable tendency to glycolipid phase separation--as indicated by coexistence of fluid phospholipid-enriched and gel phase glycolipid-enriched domains over a wide range of temperatures, and by coexistence of distinct ordered phase domains at lower temperature. In contrast, at lower glycolipid concentrations reflective of many biological membranes, the lipid components were miscible in both the liquid crystal and gel phases, with only a narrow temperature range of fluid and ordered phase coexistence. For the fluid phase at low glycolipid concentrations, spectra of the deuterated glycolipid 24-carbon fatty acid suggest that orientational order is low for a number of methylene groups near the methyl end of the chain.

Deuterium↗

PubMed1992

Cloning, expression and localization of an RNA helicase gene from a human lymphoid cell line with chromosomal breakpoint 11q23.3.

A gene encoding a putative human RNA helicase, p54, has been cloned and mapped to the band q23.3 of chromosome 11. The predicted amino acid sequence shares a striking homology (75% identical) with the female germline-specific RNA helicase ME31B gene of Drosophila. Unlike ME31B, however, the new gene expresses an abundant transcript in a large number of adult tissues and its 5' non-coding region was found split in a t(11;14)(q23.3;q32.3) cell line from a diffuse large B-cell lymphoma.

Amino Acid Sequence↗

PubMed1992

Characterization of a polyclonal anti-peptide antibody to the angiotensin II type-1 (AT1) receptor.

A polyclonal antibody has been prepared against a synthetic peptide corresponding to amino acids 14-23 of the angiotensin II type-1 (AT1) receptor. The antibody is of high titer and mono-specific. Western blot analysis of membranes from rat liver, kidney, and adrenal gland showed that the antibody specifically recognizes a protein band of MW 70,000 whose amounts are highest in the liver, followed by kidney and adrenals. In addition, a relatively less prominent band of MW 95,000 was also detected. The relative distribution of this protein correlates well with the values obtained for [3H]-DuP753 binding and AT1 receptor mRNA.

Adrenal Glands↗

PubMed1992

Differential effect of interleukin 1 on naive and memory CD4+ T cells.

Freshly derived murine CD4+ T cells are divided into naive and memory cells based on the expression of CD45 isoforms. Cross-linking the T cell receptor CD3 complex either by plastic-bound anti-CD3 antibodies or the antibody presented on non-lymphoid Fc gamma receptor type II-positive Chinese hamster ovary cells in absence of competent antigen-presenting cells fails to activate naive cells to either secrete cytokines or to proliferate. In contrast, memory cells secrete their characteristic cytokines [interleukin (IL) 2, IL4, and interferon-gamma] and show significant proliferation to this stimulus. IL 1 however, is required for their optimal clonal expansion. Differential expression of IL 1 receptor mRNA in memory cells also correlate with their responsiveness to IL 1. Thus, these data reveal a basic difference in the requirements for activation of naive and memory CD4+ T cells.

Animals↗

PubMed1992

Specific chromosomal translocations and therapy-related leukemia induced by bimolane therapy for psoriasis.

This paper reports for the first time results of cytogenetic studies on 14 consecutive secondary acute non-lymphocytic leukemia (S-ANLL) induced by bimolane therapy. They included 10 males and 4 females with ages ranging from 17 to 54 years. They had all suffered from psoriasis and received bimolane treatment before the occurrence of their leukemia. The total dose of bimolane ranged from 40 to 400 g (mean dose 194 g). The interval between the initiation of bimolane therapy and the diagnosis of leukemia was 12-96 months (median 30 months). A preleukemic phase was only found in one case. No dysplastic features in the hemopoietic series were seen in any patient. Chromosome analysis of bone marrow cells using banding techniques revealed clonal karyotypic abnormalities in all cases: t(15;17) in 8 cases of M3, of which 75% had extra abnormalities, t(8;21) in 4 cases of M2, del(7q) only in one case of M4 and one case of M5. After antileukemic therapy, complete remission was obtained in 10 out of 12 cases with specific translocations and one out of 2 cases with 7q-anomaly, respectively. The former survived 4-58 months (median 12 months), while the latter 1 and 9 months, respectively. This study indicates that: (1) bimolane is a causative factor of leukemia in this series; (2) the leukemia in our series is therapy-related leukemia (TRL) rather than de novo ANLL; (3) there exists, in fact, a new subgroup of TRL characterized by specific rearrangements, whose clinical, hematological and prognostic features and pathogenetic mechanism may be different from classical TRL characterized by chromosome abnormalities involving absence or deletion of parts of chromosome 5 and/or 7.

Adolescent↗