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Biomedical subjects

L Liu

Publications and source records attributed to L Liu.

At least 973 records · Page 54Linked to original sources
PubMed1995

Heat shock-sensitive expression of calreticulin. In vitro and in vivo up-regulation.

Calreticulin (CRT) is an ubiquitous, highly conserved, Ca(2+)-binding protein of the sarcoplasmic and endoplasmic reticulum. The precise function(s) of CRT is unknown. However, based on sequence analyses and observations that it may bind to steroid receptors and integrins and store Ca2+ within the cell, it has been postulated to play a "housekeeping" role. To determine whether the level of expression of CRT is affected by stress, we examined the heat shock response of CRT from a variety of cultured cells, including vascular endothelial, lung epithelial, and lung fibroblasts. Following exposure of the cells to 42 degrees C, CRT mRNA transiently accumulated 2.5-4.2-fold at 1-6 h. Nuclear run-on studies and mRNA stability experiments confirmed that the predominant mechanism of augmentation was transcriptional. Chloramphenicol acetyltransferase assays further indicated that the promoter region, containing a putative heat shock element between -172 and -158 of the human CRT gene, is heat shock-sensitive. Finally, we demonstrated the in vivo significance of these findings by exposing rats to hyperthermia. This resulted in accumulation of CRT mRNA and an augmentation of CRT protein in lung tissue. We hypothesize that this stress-induced up-regulation of CRT contributes to the mechanism(s) by which the vascular endothelium and lung tissue, and possibly other organ systems, maintain homeostasis when exposed to a variety of pathophysiological conditions.

Animals↗
PubMed1995

Protective effect of seminal plasma proteins on the degradation of ascorbic acid.

The objectives of this study were to determine ascorbic acid stability and its effect on antiproteinase activity of seminal plasma in the presence of an oxidant. Effect of seminal plasma, and additives: glutathione, albumin, hydrogen peroxide and Tris buffer, on ascorbic acid degradation was investigated by UV absorbance. Antiproteinase against trypsin amidase activity was measured spectrophotometrically using N-benzoyl-DL-arginine-p-nitroanilide (BAPNA) as substrate. Ascorbic acid was destroyed much more rapidly with the addition of hydrogen peroxide than in Tris buffer at pH 8.2 alone. Seminal plasma protected ascorbic acid more efficiently than glutathione and albumin alone. The protective effect of seminal plasma on ascorbic acid degradation may closely relate to the function of ascorbic acid in reproductive system of scurvy-prone animals including teleost fish. Within the range of 1-8 mM concentrations, ascorbic acid had a pro-oxidant action on seminal plasma antiproteinase activity in vitro when they were incubated with hydrogen peroxide.

Animals↗
PubMed1995

Insulin potentiates N-methyl-D-aspartate receptor activity in Xenopus oocytes and rat hippocampus.

Growth factor signal transduction pathways have recently been shown to affect voltage-gated ion channel activity. In this study we report that insulin can modulate the activity of a ligand-gated ion channel, the N-methyl-D-aspartate (NMDA) receptor. In Xenopus oocytes, brief insulin exposure rapidly potentiated NR1a/NR2A and NR1a/NR2B receptor responses 2-3 fold and weakly potentiated NR1a/NR2C and NR1a/NR2D mediated-responses. Insulin potentiation of NR1a/NR2A receptor responses was significantly blocked by staurosporine, suggesting kinase involvement in insulin action. Insulin modulation of native NMDA receptors is suggested by the observation that insulin potentiated the NMDA receptor-mediated synaptic component in hippocampal slices. Regulation of NMDA receptor activity by growth factors may account for previous observations of growth factor modulation of central nervous system excitotoxicity.

Animals↗
PubMed1995

Retroviral transduction and expression of the human alkyltransferase cDNA provides nitrosourea resistance to hematopoietic cells.

Myelosuppression is the dose-limiting toxicity for nitrosourea chemotherapy. This toxicity predominantly involves modification of the O6 position of guanine with an alkyl moiety. The enzyme responsible for repair of O6-alkylguanine adducts, O6-alkylguanine-DNA alkyltransferase (alkyltransferase), is expressed at low levels in bone marrow (BM) cells. High alkyltransferase expression prevents the cytotoxicity and carcinogenicity of nitrosoureas in several transgenic and in vitro gene transfer models. We used gene transfer using a novel myeloproliferative sarcoma virus (MPSV) based retrovirus (vM5MGMT) to express the human alkyltransferase cDNA (MGMT) in human and murine hematopoietic cells. Transduced K562 cells had very high levels of alkyltransferase expression and significantly increased resistance to 1,3-bis (2-chloroethyl) nitrosourea (BCNU) as compared with untransduced K562 cells. Primary murine BM progenitors showed a high transduction efficiency with vM5MGMT and have increased BCNU resistance in vitro. After BM transplantation with vM5MGMT-transduced BM cells and BCNU treatment of these mice, BM, spleen and thymus had a 10- to 40-fold increase in alkyltransferase expression that persisted for at least 23 weeks posttransplantation. Progenitor cells procured from mice expressing high levels of alkyltransferase also had increased resistance to BCNU. Thus, an MPSV-based retroviral vector transduces mouse and human hematopoietic cells at high efficiency and results in high levels of gene expression both in vitro and in vivo. Overexpression of the alkyltransferase protein may protect hematopoietic progenitors from nitrosourea-induced myelosuppression.

Animals↗
PubMed1995

Determination of extracellular glutathione in livers of anaesthetized rats by microdialysis with on-line high-performance liquid chromatography.

An on-line analytical system for the continuous in vivo monitoring of extracellular glutathione (gamma-glutamyl-cysteinylglycine) (GSH) concentrations in the livers of anaesthetized rats was developed. Microdialysates perfused through implanted microdialysis probes were collected with a loading loop of an on-line injector for direct and automatic injection into a high-performance liquid chromatographic system equipped with an electrochemical detector with Au-Hg electrodes. This method shortened the analysis time and circumvented the sample preparation process which is essential for accurate determination of GSH levels in biological samples. Additionally, this method provided continuous and real-time monitoring of extracellular GSH levels. Basal extracellular GSH concentrations in the livers of anaesthetized rate were found to vary over a wide range (from 4.16 to 76.5 microM). The method was applied to study the effect of global liver ischaemia on extracellular GSH concentrations and it was found that extracellular GSH levels in livers increased immediately with the onset of ischaemia and remained elevated for the 30-min ischaemic period. Ensuing reperfusion did reduce the GSH increase; however, the GSH levels did not return to the basal value.

Anesthesia↗
PubMed1995

Murine 32D c13 cells--a transfectable model of phagocyte granule formation.

Granulocytes expose phagocytized microbes to microbicidal substances that are stored in cytoplasmic granules and delivered by fusion to the phagocytic vacuoles (phagosomes). To determine if the murine myeloid cell line 32D c13 is suitable as a genetic model of protein translocation to granules and phagosomes, we permanently transduced 32D c13 cells with human HNP-1 defensin cDNA, incubated them with opsonized zymosan, and immunostained them for human defensin HNP-1. Although their phagocytic rate was much slower than that of neutrophil granulocytes, 32D c13 cells ingested zymosan into vacuoles that accumulated most of the transgenic defensin. The 32D c13 cell line should be useful for studies of the targeting of proteins to granules and phagosomes.

Animals↗
PubMed1995

Altered subcellular location of an activated and tumour-associated epidermal growth factor receptor.

The epidermal growth factor (EGF) receptor is a membrane bound tyrosine kinase whose activity is initiated by ligand binding. The malignant brain tumour glioblastoma frequently shows amplification and rearrangements of the EGF receptor gene that are associated with the synthesis of a constitutively activated tyrosine kinase, lacking amino acids 6-273 near the protein's N-terminus. When expressed in Chinese hamster ovary (CHO) cells, this mutant receptor (p140EGFR) displays ligand-independent tyrosine kinase activity, stimulates DNA synthesis, and promotes cell proliferation. Here, we investigate the subcellular location of p140EGFR in CHO cell transfectants as well as in human glioblastoma tumours. p140EGFR had an intracellular location that contrasted sharply with the plasma membrane location of the wild-type EGF receptor. Endoglycosidase H sensitivity analysis and the pattern of p140EGFR immunoreactivity suggested that the aberrant tyrosine kinase resided primarily in the endoplasmic reticulum. The half-life of p140EGFR in the endoplasmic reticulum was extended several-fold over that of the ligand-activated wild-type receptor. The altered subcellular location of p140EGFR in combination with its prolonged half-life suggest that this activated tyrosine kinase may escape the regulatory mechanisms utilized for the attenuation of wild-type receptor signaling. Therefore, the previously reported growth stimulatory property of the ligand-independent p140EGFR may be attributed to a sustained tyrosine kinase activity resulting from an altered subcellular location.

Animals↗
PubMed1995

The pro region of human neutrophil defensin contains a motif that is essential for normal subcellular sorting.

Human defensins (human neutrophil peptides) HNP 1-3 are 29-30 amino acid antibiotic and cytotoxic peptides highly abundant in the cytoplasmic granules of polymorphonuclear leukocytes. The peptides are produced from 94 amino acid (aa) prepropeptides by proteolytic cleavage of the signal sequence and stepwise removal of the 44-45 aa anionic propiece. To study the role of the propiece, we constructed five in-frame deletions in preproHNP-1 cDNA between the signal peptidase site and the amino-terminus of the mature defensin region (aa 21-64). The wild type HNP-1 cDNA and the deletion mutants were ligated into the pBabe-Neo retroviral vector, expressed in GP+E86 packaging derivative of NIH 3T3 cells, then transduced into the 32D cl3 granulocytic cell line. For each construction and both cell lines, we measured the accumulation of the various defensin forms in cells and media by 24-hour labeling or pulse-chase with 35S-cysteine- and immunoprecipitation/sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Deletions in the amino-terminal two-fifths of the propiece, delta 21-28 and delta 21-38, had only minor effects on defensin biosynthesis in both cell lines and did not interfere with the accumulation of mature defensin in the granules of 32D cl3 cells. Deletions in the carboxyterminal three-fifths of the propiece (delta 21-51 and delta 21-64) diminished net defensin synthesis, blocked constitutive secretion of prodefensin in both cell lines, and interfered with defensin accumulation in cytoplasmic granules of 32D cl3 cells. These effects were reproduced by the smaller deletion delta 40-51, which contains highly conserved secondary structure. The propiece segment 40-51 appears to be essential for the subcellular trafficking and sorting of HNP-1 defensin.

3T3 Cells↗
PubMed1995

Stilbene disulfonic acids inhibit synexin-mediated membrane aggregation and fusion.

Stilbene disulfonic acids inhibit surfactant secretion from lung epithelial type II cells by an undefined mechanism, and inhibit CD4 mediated cell-cell fusion. We have previously shown that lung synexin promotes in vitro fusion of lamellar bodies and plasma membranes, an obligatory process for surfactant secretion. This study investigates the effect of stilbene disulfonic acids, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS), and 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid (AMDS), on synexin-mediated liposome aggregation and fusion. Structurally, these three stilbene compounds differ in the number of isothiocyano groups present (DIDS = 2, SITS = 1, and AMDS = 0). At 10 micrograms synexin/ml, DIDS and SITS inhibited synexin-mediated liposome aggregation with an EC50 of 3.5 microM and 148 microM, respectively. In comparison, AMDS was least inhibitory (EC50 > 1 mM). Thus, the inhibitory potency (DIDS > SITS > AMDS) was partly dependent upon the number of isothiocyano groups. The EC50 was also dependent on synexin concentration. Stilbene disulfonic acids were also inhibitory for arachidonic acid-enhanced synexin-mediated liposome fusion. The EC50 for DIDS and SITS for fusion were similar to that for liposome aggregation. Ca(2+)-induced synexin polymerization, measured by 90 degrees light scattering, was increased by DIDS, suggesting binding of stilbene disulfonic acids to synexin. The binding of DIDS to synexin was dependent on the molar ratio of synexin to DIDS. These results indicate that stilbene disulfonic acids interact directly with synexin to inhibit membrane aggregation and fusion. Our results suggest that such inhibition of synexin activity may contribute towards inhibition of surfactant secretion by DIDS, and support a physiological role for synexin in lung surfactant secretion.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1995

Transcriptional upregulation of TGF-alpha by phenylacetate and phenylbutyrate is associated with differentiation of human melanoma cells.

The aromatic fatty acids phenylacetate (PA) and phenylbutyrate (PB) induce tumour cell differentiation in experimental models and both are currently in clinical trials. The purpose of this study was to determine the effect of these antitumour agents on the expression of transforming growth factor-alpha (TGF-alpha) in neoplastic cells. Treatment of human melanoma 1011 cultures with either PA or PB caused over 40-fold increase in TGF-alpha biosynthesis and secretion into the media. Whereas elevation in TGF-alpha mRNA steady-state levels became evident within 6-12 h and reached peak quantities the following day, the amounts of its coded protein increased gradually over a period of 5 days of treatment. Further molecular analysis revealed that regulation of TGF-alpha expression occurred at the transcriptional level. In contrast to TGF-alpha, expression of its receptor remained below detectable levels, indicating that an autocrine loop involving this growth factor is unlikely. Interestingly, the increase in TGF-alpha production paralleled drug-induced cytostasis and differentiation defined by morphological changes and increased melanogenesis. Like PA and PB, other differentiation inducers such as all-trans-retinoic acid, dimethyl sulfoxide, and 5-aza-2'-deoxycytidine, all induced TGF-alpha expression in the melanoma cells. The close association between enhanced TGF-alpha production and melanoma cell differentiation suggests that this growth factor, often linked to mitogenesis, may play a novel role in tumour differentiation by PA and PB.

Antineoplastic Agents↗
PubMed1995

Potential molecular targets mediating chemical teratogenesis: in vitro peroxidase-catalyzed phenytoin metabolism and oxidative damage to proteins and lipids in murine maternal hepatic microsomes and embryonic 9000g supernatant.

Phenytoin and related proteratogens may be bioactivated by peroxidases to a reactive free radical intermediate that initiates teratogenesis. This study evaluated the potential molecular targets mediating phenytoin teratogenicity. In vitro phenytoin-induced oxidative tissue damage following metabolic activation by prostaglandin H synthase (PHS) and lipoxygenases (LPOs) was quantified in both maternal hepatic microsomes and embryonic 9000g supernatant (S-9) from CD-1 mice, using lipid peroxidation and protein oxidation and degradation as indices. Phenytoin (0-0.2 mM) and the PHS/LPO cofactor arachidonic acid (AA, 0-1.0 mM) were incubated with maternal hepatic microsomes or embryonic S-9 (2 mg) for 0-60 min. Lipid peroxidation was measured by the formation of thiobarbituric acid-reactive substance, protein oxidation quantified by the formation of carbonyl groups in proteins, and protein degradation measured by the release of primary amines. Phenytoin was cooxidized by peroxidases in both maternal hepatic microsomes and embryonic S-9 to initiate oxidative damage to tissue lipids and proteins. The lipid peroxidation and protein oxidation and degradation were dependent on incubation time, AA concentration, and phenytoin concentration (p < 0.05), indicating that peroxidases were involved in phenytoin metabolic activation. Phenytoin-initiated oxidative tissue damage in maternal hepatic microsomes was inhibited by the cyclooxygenase inhibitor indomethacin, confirming that PHS was involved in the reactions. Phenytoin-induced oxidative damage in embryonic S-9 was not reduced by indomethacin, but was by nordihydroguaiaretic acid, an antioxidant and LPO inhibitor, indicating that additional enzymes containing peroxidase activity, such as LPO, may play a substantial role in phenytoin metabolic activation in embryonic tissues. Phenytoin-initiated lipid peroxidation and protein oxidation were early events, preceding protein degradation (10 min versus 60 min), and a lower phenytoin concentration was needed for lipid peroxidation (0.04 mM) and protein oxidation (0.01 mM) than for protein degradation (0.08 mM), suggesting that phenytoin-initiated protein and lipid oxidation may be two potential causes for protein cleavage. These results provide an in vitro basis for the in vivo evidence that embryonic lipid and protein may constitute important molecular targets mediating phenytoin teratogenicity, wherein peroxidase-catalyzed metabolic activation initiates early macromolecular oxidation by reactive oxygen species.

Animals↗
PubMed1995

Chromosome 17 abnormalities and lack of TP53 mutations in paediatric central nervous system tumours.

Central nervous system (CNS) tumours are the most common solid tumours in children. Cytogenetic and molecular genetic studies of these neoplasms have previously shown abnormalities of chromosome 17, implicating genes on this autosome in tumorigenesis. To identify mutations in the TP53 tumour suppressor gene (17p13.1), we have sequenced the five highly conserved regions of this gene in 29 mixed paediatric CNS tumors. No mutations were detected by this analysis. In order to identify other candidate disease loci on chromosome 17, we have carried out a detailed deletion mapping analysis using 16 polymorphic DNA markers on 19 of the above tumours and an additional four cases. Abnormalities of chromosome 17 occurred in nine cases (39%), six of which were primitive neuroectodermal tumour (PNET)-medulloblastomas. These findings suggest that it is unlikely that the TP53 gene is directly involved in the development of common paediatric brain tumours. This is in contrast to findings from adult brain and other tumour types. Moreover, the frequency of chromosome 17 aberrations, especially in PNET-medulloblastomas, suggests that other genes on this chromosome contribute to tumourigenesis.

Adolescent↗
PubMed1995

The bovine rod outer segment guanylate cyclase, ROS-GC, is present in both outer segment and synaptic layers of the retina.

Cyclic-GMP, which plays a pivotal role in visual transduction in the vertebrate retina, is synthesized by guanylate cyclase. The purpose of this study was to localize a rod outer segment-derived particulate guanylate cyclase (ROS-GC) to the retina of several species that have different populations of rods and cones. A rabbit antibody was raised against a synthetic peptide, corresponding to the sequence A107-L125 of bovine ROS-GC. Western blot analysis showed a single immunoreactive band at about 115 kDa with bovine rod outer segments but not with human rod outer segments. Light microscopic immunocytochemistry of tissue sections revealed immunoreactivity in the outer segment layer and in the outer and inner plexiform layers. The rod-rich rat retina showed uniform immunolabeling of outer segments; the cone-containing cat retina showed heavily labeled cone outer segments and lighter labeling of rod outer segments; the cone-rich chicken retina showed a uniformly and intensely labeled outer segment layer. Preincubation of the primary antibody with the peptide completely blocked antibody binding. Electron microscopic immunocytochemistry of the cat retina confirmed the presence of guanylate cyclase in photoreceptor outer segments and demonstrated its association with disk and plasma membranes. These data support a concept in which guanylate cyclase is much more concentrated in the outer segments of cones than rods. The immunolabeling of the plexiform layers suggests that the particulate guanylate cyclase is not unique to the photoreceptor outer segments, and may also play a role in transduction processes of retinal synapses.

Amino Acid Sequence↗
PubMed1995

Evidence for activation of the terminal pathway of complement and upregulation of sulfated glycoprotein (SGP)-2 in the hypoglossal nucleus following peripheral nerve injury.

In a previous study, we found immunoreactivity for complement factors C3, C3d, and C4d, as well as endogenous IgG in the hypoglossal nucleus following hypoglossal nerve transection, suggesting that activation of the complement cascade had taken place in the vicinity of the axotomized motorneurons. In the present study, we found increased immunoreactivity for complement factor C1 and C1q in reactive microglia, indicating an increased potential for initiation of the classical pathway by binding of IgG to C1q. Furthermore, we found immunoreactivity for C9, which contributes to the formation of C5b-9, the final lytic product of the complement cascade close to the axotomized neurons and perineuronal glia. In addition, immunoreactivity and mRNA labeling of sulfated glycoprotein (SGP-2), a putative complement inhibitor, was increased in a subpopulation of the axotomized motorneurons. SGP-2 immunoreactivity was also increased in astroglial cells ipsilateral to the nerve injury. The results lend further support to the hypothesis that the complement cascade is activated in the vicinity of axotomized neurons, which in turn may be protected by complement inhibitors. The balance between activation of complement and complement inhibitors might have an impact on the degenerative components of the axon reaction and, in particular, the events leading to nerve cell death.

Animals↗
PubMed1995

Phase-shifting human circadian rhythms with exercise during the night shift.

Appropriately timed exercise can phase shift the circadian rhythms of rodents. The purpose of this study was to determine whether exercise during the night shift could phase delay the temperature rhythm of humans to align with a daytime sleep schedule. Exercise subjects (N = 8) rode a stationary cycle ergometer for 15 min every h during the first 3 of 8 consecutive night shifts, whereas control subjects (N = 8) remained sedentary. All subjects wore dark welder's goggles when outside after the night shift until bedtime, and then slept in dark bedrooms. Sleep was delayed 9 h from baseline. Rectal temperature was continuously measured. There were fewer evening-types and more morning-types in the exercise group than in the control group, which should have made phase delay shifts more difficult for the exercise group. Nevertheless, a majority of the exercise subjects (63%) had large temperature rhythm phase delay shifts ( > 6 h in the last 4 days relative to baseline), whereas only 38% of the control subjects had large shifts. An ANCOVA showed that, when morningness-eveningness was accounted for (as the covariate), the exercise group had a significantly larger temperature rhythm phase shift than the control group. As expected, there was a correlation between the temperature rhythm phase shift and morningness-eveningness in the control group, with greater eveningness resulting in larger phase shifts. However, there was no such relationship in the exercise group; exercise facilitated temperature rhythm phase shifts regardless of circadian type. These results suggest that exercise might be used to promote circadian adaptation to night shift work.

Adult↗