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W Liu

Publications and source records attributed to W Liu.

At least 811 records · Page 45Linked to original sources
PubMed1996

Calcium channel blockade and anti-free-radical actions of panaxatriol saponins in cultured myocardiocytes.

AIM: To identify the calcium channel blockade and anti-free-radical actions of panaxatriol saponins Re, Rf, Rg1, Rg2, Rh1, and oleanolic acid saponin Ro. METHODS: On ventricular myocytes of Wistar rats, single channel activities of B, L, and T type calcium channels were recorded with the cell-attached configuration of patch-clamp technic, and free radical contents were measured with electron spin resenance method. RESULTS: Re, Rg1, Rg2, and Rh1 shortened the open times, prolonged the close times, and reduced the open-state probabilities of B, L, and T type calcium channels; Rf shortened the open time, prolonged the close time and reduced the open-state probability of L type calcium channel; Ro did not influence the activity of calcium channels (60 mumol.L-1). Re, Rg1, Rg2, and Rh1 antagonized the increase of free radical content induced by xanthine 0.42 mmol.L-1-xanthine oxidase 5.3 nmol.L-1; Ro and Rf had no effect (30 mumol.L-1). CONCLUSION: Re, Rg1, Rg2, and Rh1 had both the calcium channel blockade and antifree-radical actions. Rf had blockade action on L type calcium channel.

Animals↗
PubMed1995

Steroid receptor heterodimerization demonstrated in vitro and in vivo.

The mineralocorticoid and glucocorticoid receptors (MR and GR, respectively) are members of the intracellular receptor superfamily that bind as homodimers to the same hormone response elements (HREs). Physiological evidence suggests that MR and GR interact with each other in cells that express both receptors, implying that they might directly interact in the regulation of transcription initiation. Indeed, we have found that coexpressed MR and GR interact functionally at the transcriptional level and furthermore that they interact physically through heterodimer formation at a shared HRE in vitro and in vivo. We suggest from these findings that heterodimerization may play an important role in steroid receptor transcriptional regulation.

Alcohol Dehydrogenase↗
PubMed1995

Proton/peptide cotransporter (PEPT 2) from human kidney: functional characterization and chromosomal localization.

We report here on the functional characterization of the H+/peptide cotransporter PEPT 2 cloned from human kidney and on the chromosomal localization of the PEPT 2 gene. PEPT 2, when functionally expressed in HeLa cells, induces the transport of the neutral dipeptide glycylsarcosine. The induced transport activity is markedly influenced by extracellular pH. The optimum pH for the transport process is 6.0-7.0. Kinetic analysis has revealed that PEPT 2 is a high-affinity transporter, the Michaelis-Menten constant for glycylsarcosine being 74 +/- 14 microM. The human intestinal H+/peptide cotransporter PEPT 1 has 4-fold less affinity for the dipeptide under identical experimental conditions. Studies with other chemically diverse dipeptides have established that PEPT 2 possesses higher affinity than PEPT 1 not only for neutral peptides but also for peptides consisting of anionic and/or cationic amino acids. Somatic cell hybrid analysis and in situ hybridization have shown that the gene encoding PEPT 2 maps to human chromosome 3q13.3-q21.

Carrier Proteins↗
PubMed1995

Induction of gene expression by intracellular interferon-gamma: abrogation of the species specificity barrier.

We reported previously that murine L-929 cells expressing a human interferon (IFN)-gamma cDNA lacking a signal peptide sequence synthesize but fail to secrete human IFN-gamma and support viral replication at a reduced level. These cells also had elevated levels of IFN-inducible gene products. We show here that a similar response is seen in human cells expressing a mutated murine IFN-gamma cDNA. The ability of human IFN-gamma to induce gene expression in murine cells is shown to be due to the intracellular IFN-gamma rather than to clonal variation, induction of endogenous murine IFN, or alternative mediators of antiviral activity. We have used a murine cell line, Ltk-aprt-, which is resistant to both type I and II IFNs but responsive to combined treatment with both. Ltk-aprt- cells transfected with human IFN-gamma cDNA lacking a signal sequence support virus replication at the same level as control cells. However, unlike transfectants containing only the neoR selection gene, clones expressing the mutated human IFN-gamma gene show strong protection against viral infection and elevated levels of 2,5 A synthetase mRNA and MHC class I protein after treatment with IFN-beta alone. Reverse transcriptase-PCR rules out the induction of endogenous murine IFN expression as a mediator of these effects. Thus, expression of intracellular human IFN-gamma mimics treatment with extracellular murine IFN-gamma in permitting a synergistic response to IFN-beta. Given the inability of human IFN-gamma to bind to the murine cell-surface receptor our results show that intracellular IFN-gamma can activate certain responses independent of cell-surface binding.

Animals↗
PubMed1995

NT-3 combined with CNTF promotes survival of neurons in modiolus-spiral ganglion explants.

Auditory neurons depend upon the integrity of both their peripheral (auditory hair cells) and central (cochlear nucleus) targets for survival. One proposed trophic mechanism is the production of neurotrophin-3 (NT-3) by auditory hair cells. Modiolus-spiral ganglion explants from adult rats that closely mirror cell-cell interactions and in vivo tissue relationships within this ganglion provide a model for testing trophic factors. Brain derived neurotrophic factor (BDNF), NT-3 and ciliary neurotrophic factor (CNTF) were tested for their ability, both individually and in combination, to support neuronal survival. NT-3 was the strongest individual promoter of survival, while CNTF (a cytokine) with NT-3 (a neurotrophin) was the most effective combination for promoting the survival of auditory neurons.

Animals↗
PubMed1995

Genetic analysis of cinnamyl alcohol dehydrogenase in loblolly pine: single gene inheritance, molecular characterization and evolution.

The gene encoding the monolignol biosynthetic enzyme cinnamyl alcohol dehydrogenase (CAD, E.C. 1.1.1.195) can be expressed in response to different developmental and environmental cues. Control of Cad gene expression could involve either differential regulation of more than one Cad gene or, alternatively combinatorial regulation of a single Cad gene. In loblolly pine (Pinus taeda L.), we found several electrophoretic variants (allozymes) of CAD and a high level of heterozygosity (he = 0.46). Analysis of inheritance patterns of pine CAD allozymes gave segregation ratios that were consistent with Mendelian expectations for a single functional gene. The identity of the full-length Cad cDNA sequence was confirmed by alignment with peptide sequences obtained from purified active enzyme and by extensive similarity to Cad sequences from other species. Southern blot analysis of genomic DNA using the Cad cDNA as a hybridization probe gave simple patterns, consistent with our interpretation that pine Cad is a single-copy gene. Phylogenetic analysis and evolution rate estimates showed that Cad sequences are diverging less rapidly in the gymnosperms than in the angiosperms. The Cad mRNA was present in both lignifying tissues and a non lignifying tissue (the megagametophyte) of pine. The presence of a single gene suggests that different regulatory mechanisms for a single Cad gene, rather than differential regulation of several genes, can account for its expression in response to different cues.

Alcohol Oxidoreductases↗
PubMed1995

Effects of oligosaccharide binding on glycogen debranching enzyme activity and conformation.

Glycogen debranching enzyme contains two catalytic activities (4-alpha-glucanotransferase and amylo-1,6-glucosidase) on its single polypeptide chain, and they are affected differently by the binding of oligosaccharides. Glucose, maltose, and maltotriose are competitive inhibitors of the amylo-1,6-glucosidase activity measured by the hydrolysis of alpha-glucosyl fluoride, whereas saccharides with four or more glucose units are activators of the same activity, showing apparent "uncompetitive" kinetics. This suggests that they do not bind until the alpha-glucosyl fluoride is bound. In either case the potency of the effect increases with the length of the oligosaccharide chain. On the other hand, all oligosaccharides tested (maltose to maltohexaose, alpha-cyclodextrin, and beta-cyclodextrin) are competitive inhibitors of the transferase activity and also cause a decrease in the intrinsic fluorescence, both functions again increased by chain length, thus indicating that these saccharides do bind to the free enzyme. These interesting results can be reconciled if the extended main chain resulting from the transferase reaction has to be reoriented into a different binding mode in order to position the alpha-1,6-linked side-chain glucose into the correct position for the glucosidase reaction. Therefore, activating oligosaccharides behave kinetically as if they had not been previously bound. It is concluded that the main chain of the natural limit dextrin substrate has a different mode of binding for the two catalytic reactions in order to position properly first the maltotetraosyl side chain in the transferase catalytic site and then the glucosyl side chain in the glucosidase catalytic site.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Association of Polyomavirus middle tumor antigen with phospholipase C-gamma 1.

Middle tumor antigen (MT) is the primary transforming protein of murine Polyomavirus. MT transforms by associating with and modulating the activities of cellular proteins involved in control of cell proliferation. MT binds to and is phosphorylated by cellular tyrosine kinases. The phosphorylated tyrosines become docking sites for SH2 (Src homology 2) domain-containing molecules. Tyrosine 322 of MT is known to be phosphorylated but has no known binding protein. We have found that phospholipase C-gamma 1 (PLC-gamma 1), a SH2 domain-containing protein, coimmunoprecipitates with MT. Tyrosine phosphorylation of PLC-gamma 1 is elevated in cells expressing MT, suggesting activation of this enzyme by MT. A Tyr-322-->Phe mutation in MT renders it defective in MT-PLC-gamma 1 interaction and in transformation. From the correlation between transformation and MT-PLC-gamma 1 interaction, we suggest that PLC-gamma 1 may play a role in transformation.

3T3 Cells↗
PubMed1995

Molecular characterization of two Drosophila guanylate cyclases expressed in the nervous system.

We have isolated, by interspecies hybridization, two classes of Drosophila cDNA each encoding a different guanylate cyclase (GC). One of them encodes an alpha subunit homolog of soluble GC, designated DGC alpha 1, and the other encodes a receptor-type GC, designated DrGC. The dgc alpha 1 cDNA encodes a protein of 676 amino acids and maps to 99B. In situ hybridization to adult tissue sections showed that dgc alpha 1 mRNA is found mainly in the cell bodies of the optic lobe, central brain, and thoracic ganglia. The DGC alpha 1 protein was also localized primarily to the nervous system by immunocytochemical staining, consistent with results of in situ hybridization. However, no detectable expression of this protein was found in the retina. The other class of cDNA, drgc, maps to 76C and encodes a 1525-amino acid protein displaying structural features similar to other known receptor-type guanylate cyclases. However, it has a C-terminal 430 amino acid region that has no homology to any known proteins. drgc RNA is expressed at low levels throughout development and in adult heads and bodies. In situ hybridizations to adult tissue sections showed that drgc mRNA is expressed in a wide range of tissues, including the optic lobe, central brain, thoracic ganglia, digestive tract, and the oocyte.

Amino Acid Sequence↗
PubMed1995

Molecular cloning of PEPT 2, a new member of the H+/peptide cotransporter family, from human kidney.

Mammalian kidney is known to express a transport system specific for small peptides and pharmacologically active aminocephalosporins. This system is energized by a transmembrane electrochemical H+ gradient. Recently, a H(+)-coupled peptide transporter has been cloned from rabbit and human intestine (Fei et al. (1994) Nature 368, 563-566; Liang et al., J. Biol. Chem., in press). Functional studies have established that the renal peptide transport system is similar but not identical to its intestinal counterpart. Therefore, in an attempt to isolate the renal H+/peptide cotransporter cDNA, we screened a human kidney cDNA library with a probe derived from the rabbit intestinal H+/peptide cotransporter cDNA. This has resulted in the isolation of a positive clone with a 2190 bp long open reading frame. The predicted protein consists of 729 amino acids. Hydropathy analysis of the amino acid sequence indicates the presence of twelve putative transmembrane domains. The primary structure of this protein exhibits 50% identity and 70% similarity to the human intestinal H+/peptide cotransporter. Functional expression of the kidney cDNA in HeLa cells results in the induction of a H(+)-coupled transport system specific for small peptides and aminocephalosporins. Reverse transcription-coupled polymerase chain reaction demonstrates that the cloned transporter is expressed in human kidney but not in human intestine. This transporter, henceforth called PEPT 2, represents a new member in the growing family of H(+)-coupled transport systems in the mammalian plasma membrane.

Amino Acid Sequence↗
PubMed1995

Noradrenergic enhancement of GABA-induced input resistance changes in layer V regular spiking pyramidal neurons of rat somatosensory cortex.

Previous in vivo studies have shown that microiontophoretic application of norepinephrine (NE) and isoproterenol (ISO) can enhance gamma-aminobutyric acid (GABA)-induced depressant responses of rat somatosensory cortical neurons. In the present investigation we have examined the transmembrane electrophysiological events which are associated with interactions between NE and GABA in layer V pyramidal neurons of rat barrel field cortex. Intracellular recordings were made from electrophysiologically identified cells in a superfused cortical tissue slice preparation before, during and after bath or microdrop application of GABA, NE and ISO, alone or in combination. GABA application produced a small depolarization from resting membrane potential associated with a reduction (22%) in input resistance. NE and ISO (10-100 microM) also produced in some cases small membrane depolarizations (1-4 mV) but little concomitant changes in input resistance. Simultaneous application of NE with GABA potentiated amino acid-induced changes in input resistance in 4 cases and antagonized (n = 4) or had no effect (n = 4) on GABA-associated membrane events in 8 other cases. When the alpha-blocker, phentolamine (20 microM), was added to the medium, NE-induced enhancement of the GABA response was observed in 3 of 5 cases (60%), suggesting both, a beta-adrenergic mediation and a possible alpha-receptor masking of this noradrenergic-potentiating action. Consistent with this interpretation was the finding that the beta-agonist, ISO (10-100 microM), produced net increases in GABA-induced input resistance changes in 64% of cases tested (9 of 14). The potentiating effect of NE and ISO was mimicked by the adenyl cyclase activator, forskolin (n = 2), and a membrane permeant analog of cyclic-AMP, 8-bromo-cyclic AMP (n = 3); and could also be demonstrated when the GABAA agonist muscimol (0.5-1 microM) was substituted for GABA. The reversal potential for GABA and GABA + NE remained the same. These findings suggest that previous demonstrations of NE-potentiating effects on GABA inhibition may be mediated by beta-receptor/cyclic-AMP-linked actions on mechanisms which regulate GABAA receptor-induced membrane conductance changes.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1995

Heparan sulfate-mediated cell aggregation. Syndecans-1 and -4 mediate intercellular adhesion following their transfection into human B lymphoid cells.

Because syndecans are present at sites of cell-cell contact in vivo it has been hypothesized that they play a role in mediating cell-cell adhesion. However, there has been no direct evidence to support this notion. To address this question, B lymphoid (ARH-77) cells were transfected with the cDNA for murine syndecan-1. Unlike the parental cells, the transfectants form large multicellular aggregates in suspension cultures and stain intensely for syndecan-1 at sites of cell-cell contact. Using rotation-mediated aggregation assays, we find that aggregation of syndecan-1-transfected cells is dependent on divalent cations and is inhibited by the following: (i) addition of heparin and heparin-like glycosaminoglycans, (ii) removal of heparan sulfate from the cell surface, or (iii) addition of exogenous purified syndecan-1. Mixing of syndecan-1-transfected and control-transfected cells results in aggregates containing both cell types indicating that aggregation occurs through a heterophilic adhesion mechanism in which heparan sulfate chains bind to a counter-receptor present on these cells. Importantly, syndecan-4-transfected cells also aggregate in a heparan sulfate-dependent manner, while in contrast, betaglycan-transfected cells aggregate poorly. Thus, syndecans may be important mediators of cell-cell adhesion, but this function may not be common to all transmembrane heparan sulfate-bearing proteoglycans.

Animals↗
PubMed1995

Analysis of the interrelationship between IL-12, TNF-alpha, and IFN-gamma production during murine listeriosis.

IL-12, a recently described cytokine, is an important mediator in the early production of IFN-gamma during infection. To evaluate the timing of IL-12 production, and its relationship to TNF-alpha, and IFN-gamma production during primary murine listeriosis, we measured cytokine mRNA and protein levels in C57B1/6 mice infected intravenously with Listeria monocytogenes (LM). IL-12 is a disulfide-linked heterodimer containing two chains (designated P35 and P40); however, bioactive cytokine production has been more closely linked with P40 expression. Consequently, we monitored mRNA and protein levels of P40 in the spleen as a marker for IL-12 production in vivo. Splenic P40 mRNA levels (assayed using RNase protection methods) were low in uninfected animals, but increased markedly beginning 15 to 18 hr after LM infection. In sublethally infected animals, P40 mRNA levels remained elevated for 5 days, returning to baseline with the resolution of infection. P40 protein (assayed using an antibody capture ELISA) could be detected in the spleens of LM-infected animals beginning around 18 hr postinfection confirming linkage between P40 mRNA accumulation and the generation of a protein product. In comparing P40 and IFN-gamma mRNA levels in vivo, we found in each case that substantial increases in mRNA accumulation did not appear until 15-18 hr postinfection. In comparable studies using BALB/c animals, cytokine production began slightly earlier (between 12 and 15 hr) but once again P40 and IFN-gamma mRNA levels increased in a coordinated manner. P40 mRNA (like IFN-gamma and TNF-alpha mRNA) only accumulated in animals infused with live, virulent bacteria. Although we could detect no obvious lag between the time of onset of IL-12 and IFN-gamma accumulation in vivo, infusions of anti-IL-12 antibodies markedly reduced IFN-gamma expression implying that IL-12 production precedes and directs IFN-gamma production. TNF-alpha production, on the other hand, was not diminished by anti-IL-12 treatment. Our studies demonstrate that IL-12 generation is an essential step in normal IFN-gamma production during listeriosis, and suggest that IL-12, once produced, may begin enhancing IFN-gamma production in vivo in less than 3 hr.

Animals↗
PubMed1995

Fracture pattern and instability of thoracolumbar injuries.

Spinal fractures are common in the thoracolumbar region. Assessment of fracture instability is often made from fracture pattern seen on plain radiographs or CT scans. The purpose of this in vitro study was to correlate three-dimensional flexibility to each fracture type, i.e., endplate, wedge, and burst. Ten fresh cadaveric human spine specimens (T11-L1) were incrementally impacted in a high-speed trauma apparatus until a fracture occurred. All fractures were produced by the same mechanism (axial compression/flexion load). The occurrence of a fracture was monitored by lateral radiographs of the specimen, whose canal was lined with 1.6-mm steel balls. After each impact, the specimen was studied for its flexibility in flexion, extension, left and right lateral bindings, and left and right axial rotations. The flexibility was determined in response to the application of maximum pure moments of 7.5 Nm. Each moment was applied individually and in three load cycles. Parameters of neutral zone (NZ) and range of motion (ROM) were computed. Average flexion-extension ROM (and NZ) for intact, endplate, wedge, and burst fracture were respectively, 12.7 degrees (1.3 degrees), 13.9 degrees (1.7 degrees), 19.2 degrees (3.2 degrees), 22.0 degrees (6.0 degrees). The average lateral bending ROM (NZ) were 12.6 degrees (1.2 degrees), 13.6 degrees (1.9 degrees), 19.1 degrees (3.7 degrees), 27.2 degrees (9.8 degrees). The average axial rotation ROM (NZ) were 4.7 degrees (0.4 degree), 6.1 degrees (0.7 degrees), 7.1 degrees (1.0 degree), 12.9 degrees (3.1 degrees).(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1995

Increased mRNA expression of manganese superoxide dismutase in psoriasis skin lesions and in cultured human keratinocytes exposed to IL-1 beta and TNF-alpha.

Because reactive oxygen species have been implicated in the pathogenesis of various hyperproliferative and inflammatory diseases, the mRNA expression of the antioxidant enzyme superoxide dismutase was studied in psoriatic skin tissue. By using reverse transcription-PCR we found similar expression of copper, zinc superoxide dismutase (CuZnSOD) in the involved vs. uninvolved psoriatic skin. In contrast, the level of the manganese superoxide dismutase (MnSOD) mRNA message was consistently higher in lesional psoriatic skin as compared to adjacent uninvolved skin and healthy control skin. Parallel investigation of those cytokines that are thought to be direct or indirect inducers of the MnSOD activity revealed an increased mRNA expression of IL-1 beta, TNF-alpha, and GM-CSF in lesional psoriatic skin. To study if these cytokines exert a direct effect on dismutase expression in epidermal cells, human keratinocytes in culture were challenged with IL-1 beta, TNF-alpha, and GM-CSF. It was found that IL-1 beta and TNF-alpha, but not GM-CSF, induced the mRNA expression of MnSOD, and an additive effect was demonstrated for the two former cytokines. Further, the expression of both CuZnSOD and MnSOD transcripts was similar in cultured keratinocytes maintained at low differentiation (low Ca2+ medium) and cells forced to terminal differentiation (by high Ca2+ medium). Our results indicate that the abnormal expression of MnSOD mRNA in lesional psoriatic skin is not directly linked to the pathologic state of keratinocyte differentiation in the skin. It seems more likely that the cutaneous overexpression of MnSOD in psoriatic epidermis represents a protective cellular response evoked by cytokines released from inflammatory cells invading the diseased skin.

Adult↗
PubMed1995

Gene expression after uninephrectomy in the rat: simultaneous expression of positive and negative growth control elements.

PURPOSE: Compensatory renal growth after contralateral nephrectomy consists largely of hypertrophy of the renal cortex. The signals initiating compensatory renal growth are as yet unknown. MATERIALS AND METHODS: Using a technique of quantitative dot-blot hybridization, we examined the expression of a number of genes after contralateral nephrectomy in the rat in an effort to establish a characteristic "fingerprint" which might shed light on the mechanism of compensatory renal growth. RESULTS: Negative growth control elements, including transcriptional repressors (WT-1, junB, myn, HNF-1, p53, RB, Dr1, gas2, gadd153) were induced 1.7 to 4.7-fold within 1 hour after uninephrectomy, as were positive growth control elements (egr-1, c-jun, cyclin C, cyclin E; 1.9- to 4.2-fold induction). The steady state mRNA levels for heat shock genes hsp70, hsp86 and hsp90 beta, as well as extracellular matrix genes fibronectin and collagen I (alpha 1 chain) were also increased 1 hour after uninephrectomy. In addition, 2 Na(+)-specific exchangers (NHE-1 and Na/Ca) were induced within 1 hour after uninephrectomy. CONCLUSIONS: These results suggest the coordinate expression of positive and negative growth control elements early in this physiologic model of organ growth.

Animals↗