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Biomedical subjects

L Liu

Publications and source records attributed to L Liu.

At least 397 records · Page 22Linked to original sources
PubMed2000

Noninvasive measurement of potassium efflux as an early indicator of cell death in mouse embryos.

Programmed cell death (apoptosis) occurs in nearly all cell types examined, including mammalian oocytes and embryos, where it may underlie some forms of infertility in humans. Although the molecular machinery participating in apoptosis have been intensely investigated, the accompanying physiological changes have not received similar attention. In this study, a novel electrophysiology technique has been employed to monitor real-time perturbations in the physiology of mouse embryos undergoing apoptosis evoked by hydrogen peroxide, diamide, and staurosporine. Despite differences in their mode of action, these agents evoked a similar early change in cellular physiology; namely, a pronounced, transient, potassium efflux through tetraethylammonium-sensitive potassium channels accompanied by cell shrinkage. Mouse zygotes exposed to 200 microM H(2)O(2) exhibited potassium efflux that elevated the potassium concentration of the media surrounding embryos by 1.4 +/- 0.1 microM. Pretreatment with tetraethylammonium inhibited this increase (0.2 +/- 0.1 microM). Our results indicate that potassium efflux through potassium channels and concurrent cell shrinkage are early indicators of cell death in embryos and that noninvasive measurements of potassium pathophysiology may identify embryos undergoing cell death prior to the manifestation of other morphological or molecular hallmarks of cell death.

Animals↗
PubMed2000

The Egr-1 promoter contains information for constitutive and inducible expression in transgenic mice.

Egr-1 is an immediate early gene that couples short-term changes in the extracellular milieu to long-term changes in gene expression. Under in vitro conditions, the Egr-1 gene is expressed in many cell types and is induced by a wide variety of extracellular signals. The mechanisms by which the Egr-1 gene is regulated in vivo remain poorly understood. In this study, we have generated transgenic mice with a construct containing 1200 bp of the mouse Egr-1 promoter coupled to nuclear localized LacZ. In multiple independent lines of mice, reporter gene expression was detected in subsets of endothelial cells, vascular smooth-muscle cells, cardiomyocytes, neurons, and hepatocytes. This pattern closely resembled that of the endogenous gene. After partial hepatectomy, reporter gene activity was upregulated between two- and fivefold in regenerating livers. Taken together, these findings suggest that the Egr-1 promoter contains information for appropriate spatial and temporal expression in vivo.

Animals↗
PubMed2000

cis,cis,cis-tetramethyl 1,2,4,5-cyclohexanetetracarboxylate

The title compound, C(14)H(20)O(8), was synthesized from the hydrogenation of tetramethyl 1,4-cyclohexadiene-1,2,4, 5-tetracarboxylate with a catalytic amount of palladium/carbon. All four carbonyl moieties of the methyl ester groups are on the same face of the chair-conformed ring. The substantial ring distortion associated with the 1,3-diaxial methoxycarbonyl substituents is reflected in the large difference between bond angles as well as torsion angles, respectively, that in undistorted cyclohexanes would be approximately the same.

Journal Article↗
PubMed2000

Effects of ipsilateral and contralateral precursors on overshoot.

Overshoot is defined as the decrease in threshold as a brief signal is moved from the beginning to near the temporal center of a longer duration, broadband noise masker. Overshoot can be reduced when another noise (a precursor) is presented just prior to the masker. The purpose of the present investigation was to follow up on a recent psychophysical study which showed that overshoot could be reduced by a precursor presented to the ear contralateral to that receiving the masker and signal. The signal was a 20-ms, 4000-Hz tone that was presented at the beginning or in the temporal center of a 400-ms broadband noise masker. In the first experiment, a 200-ms broadband precursor was presented either to the ipsilateral or to the contralateral ear. The ipsilateral precursor reduced overshoot for all ten subjects, but the contralateral precursor reduced overshoot for only four of the ten subjects. In a supplementary experiment, the contralateral precursor failed to reduce overshoot in a new group of five subjects, both when tested with supra-aural headphones and with insert earphones. In the second experiment, the four subjects who showed an effect of the contralateral precursor in experiment 1 were tested under conditions where the bandwidth of the precursor was manipulated, resulting in either a narrow-band precursor centered at 4000 Hz, a low-band precursor with energy primarily below 4000 Hz, or a high-band precursor with energy primarily above 4000 Hz. There was a tendency for the effectiveness of the ipsilateral and contralateral precursors to be affected similarly (though to different degrees) by changes in the spectral content of the precursor. These results suggest that the effect of the contralateral precursor is not due to a timing cue, and that the processing underlying the effectiveness of ipsilateral and contralateral precursors may be largely the same.

Adult↗
PubMed2000

GAA trinucleotide repeat region regulates M9/pMGA gene expression in Mycoplasma gallisepticum.

Mycoplasma gallisepticum, the cause of chronic respiratory infections in the avian host, possesses a family of M9/pMGA genes encoding an adhesin(s) associated with hemagglutination. Nucleotide sequences of M9/pMGA gene family members indicate extensive sequence similarity in the promoter regions of both the transcribed and silent genes. The mechanism that regulates M9/pMGA gene expression is unknown, but studies have revealed an apparent correlation between gene expression and the number of tandem GAA repeat motifs located upstream of the putative promoter. In this study, transposon Tn4001 was used as a vector with the Escherichia coli lacZ gene as the reporter system to examine the role of the GAA repeats in M9/pMGA gene expression in M. gallisepticum. A 336-bp M9 gene fragment (containing the GAA repeat region, the promoter, and the translation start codon) was amplified by PCR, ligated with a lacZ gene from E. coli, and inserted into the Tn4001-containing plasmid pISM2062. This construct was transformed into M. gallisepticum PG31. Transformants were filter cloned on agar supplemented with 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal) to monitor lacZ gene expression on the basis of blue/white color selection. Several cycles of filter cloning resulted in cell lineages in which lacZ gene expression alternated between the On and Off states in successive generations of progeny clones. The promoter regions of the M9-lacZ hybrid genes of individual progeny clones were amplified by PCR and sequenced. The only differences between the promoter regions of the blue and white colonies were in the number of GAA repeats. Clones that expressed lacZ had exactly 12 tandem copies of the GAA repeat. Clones that did not express lacZ invariably had either more than 12 (14 to 16) or fewer than 12 (5 to 11) GAA repeats. Southern analysis of M. gallisepticum chromosomal DNA confirmed that the phase-variable expression of the lacZ reporter gene was not caused by Tn4001 transposition. These data strongly indicate that changes in the length of the GAA repeat region are responsible for regulating M9/pMGA gene expression.

Amino Acid Sequence↗
PubMed2000

Candida albicans and Candida krusei differentially induce human blood mononuclear cell interleukin-12 and gamma interferon production.

Protection against Candida infection involves both innate and acquired immune responses, and cytokines produced by monocytes during the innate response may modify the acquired immune response by T cells. We hypothesized that Candida species which differ in pathogenicity can differentially induce production of immunoregulatory cytokines by human monocytes, which in turn modify T cells for immune responses to Candida. To test this hypothesis, we examined the effects of Candida albicans and Candida krusei on immunoregulatory cytokine production by human monocytes and gamma interferon (IFN-gamma) production by peripheral blood mononuclear cells (PBMC). Purified monocytes were incubated with live or heat-killed strains of C. albicans and C. krusei at the optimal Candida/monocyte ratio of 0.5. Cytokines in the supernatants were measured by enzyme-linked immunosorbent assay. Our data demonstrated that live C. albicans and C. krusei significantly induced interleukin-10 (IL-10), monocyte chemotactic factor 1, IL-1beta, and tumor necrosis factor alpha production by monocytes relative to unstimulated monocytes. In contrast, unlike C. krusei, pathogenic live strains of C. albicans induced no or only a minimal level of IL-12. The expression of IL-12 p40 mRNA levels by reverse transcription-PCR corroborated the IL-12 protein (p70) findings. In human PBMC, human blood monocytes were the major source of both IL-10 and IL-12 production in response to C. albicans and C. krusei. Upon activation of T cells in the presence of Candida-modified monocytes and antigen-presenting cells, IL-12 production by PBMC treated with Candida organisms correlated strongly with the level of IFN-gamma production by T cells. These results indicate that the virulence of C. albicans may be related to its ability to induce the monocytic type II cytokine IL-10, with a selective inhibition of IL-12 production, which may be responsible for the observed lack of T-cell IFN-gamma and may restrain an effective type I immune response to Candida.

Candida↗
PubMed2000

Regulation of human beta-defensins by gastric epithelial cells in response to infection with Helicobacter pylori or stimulation with interleukin-1.

Gastric epithelial cells in vitro and in vivo are shown to constitutively express the peptide antibiotic human beta-defensin type 1 (hBD-1). In contrast, hBD-2 expression is regulated in gastric epithelial cells and increases in response to infection with Helicobacter pylori or stimulation with the proinflammatory cytokine interleukin-1. These data suggest that hBD-2 is a component of the regulated host gastric epithelial cell response to H. pylori infection and proinflammatory mediators.

Defensins↗
PubMed2000

Incorporation of tumor vasculature targeting motifs into moloney murine leukemia virus env escort proteins enhances retrovirus binding and transduction of human endothelial cells.

Adhesion receptors expressed on the surfaces of tumor-activated endothelial cells provide an advantageous locus for targeting gene therapy vectors to angiogenic tissues and/or tumor vasculature. In this study, we engineered a series of Asn-Gly-Arg (NGR)-containing congeners of the presumptive cell binding motif contained within the ninth type III repeat of fibronectin and displayed these tumor vasculature targeting motifs (TVTMs) within the context of Moloney murine leukemia envelope "escort" proteins. Comparative studies of envelope incorporation into viral particles and evaluation of the cell binding properties of the targeted vectors revealed critical structural features, thus identifying a subset of optimal TVTMs. Utilizing a modified ELISA to evaluate viral binding to target cells, we observed a significant down-regulation of TVTM-virion binding to human endothelial cells following sustained (48-h) exposure to VEGF. Normalized for equivalent titers (10(6) CFU/ml), as assayed on NIH 3T3 cells, vectors displaying TVTM escort proteins significantly enhanced the transduction efficiency from 12.2 to 37.4% in human KSY-1 endothelial cell cultures (P < 0.001) and from 0.4 to 4.1% in human umbilical vein endothelial cell (HUVEC) cultures (P < 0.001). In summary, these studies utilized an engineering approach to identify a subset of TVTMs that are stably incorporated as envelope "escort" proteins into retroviral vectors and that, by functioning to improve the binding efficiency and transduction of both HUVEC and KSY1 endothelial cells, may have therapeutic potential for targeting gene delivery to the tumor-associated vasculature.

3T3 Cells↗
PubMed2000

Multiple homing pathways used by yeast mitochondrial group II introns.

The yeast mitochondrial DNA group II introns aI1 and aI2 are retroelements that insert site specifically into intronless alleles by a process called homing. Here, we used patterns of flanking marker coconversion in crosses with wild-type and mutant aI2 introns to distinguish three coexisting homing pathways: two that were reverse transcriptase (RT) dependent (retrohoming) and one that was RT independent. All three pathways are initiated by cleavage of the recipient DNA target site by the intron-encoded endonuclease, with the sense strand cleaved by partial or complete reverse splicing, and the antisense strand cleaved by the intron-encoded protein. The major retrohoming pathway in standard crosses leads to insertion of the intron with unidirectional coconversion of upstream exon sequences. This pattern of coconversion suggests that the major retrohoming pathway is initiated by target DNA-primed reverse transcription of the reverse-spliced intron RNA and completed by double-strand break repair (DSBR) recombination with the donor allele. The RT-independent pathway leads to insertion of the intron with bidirectional coconversion and presumably occurs by a conventional DSBR recombination mechanism initiated by cleavage of the recipient DNA target site by the intron-encoded endonuclease, as for group I intron homing. Finally, some mutant DNA target sites shift up to 43% of retrohoming to another pathway not previously detected for aI2 in which there is no coconversion of flanking exon sequences. This new pathway presumably involves synthesis of a full-length cDNA copy of the inserted intron RNA, with completion by a repair process independent of homologous recombination, as found for the Lactococcus lactis Ll.LtrB intron. Our results show that group II intron mobility can occur by multiple pathways, the ratios of which depend on the characteristics of both the intron and the DNA target site. This remarkable flexibility enables group II introns to use different recombination and repair enzymes in different host cells.

Base Sequence↗
PubMed2000

The Egr-1 gene is induced by epidermal growth factor in ECV304 cells and primary endothelial cells.

The early growth response (Egr)-1 transcription factor serves to couple changes in the extracellular environment to alterations in gene expression. An understanding of the mechanisms that underlie Egr-1 gene regulation should provide important insights into how environmental signals are transduced by endothelial cells. The aim of the present study was to determine whether epidermal growth factor (EGF) induces Egr-1 expression in endothelial cells. In ECV304 cells, Egr-1 mRNA and protein levels were increased in response to EGF. In stable transfection assays, the 1,200-bp promoter of the mouse Egr-1 gene contained information for EGF response via a protein kinase C-independent, mitogen-activated protein kinase-dependent pathway. The endogenous Egr-1 gene was similarly responsive to EGF in primary human umbilical vein endothelial cells, human coronary artery endothelial cells, and rat fat pad endothelial cells, but not in bovine aortic endothelial cells, calf pulmonary artery endothelial cells, or PY-4-1 endothelial cells. Together, these results suggest that the Egr-1 gene is responsive to EGF in a subset of endothelial cells.

3T3 Cells↗
PubMed2000

Metabolic regulation of Na(+)/P(i)-cotransporter-1 gene expression in H4IIE cells.

We showed that the rat Na(+)/P(i) cotransporter-1 (RNaPi-1) gene was regulated by insulin and glucose in rat hepatocytes. The aim of this work was to elucidate signaling pathways of insulin-mediated metabolic regulation of the RNaPi-1 gene in H4IIE cells. Insulin increased RNaPi-1 mRNA abundance in the presence of glucose and decreased RNaPi-1 mRNA in the absence of glucose, clearly establishing an involvement of metabolic signals for insulin-induced upregulation of the RNaPi-1 gene. Pyruvate and insulin increased RNaPi-1 expression but downregulated L-pyruvate kinase, indicating the existence of gene-specific metabolic signals. Although fructose, glycerol, and lactate could support insulin-induced upregulation of the RNaPi-1 gene, compounds entering metabolism beyond pyruvate oxidation, such as acetate and citrate, could not, suggesting that RNaPi-1-specific metabolic signals are generated at or above pyruvate oxidation. Wortmannin, LY-294002, and rapamycin abolished the insulin effect on the RNaPi-1 gene, whereas expression of dominant negative Asn(17) Ras and mitogen-activating protein kinase (MAPK) kinase (MEK) inhibitor PD-98059 exhibited no effect. Thus we herein propose that metabolic regulation of RNaPi-1 expression by insulin is mediated through the phosphatidylinositol 3-kinase/p70 ribosomal S6 kinase pathways, but not the Ras/MAPK pathway.

Adenoviridae↗
PubMed2000

Aging alters gastric mucosal responses to epidermal growth factor and transforming growth factor-alpha.

Administration of pharmacological doses of epidermal growth factor (EGF) or transforming growth factor-alpha (TGF-alpha) in young rats stimulates gastric mucosal proliferation, but, in aged rats, the same treatment inhibits proliferation. This may be due to enhanced ligand-induced internalization of EGF receptor (EGFR). In support of this, we demonstrated that although a single injection of EGF (10 microg/kg) or TGF-alpha (5 microg/kg) in young (4-6 mo old) rats greatly increased membrane-associated EGFR tyrosine kinase activity, the same treatment slightly inhibited the enzyme activity in aged (24 mo old) rats. This treatment also produced a greater abundance of punctate cytoplasmic EGFR staining in gastric epithelium of aged rats, consistent with EGFR internalization. In vitro analyses demonstrated that exposure of isolated gastric mucosal cells from aged but not young rats to 100 pM TGF-alpha resulted in marked increases in intracellular EGFR tyrosine kinase activity and that induction of EGFR tyrosine kinase activity in mucosal membranes from aged rats occurred at doses 1,000-fold less than those required in young rats. Our data suggest that aging enhances sensitivity of the gastric mucosa to EGFR ligands. This may partly explain EGFR-mediated inhibition of gastric mucosal proliferation in aged rats.

Aging↗
PubMed2000

Regulation of DHP receptor expression by elements in the 5'-flanking sequence.

The alpha(1)-subunit of the cardiac/vascular Ca(2+) channel, which is the dihydropyridine (DHP)-binding site (the DHP receptor), provides the pore structure for Ca(2+) entry. It contains the binding sites for multiple classes of drugs collectively known as Ca(2+) antagonists. As an initial step toward understanding the mechanisms controlling transcription of the rat cardiac alpha(1C)-subunit gene, we have cloned a 2.3-kb fragment containing the 5'-flanking sequences and identified the alpha(1C)-subunit gene transcription start site. The rat alpha(1C)-subunit gene promoter belongs to the TATA-less class of such basal elements. Using deletion analysis of alpha(1C)-subunit promoter-luciferase reporter gene constructs, we have characterized the transcriptional modulating activity of the 5'-flanking region and conducted transient transfections in cultured neonatal rat cardiac ventricular myocytes and vascular smooth muscle cells. Sequence scanning identified several potential regulatory elements, including five consensus sequences for the cardiac-specific transcription factor Nkx2.5, an AP-1 site, a cAMP response element, and a hormone response element. Transient transfection experiments with the promoter-luciferase reporter fusion gene demonstrate that the 2-kb 5'-flanking region confers tissue specificity and hormone responsiveness to expression of the Ca(2+) channel alpha(1C)-subunit gene. Electrophoretic mobility shift assays identified a region of the alpha(1C)-subunit gene promoter that can bind transcription factors and appears to be important for gene expression.

5' Untranslated Regions↗
PubMed2000

Effect of high Ba(2+) on norepinephrine-induced inhibition of N-type calcium current in bullfrog sympathetic neurons.

The voltage-dependent inhibition of N-type calcium current by neurotransmitters is the best-understood example of neuronal calcium channel inhibition. One of the mechanisms by which this pathway is thought to inhibit the calcium current is by reducing the permeation of divalent cations through the channel. In this study one prediction of this hypothesis was examined, that high concentrations of divalent cations reduce the maximum neurotransmitter-induced inhibition. Norepinephrine (NE)-induced inhibition was compared in external solutions containing either 2 or 100 mM Ba(2+). Initially, NE dose-response curves were generated by averaging data from many neurons, and it was found that the relationship was right shifted in the high-Ba(2+) external solution without an effect on maximum inhibition. The IC(50) was 0.6 and 3 microM in 2 and 100 mM Ba(2+), respectively. This shift was verified by comparing the effect of NE on single neurons exposed to both 2 and 100 mM Ba(2+). The inhibition induced by 1 microM NE was reduced in 100 mM Ba(2+) compared with that in 2 mM Ba(2+). However, the response to 100 microM NE was identical between high and low Ba(2+). Thus, divalent cations appear to act as a competitive inhibitor of NE binding, which likely results from these ions' interacting with negatively charged amino acids that are important for catecholamine binding to adrenergic receptors. Because the maximum inhibition induced by NE was similar in low and high Ba(2+), the effect of inhibition on single N-type calcium channels was not altered by the divalent cation concentration.

Animals↗
PubMed2000

Stereotactic biopsy for intracranial space-occupying lesions: clinical analysis of 550 cases.

OBJECT: To investigate the role of stereotactic biopsy in planning the optimal management of intracranial space-occupying lesions. PATIENTS AND METHODS: Between December 1989 and December 1999, stereotactic biopsy was performed in 550 patients with intracranial mass lesions that were deep-seated or located in the functional area. There were 340 males and 210 females, and their ages ranged from 4 to 75 years. All the procedures were done under local anesthesia with a Leksell stereotactic system. A CT scan was used to determine the coordinates in the first 420 cases and the Aero Tech Stereotactic Surgical Plan System in the subsequent 130 patients. RESULTS: Brain tumors were diagnosed pathologically in 475 patients (86.4%), inflammatory process in 44 (8.0%), other lesions in 12 (2.2%) and no conclusive diagnosis was found in 19 (3.4%). The overall positive rate of biopsy was 96.6%, and the positive rate for brain tumor was 86.4%. Intracranial hematomas after biopsy were found in 13 cases (2.4%). Seizures occurred during the operation in 7 cases (1.2%), and slight and transient neurological deficits were found in 23 cases (4.2%). There were no deaths or other serious complications. CONCLUSIONS: The results suggest that the stereotactic biopsy is a reliable method to obtain histopathological diagnosis of intracranial mass lesions, and it is also of great help in selecting the appropriate management.

Adolescent↗
PubMed2000

The viral anti-inflammatory chemokine-binding protein M-T7 reduces intimal hyperplasia after vascular injury.

Chemokines and IFN-gamma function as central regulators of inflammatory responses to vascular injury. Both classes of cytokines are upregulated during restenosis, a response to vascular injury that leads to recurrent atherosclerotic plaque growth, but the relative impact of each class of cytokines remains undetermined. M-T7 is a secreted myxoma viral immunomodulatory glycoprotein that functions both as a species-specific inhibitor of rabbit IFN-gamma and as a chemokine-binding protein, interacting with a wide range of C, C-C, and C-X-C chemokines in a species-nonspecific fashion. We wished to (a) assess the efficacy of purified M-T7 protein in inhibiting intimal hyperplasia after angioplasty injury and (b) exploit unique species-specific functions of M-T7 in order to judge the relative importance of each cytokine class on plaque growth. Anesthetized New Zealand white rabbits and Sprague-Dawley rats received either M-T7 or control at the time of arterial angioplasty injury. Histological analysis at 28 days demonstrated significant reductions in intimal hyperplasia with M-T7 treatment in both models, with an associated early inhibition of inflammatory cell invasion. Purified M-T7 protein inhibits intimal hyperplasia after angioplasty injury in a species-nonspecific fashion, thus implicating the chemokine-binding activity as more critical for prevention of plaque growth after vascular injury.

Animals↗
PubMed2000

Identification of ADAM 31: a protein expressed in Leydig cells and specialized epithelia.

A family of proteins containing a disintegrin and metalloproteinase domain (ADAMs) has been identified recently. Here, we report the identification of a novel member of the ADAM protein family from mouse. This protein is designated ADAM 31. The complementary DNA sequence of ADAM 31 predicts a transmembrane protein with metalloproteinase, disintegrin, cysteine-rich, and cytoplasmic domains. Messenger RNA encoding ADAM 31 was most abundant in testes, but was also detected in many other tissues. More significantly, the antibodies raised against ADAM 31 reveal that the protein has a unique and restricted expression pattern. ADAM 31 is expressed in Leydig cells of the testes, but unlike many other ADAMs, it is not found on developing sperm. Furthermore, ADAM 31 is highly expressed on four types of specialized epithelia: the cauda epididymidis, the vas deferens, the convoluted tubules of the kidney, and the parietal cells of the stomach.

ADAM Proteins↗