Biomedical subjects

Y Liu

Publications and source records attributed to Y Liu.

At least 937 records · Page 52Linked to original sources

PubMed2000

High-titer, wild-type free recombinant adeno-associated virus vector production using intron-containing helper plasmids.

Recombinant adeno-associated virus (rAAV) is capable of directing long-term, high-level transgene expression without destructive cell-mediated immune responses. However, traditional packaging methods for rAAV vectors are generally inefficient and contaminated with replication-competent AAV (rcAAV) particles. Although wild-type AAV is not associated with any known human diseases, contaminating rcAAV particles may affect rAAV gene expression and are an uncontrolled variable in many AAV gene transfer studies. In the current study, a novel strategy was designed to both optimize AAV rep gene expression and increase vector yield, as well as simultaneously to diminish the potential of generating rcAAV particles from the helper plasmid. The strategy is based on the insertion of an additional intron in the AAV genome. In the AAV infectious clone, the intron insertion had no effects on the properties of Rep proteins expressed. Normal levels of both Rep and Cap proteins were expressed, and the replication of the AAV genome was not impaired. However, the generation of infectious rcAAV particles using intronized AAV helper was greatly diminished, which was due to the oversized AAV genome caused by the insertion of the artificial introns. Moreover, the rAAV packaging was significantly improved with the appropriate choice of intron and insertion position. The intron is another element that can regulate the rep and cap gene expression from the helper plasmid. This study provides for a novel AAV packaging system which is highly versatile and efficient. It can not only be combined with other AAV packaging systems, including rep-containing cell lines and herpes simplex virus hybrid packaging methods, but also be used in other vector systems as well.

Dependovirus↗

PubMed2000

Adenovirus E1B 55-kilodalton oncoprotein inhibits p53 acetylation by PCAF.

The adenovirus E1B 55-kDa protein binds to cellular tumor suppressor p53 and inactivates its transcriptional transactivation function. p53 transactivation activity is dependent upon its ability to bind to specific DNA sequences near the promoters of its target genes. It was shown recently that p53 is acetylated by transcriptional coactivators p300, CREB bidning protein (CBP), and PCAF and that acetylation of p53 by these proteins enhances p53 sequence-specific DNA binding. Here we show that the E1B 55-kDa protein specifically inhibits p53 acetylation by PCAF in vivo and in vitro, while acetylation of histones and PCAF autoacetylation is not affected. Furthermore, the DNA-binding activity of p53 is diminished in cells expressing the E1B 55-kDa protein. PCAF binds to the E1B 55-kDa protein and to a region near the C terminus of p53 encompassing Lys-320, the specific PCAF acetylation site. We further show that the E1B 55-kDa protein interferes with the physical interaction between PCAF and p53, suggesting that the E1B 55-kDa protein inhibits PCAF acetylase function on p53 by preventing enzyme-substrate interaction. These results underscore the importance of p53 acetylation for its function and suggest that inhibition of p53 acetylation by viral oncoproteins prevent its activation, thereby contributing to viral transformation.

Acetylation↗

PubMed2000

Deficiency of PTEN in Jurkat T cells causes constitutive localization of Itk to the plasma membrane and hyperresponsiveness to CD3 stimulation.

Pleckstrin homology (PH) domain binding to D3-phosphorylated phosphatidylinositides (PI) provides a reversible means of recruiting proteins to the plasma membrane, with the resultant change in subcellular localization playing a key role in the activation of multiple intracellular signaling pathways. Previously we found that the T-cell-specific PH domain-containing kinase Itk is constitutively membrane associated in Jurkat T cells. This distribution was unexpected given that the closely related B-cell kinase, Btk, is almost exclusively cytosolic. In addition to constitutive membrane association of Itk, unstimulated JTAg T cells also exhibited constitutive phosphorylation of Akt on Ser-473, an indication of elevated basal levels of the phosphatidylinositol 3-kinase (PI3K) products PI-3,4-P(2) and PI-3,4,5-P(3) in the plasma membrane. Here we describe a defect in expression of the D3 phosphoinositide phosphatase, PTEN, in Jurkat and JTAg T cells that leads to unregulated PH domain interactions with the plasma membrane. Inhibition of D3 phosphorylation by PI3K inhibitors, or by expression of PTEN, blocked constitutive phosphorylation of Akt on Ser-473 and caused Itk to redistribute to the cytosol. The PTEN-deficient cells were also hyperresponsive to T-cell receptor (TCR) stimulation, as measured by Itk kinase activity, tyrosine phosphorylation of phospholipase C-gamma1, and activation of Erk compared to those in PTEN-replete cells. These data support the idea that PH domain-mediated association with the plasma membrane is required for Itk activation, provide evidence for a negative regulatory role of PTEN in TCR stimulation, and suggest that signaling models based on results from Jurkat T-cell lines may underestimate the role of PI3K in TCR signaling.

Animals↗

PubMed2000

Telomerase-associated protein TEP1 is not essential for telomerase activity or telomere length maintenance in vivo.

TEP1 is a mammalian telomerase-associated protein with similarity to the Tetrahymena telomerase protein p80. Like p80, TEP1 is associated with telomerase activity and the telomerase reverse transcriptase, and it specifically interacts with the telomerase RNA. To determine the role of mTep1 in telomerase function in vivo, we generated mouse embryonic stem (ES) cells and mice lacking mTep1. The mTep1-deficient (mTep1(-/-)) mice were viable and were bred for seven successive generations with no obvious phenotypic abnormalities. All murine tissues from mTep1(-/-) mice possessed a level of telomerase activity comparable to that in wild-type mice. In addition, analysis of several tissues that normally lack telomerase activity revealed no reactivation of telomerase activity in mTep1(-/-) mice. Telomere length, even in later generations of mTep1(-/-) mice, was equivalent to that in wild-type animals. ES cells deficient in mTep1 also showed no detectable alteration in telomerase activity or telomere length with increased passage in culture. Thus, mTep1 appears to be completely dispensable for telomerase function in vivo. Recently, TEP1 has been identified within a second ribonucleoprotein (RNP) complex, the vault particle. TEP1 can also specifically bind to a small RNA, vRNA, which is associated with the vault particle and is unrelated in sequence to mammalian telomerase RNA. These results reveal that TEP1 is an RNA binding protein that is not restricted to the telomerase complex and that TEP1 plays a redundant role in the assembly or localization of the telomerase RNP in vivo.

Animals↗

PubMed2000

HuR regulates p21 mRNA stabilization by UV light.

Expression of the cyclin-dependent kinase inhibitor p21 is highly induced by many stresses, including exposure to short-wavelength UV light (UVC), which increases p21 mRNA stability. Investigation into the mechanisms underlying this stabilization process revealed that proteins present in cytoplasmic lysates of human RKO colorectal carcinoma cells formed complexes with p21 mRNA that were inducible by treatment with UVC and other stress agents. The ubiquitous Elav-type RNA-binding protein HuR was identified within the p21 mRNA-protein complexes, as antibodies recognizing HuR supershifted these complexes and revealed HuR-immunoreactive proteins complexing with p21 mRNA on Western blots. Lowering of endogenous HuR levels through expression of antisense HuR decreased p21 RNA-protein complexes, greatly reduced the UVC inducibility and half-life of p21 mRNA, and prevented UVC-mediated induction of luciferase activity in p21 3' untranslated region-containing reporter constructs. Our findings indicate that HuR plays a major role in regulating stress-induced p21 expression by enhancing p21 mRNA stability and that these effects are coupled to HuR's elevated presence in the cytoplasm.

Antigens, Surface↗

PubMed2000

Promoter-proximal pausing on the hsp70 promoter in Drosophila melanogaster depends on the upstream regulator.

RNA polymerase II pauses in the promoter-proximal region of many genes during transcription. In the case of the hsp70 promoter from Drosophila melanogaster, this pause is long-lived and occurs even when the gene is not induced. Paused polymerase escapes during heat shock when the transcriptional activator heat shock factor associates with the promoter. However, pausing is still evident, especially when induction is at an intermediate level. Yeast Gal4 protein (Gal4p) will induce transcription of the hsp70 promoter in Drosophila when binding sites for Gal4p are positioned upstream from the hsp70 TATA element. To further our understanding of promoter-proximal pausing, we have analyzed the effect of Gal4p on promoter-proximal pausing in salivary glands of Drosophila larvae. Using permanganate genomic footprinting, we observed that various levels of Gal4p induction resulted in an even distribution of RNA polymerase throughout the first 76 nucleotides of the transcribed region. In contrast, promoter-proximal pausing still occurs on endogenous and transgenic hsp70 promoters in salivary glands when these promoters are induced by heat shock. We also determined that mutations introduced into the region where the polymerase pauses do not inhibit pausing in a cell-free system. Taken together, these results indicate that promoter-proximal pausing is dictated by the regulatory proteins interacting upstream from the core promoter region.

Animals↗

PubMed2000

Seeds induced to germinate rapidly by mentally projected 'qi energy' are apparently genetically altered.

Mentally controlled qi energy can induce crop seeds to sprout and root for several cm within about 20 min. The RAPD method was used to compare treated groups of wheat and pea seeds and their controls using 11 selected primers. Seven primers amplified polymorphisms in wheat seeds and 5 in pea seeds. It was thought preliminarily that qi energy changed the structure of a germination-correlated gene site speeding up expression and advancing it in time.

Germination↗

PubMed2000

Unnatural ligands for engineered proteins: new tools for chemical genetics.

Small molecules that modulate the activity of biological signaling molecules can be powerful probes of signal transduction pathways. Highly specific molecules with high affinity are difficult to identify because of the conserved nature of many protein active sites. A newly developed approach to discovery of such small molecules that relies on protein engineering and chemical synthesis has yielded powerful tools for the study of a wide variety of proteins involved in signal transduction (G-proteins, protein kinases, 7-transmembrane receptors, nuclear hormone receptors, and others). Such chemical genetic tools combine the advantages of traditional genetics and the unparalleled temporal control over protein function afforded by small molecule inhibitors/activators that act at diffusion controlled rates with targets.

Alleles↗

PubMed2000

Subsequent pulmonary embolism: risk after a negative helical CT pulmonary angiogram--prospective comparison with scintigraphy.

PURPOSE: To determine whether a helical computed tomographic (CT) scan that is negative for pulmonary embolism (PE) is a sufficiently reliable criterion to safely withhold anticoagulation therapy. MATERIALS AND METHODS: Patients with negative helical CT scans were prospectively compared with patients with negative or low-probability scintigrams. In a 460-bed university hospital and clinic, 1,015 adult patients underwent either scintigraphy or helical CT for possible PE for 25 months. Five hundred forty-eight patients who had negative images and were not receiving anticoagulation therapy were prospectively followed up for 3 months for clinical, new imaging, death certificate, or autopsy evidence of subsequent PE. Ninety-seven patients were lost to follow-up. RESULTS: Subsequent PE was found in two (1.0%) of 198 patients with negative CT scans, none of 188 patients with negative ventilation-perfusion (V-P) scans, and five (3.1%) of 162 patients with low-probability V-P scans (not statistically significant). Patients in the helical CT group were hospitalized more often, had more severe disease, had more substantial PE risk factors, and had a higher death rate. No deaths were attributed to PE in either group. CONCLUSION: The frequency of clinical diagnoses of PE after a negative CT scan was low and similar to that after a negative or low-probability V-P scan. Helical CT is a reliable imaging tool for excluding clinically important PE.

Adult↗

PubMed2000

CT angiographic measurement of the carotid artery: optimizing visualization by manipulating window and level settings and contrast material attenuation.

PURPOSE: To evaluate a broad range of window and level settings for various contrast material attenuation coefficients and degrees of vascular stenosis to obtain the most accurate computed tomographic (CT) angiographic measurements. MATERIALS AND METHODS: A total of 25, 480 measurements were made transversely (perpendicular to the lumen) and by means of maximum intensity projection (MIP) in a phantom with stenoses of 0%-100%, contrast material with attenuation coefficients of 150-350 HU, and 14 window and 13 level settings. Edge definition was also evaluated. RESULTS: There was an inherent relationship between the contrast material attenuation coefficient and the optimal window and level settings in the measurement of stenoses at both transverse and MIP CT angiography. This relationship between the contrast material attenuation coefficient D: and the optimal settings for window W: and level L: was represented by the following simple equations: W:/D: = [-2 x (L:/D:)] + 1.3, where -0.2 < L:/D: < 0.5, and W:/D: = [3.3 x (L:/D:)] - 1.3, where 0.5 < L:/D: < 1.0. With a vascular contrast material attenuation coefficient of 250-350 HU, the best transverse and MIP display settings for the window and level were 96 and 150 HU, respectively. CONCLUSION: The use of optimized window and level settings at CT angiography reduces measurement variability.

Carotid Arteries↗

PubMed2000

Combined perfusion- and diffusion-weighted MR imaging in acute ischemic stroke during the 1st week: a longitudinal study.

PURPOSE: To compare findings with different magnetic resonance (MR) perfusion maps in acute ischemic stroke. MATERIALS AND METHODS: Combined diffusion-weighted (DW) and perfusion-weighted (PW) MR imaging was performed in 49 patients with acute (<24 hours) stroke, on the 1st and 2nd days and 1 week after stroke. Volumes of hypoperfused tissue on maps of relative cerebral blood volume (rCBV), relative cerebral blood flow (rCBF), and mean transit time (MTT) were compared with the volume of infarcted tissue at DW imaging. RESULTS: The mean infarct volume increased from 41 to 65 cm(3) between the 1st and 2nd days (P: <.001; n = 49). On the 1st day, all perfusion maps on average showed hypoperfusion lesions larger than the infarct at DW imaging (P: <.001; n = 49). MTT maps showed significantly (P: <.001) larger hypoperfusion lesions than did rCBF maps, which showed significantly (P: <.001) larger hypoperfusion lesions than did rCBV maps. The sizes of the initial perfusion-diffusion mismatches correlated significantly with the extent of infarct growth (0.479 < r < 0.657; P: </=.001). The hypoperfusion volume on the initial rCBV maps correlated best with the final infarct size at 1 week (r = 0.891; P: <.001). CONCLUSION: Combined DW and PW imaging is a powerful tool in evaluating the hemodynamics of acute ischemic stroke.

Acute Disease↗

PubMed2000

Human coronary arteriolar dilation to adrenomedullin: role of nitric oxide and K(+) channels.

Adrenomedullin (ADM) is a vasodilator produced by vascular endothelium and smooth muscle cells. Although plasma ADM levels are increased in patients with hypertension, heart failure, and myocardial infarction, little information exists regarding the microvascular response to ADM in the human heart. In the present study we tested the hypothesis that ADM produces coronary arteriolar dilation in humans and examined the mechanism of this dilation. Human coronary arterioles were dissected and cannulated with micropipettes. Internal diameter was measured by video microscopy. In vessels constricted with ACh, the diameter response to cumulative doses of ADM (10(-12)-10(-7) M) was measured in the presence and absence of human ADM-(22-52), calcitonin gene-related peptide-(8-37), N(omega)-nitro-L-arginine methyl ester (L-NAME), indomethacin (Indo), (1)H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one, SQ-22536, or KCl (60 mM). ADM dilated human coronary arterioles through specific ADM receptors (maximum dilation = 69 +/- 11%). L-NAME or N-monomethyl-L-arginine attenuated dilation to ADM (for L-NAME, maximum dilation = 66 +/- 7 vs. 41 +/- 13%, P < 0.05). Thus the mechanism of ADM-induced dilation involves generation of nitric oxide. However, neither (1)H-[1,2,4]oxadiazolo-[4, 3-a]quinoxalin-1-one, SQ-22536, nor Indo alone altered dilation to ADM. High concentrations of KCl blocked dilation to ADM. The magnitude of ADM dilation was reduced in subjects with hypertension. We propose that, in human coronary arterioles, ADM elicits vasodilation in part through production of nitric oxide and in part through activation of K(+) channels, with little contribution from adenylyl cyclase. The former dilator mechanism is independent of the more traditional pathway involving activation of soluble guanylate cyclase.

Adenine↗

PubMed2000

The human inward rectifier K(+) channel subunit kir5.1 (KCNJ16) maps to chromosome 17q25 and is expressed in kidney and pancreas.

A novel human Kir5.1 (inward rectifier K+ channel subunit, gene name KCNJ16) was identified through database searches. This human KCNJ16 was mapped to chromosome 17q25. The full-length cDNA was identified and its genomic structure was determined. Tissue distribution studies showed that human KCNJ16 is significantly expressed in human kidney, pancreas and thyroid gland. In situ hybridization revealed expression in convoluted tubule cells of kidney and in the acinar and ductal cells of pancreas. These suggest that human Kir5.1 may be involved in the regulation of fluid and pH balance, thus making it a potential therapeutic target for hypertension, renal failure, or pancreatic disease.

Amino Acid Sequence↗

PubMed2000

Modulation of early immune responses and suppression of Trypanosoma brucei brucei infections by surgical denervation of the spleen.

OBJECTIVE: To examine critical interactions between the nervous system and the immune system during experimental African trypanosomiasis. METHODS AND RESULTS: Inoculation of Trypanosoma brucei brucei resulted in early interferon (IFN)-gamma production, elevated corticosterone and prostaglandin E(2) (PGE(2)) levels and increased splenocyte proliferation, as measured by enzyme-linked immunospot assay, radioimmunoassay and thymidine incorporation assay, respectively. Splenic denervation suppressed IFN-gamma, corticosterone and PGE(2) production, enhanced splenocyte proliferation, and significantly reduced parasitemia and prolonged rat survival. CONCLUSIONS: Our data show substantial effects of the nervous system on early immune responses that may influence the outcome of this disease. These effects were not dependent on cytokine inhibitory mediators such as prostaglandins or stress hormones. More investigations are required to understand the evident neural control over the immune system during infectious challenges, which may assist in novel therapeutic approaches.

Animals↗

PubMed2000

Analysis of related factors in complications of stereotactic radiosurgery in intracranial tumors.

OBJECT: To investigate the related factors in complications of stereotactic radiosurgery for intracranial tumors. METHODS: A retrospective review of 146 patients with intracranial tumors treated with stereotactic radiosurgery was conducted. Sixty-five patients received single-dose treatment and the rest received fractionated stereotactic radiosurgery. Ninety-five patients received conventional radiotherapy in the meantime. RESULTS: Follow-up period was 18-54 months. Follow-up rate was 92.5% and 39 patients (26.7%) had different complications. The Cox statistics showed that target volume, target peripheral dose, target maximal dose, and ratio of maximal dose to peripheral dose are related to the complications. Conversely, neither type of tumor disease, gender, radiation schedule with or without conventional radiotherapy, target minimal dose, nor ratio of target peripheral isodose volume to target volume were found to be related to complications. CONCLUSION: Target volume and dose are the major factors causing complications, and the optimization of the therapeutic planning can play a significant role in reducing them.

Brain Edema↗

PubMed2000

Edg-1, the G protein-coupled receptor for sphingosine-1-phosphate, is essential for vascular maturation.

Sphingolipid signaling pathways have been implicated in many critical cellular events. Sphingosine-1-phosphate (SPP), a sphingolipid metabolite found in high concentrations in platelets and blood, stimulates members of the endothelial differentiation gene (Edg) family of G protein-coupled receptors and triggers diverse effects, including cell growth, survival, migration, and morphogenesis. To determine the in vivo functions of the SPP/Edg signaling pathway, we disrupted the Edg1 gene in mice. Edg1(-/-) mice exhibited embryonic hemorrhage leading to intrauterine death between E12.5 and E14.5. Vasculogenesis and angiogenesis appeared normal in the mutant embryos. However, vascular maturation was incomplete due to a deficiency of vascular smooth muscle cells/pericytes. We also show that Edg-1 mediates an SPP-induced migration response that is defective in mutant cells due to an inability to activate the small GTPase, Rac. Our data reveal Edg-1 to be the first G protein-coupled receptor required for blood vessel formation and show that sphingolipid signaling is essential during mammalian development.

Animals↗

PubMed2000

Delayed catabolism of apoB-48 lipoproteins due to decreased heparan sulfate proteoglycan production in diabetic mice.

We used wild-type (WT) mice and mice engineered to express either apoB-100 only (B100 mice) or apoB-48 only (B48 mice) to examine the effects of streptozotocin-induced diabetes (DM) on apoB-100- and apoB-48-containing lipoproteins. Plasma lipids increased with DM in WT mice, and fat tolerance was markedly impaired. Lipoprotein profiles showed increased levels and cholesterol enrichment of VLDL in diabetic B48 mice but not in B100 mice. C apolipoproteins, in particular apoC-I in VLDL, were increased. To investigate the basis of the increase in apoB-48 lipoproteins in streptozotocin-treated animals, we characterized several parameters of lipoprotein metabolism. Triglyceride and apoB production rates were normal, as were plasma lipase activity, VLDL glycosaminoglycan binding, and VLDL lipolysis. However, beta-VLDL clearance decreased due to decreased trapping by the liver. Whereas LRP activity was normal, livers from treated mice incorporated significantly less sulfate into heparan sulfate proteoglycans (HSPG) than did controls. Hepatoma (HepG2) cells and endothelial cells cultured in high glucose also showed decreased sulfate and glucosamine incorporation into HSPG. Western blots of livers from diabetic mice showed a decrease in the HSPG core protein, perlecan. Delayed clearance of postprandial apoB-48-containing lipoproteins in DM appears to be due to decreased hepatic perlecan HSPG.

Animals↗

PubMed2000

The heat-stable antigen determines pathogenicity of self-reactive T cells in experimental autoimmune encephalomyelitis.

Induction of myelin-specific CD4 T cells is a pivotal event in the development of experimental autoimmune encephalomyelitis (EAE). Other checkpoints in EAE pathogenesis have not been clearly defined, although multiple genetic loci are known to influence EAE development. We report here that targeted mutation of the heat-stable antigen (HSA) abrogates development of EAE despite a complete lack of effect on induction of autoimmune T cells. To test whether T-cell expression of HSA is sufficient, we created transgenic mice in which HSA is expressed exclusively in the T-cell lineage. We found that these mice remain resistant to EAE induction. Adoptive transfer studies demonstrate that both T cells and non-T cells must express HSA in order for the pathogenic T cells to execute their effector function. Moreover, HSAIg, a fusion protein consisting of the extracellular domain of the HSA and the Fc portion of immunoglobulin, drastically ameliorates the clinical sign of EAE even when administrated after self-reactive T cells had been expanded. Thus, identification of HSA as a novel checkpoint, even after activation and expansion of self-reactive T cells, provides a novel approach for immunotherapy of autoimmune neurologic diseases, such as multiple sclerosis.

Adoptive Transfer↗