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Biomedical subjects

B Liu

Publications and source records attributed to B Liu.

At least 307 records · Page 17Linked to original sources
PubMed1999

Generation of tumor-specific cytotoxic T lymphocytes by stimulation with HPV type 16 E7 peptide-pulsed dendritic cells: an approach to immunotherapy of cervical cancer.

OBJECTIVE: The aim of this study was to generate HPV-16 E7 peptide-specific cytotoxic T lymphocytes (CTLs) in vitro for future adoptive immunotherapy of cervical cancer. METHODS: Peripheral blood mononuclear cells (PBMC) were isolated from HLA-A2+ healthy donors. The PBMCs were incubated with HPV-16 E7(11-20) peptide and varying cytokines in the primary culture. Restimulation was performed weekly with peptide-pulsed, irradiated autologous PBMCs. Alternatively, the PBMCs were depleted of abundant CD4+ cells and stimulated with HPV-16 E7(11-20) peptide-pulsed dendritic cells. Cytolytic activity was determined by a standard 4-h (51)Cr-release assay. RESULTS: After 6 weeks in culture, we were able to establish peptide-specific CTL lines in one of seven donors by incubating PBMCs with HPV-16 E7(11-20) peptide. When we employed autologous peptide-pulsed dendritic cells to stimulate CD8+ cell-enriched PBMCs, we obtained CTL lines in four of seven donors. The primed CTLs were able to lyse the HLA-A2+ and HPV-16+ cervical cancer cell line Caski. SiHa, an HLA-A2-, but HPV 16+, cervical cancer cell line could be lysed only after transfection with HLA-A2. In addition, a high cytotoxicity (>80%) was obtained against peptide-pulsed, but not unpulsed, targets such as autologous Ebstein-Barr virus-immortalized B cells or allogeneic lipopolysaccaride-stimulated PBMCs. DCs were clearly the most potent of all tested antigen presenting cells to stimulate a CTL response in a proliferation assay. CONCLUSION: HPV-16 E7 peptide-specific CTLs could be generated in vitro. A practical protocol to expand the CTLs to a sufficient number for an application in a clinical trial is in progress.

Dendritic Cells↗
PubMed1999

P21WAF1/Cip1 gene expression in primary human hepatocellular carcinoma and its relationship with P53 gene mutation.

P21WAF1/Cip1, an inhibitor of cyclin-dependent kinases, is a critical downstream effector in the P53-specific pathway of growth control. Increased expression of P21WAF1/Cip1 has been found to reflect the status of the P53 tumor-suppressor pathway. We investigated the expression of P21WAF1/Cip1 in a relatively small, but well-characterized group consisting of 28 hepatocellular carcinomas. The samples were previously studied for P53 gene mutation. P21WAF1/Cip1 expression were identified by in situ hybridization and immunohistochemistry. Positive ISH for P21WAF1/Cip1 transcripts was found in 18 of 28 cases (64.3%). All positive cases by ISH showed detectable P21WAF1/Cip1 protein reactivity by IHC. No relationship was found between P21WAF1/Cip1 staining and P53 mutational status. No associations were seen with tumor metastasis, size and tumor grade, except for tumor differentiation status which showed higher frequency of P21WAF1/Cip1 expression in moderate-well differentiated HCCs than poorly differentiated tumors (P < 0.05). It is concluded that expression of P21WAF1/Cip1 is common in HCCs, but does not correlate with P53 mutational status or pathological parameters investigated except for tumor differentiation. Also, there may be other factors beside P53 that regulate P21WAF1/Cip1 gene expression in HCCs.

Adult↗
PubMed1999

Molecular characterization of a new excitatory insect neurotoxin with an analgesic effect on mice from the scorpion Buthus martensi Karsch.

Besides the neurotoxins active on mammals, a new excitatory insect selective toxin with a mice analgesic activity was found and purified from the venom of the scorpion Buthus martensi Karsch (BmK) (Ji, Y.H., Mansuelle, P., Terakawa, S., Kopeyan, C., Yanaihara, N., Hsu, K., Rochat, H., 1996. Toxicon 34, 987; Luo, M.J., Xiong, Y.M., Wang, M., Wang, D.C., Chi, C.W., 1997. Toxicon 35, 723.). This peptide (designated as BmK IT-AP) is composed of 72 amino acid residues. Its primary structure was determined by automated Edman degradation of the N-terminal part of the reduced and S-carboxamidemethylated protein and its lysylendopeptidase degraded fragments. Based on the determined sequence, the gene specific primers were designed and synthesized for 3' and 5' RACE (rapid amplification of cDNA ends). Their partial cDNA fragments obtained by 3' and 5' RACEwere cloned and sequenced and the full length cDNA sequence of BmK IT-AP was then completed by overlapping their two partial cDNA sequences. It encodes a precursor of 90 amino acid residues: a signal peptide of 18 residues and a mature peptide of 72 residues which are consistent with the determined protein sequence of BmK IT-AP. The genomic DNA of the peptide was also amplified by PCR from the scorpion genomic DNA and sequenced, which is a first report on the genomic structure of a scorpion toxin specific for insects. Its sequence revealed an intron of 590 bp inserted in the end part of the signal peptide. The peptide caused a fast excitatory contraction paralysis on house fly larvae. Furthermore, the peptide also showed an obvious analgesic effect on mice, as assayed by using a twisting test model. This effect of BmK IT-AP well characterized at molecular level is first reported among the known scorpion insect neurotoxins.

Amino Acid Sequence↗
PubMed1999

Spectrogram enhancement algorithm: a soft thresholding-based approach.

Enhancing the spectrogram by denoising the Doppler ultrasound signal is a preliminary step, and important for further processing. Because the spectrogram may be based on the short-time fast Fourier transform (FFT) of the Doppler ultrasound signal, whose power spectrum density is time-varying, traditional denoising algorithms that simply optimize the mean-squared error are not appropriate, and they may exhibit considerable undesirable, noise-induced frequency components. A soft thresholding-based denoising algorithm is put forward in this paper, that achieves almost the minimax mean square error (MSE) over a wide range of function classes having norms measuring smoothness (i.e., it meets both the requirement of smoothness and MSE). Due to the importance of noise level estimation while applying this method, several robust L-estimators are compared and the median absolute deviation (MAD) method is chosen to estimate the noise level. The simulation study shows better performance of the later algorithm under various quantification measures, compared to the FFT thresholding and the hard thresholding wavelet method, and the results of clinical data also confirm it.

Algorithms↗
PubMed1999

Structure of the human gene for lysosomal di-N-acetylchitobiase.

Chitobiase is a lysosomal glycosidase that acts during the ordered degradation of asparagine-linked glycoproteins to cleave the core chitobiose unit at its reducing end. Human chitobiase is expressed in significant amounts, while bovine chitobiase is produced at extremely low levels. To begin to understand this species-dependent expression, we determined the gene structure of human chitobiase. The human chitobiase gene ( CTB S) is approximately 20 kb comprising seven exons varying from 0.1 to 2.3 kb and six introns of 0.3 to 8 kb. The previously characterized partial bovine chitobiase gene structure is similarly organized including exon and intron sizes and locations, but the human and bovine 5'-flanking regions differ significantly. 5'-RACE analysis of human chitobiase cDNA revealed only one transcriptional start site 45 bp upstream of the ATG translation initiation site. Computer analysis of the human chitobiase gene 5'-flanking region shows characteristics of a typical housekeeping gene. The putative promoter region contains a distal TATA box, and there are several Sp-1 and AP-2 cis elements. In contrast, bovine chitobiase gene 5'-flanking region shows totally different structures and may contain several silencers. A partial art-2 segment which is an artiodactyl Alu -like repetitive sequence, is also present. These evolutionary differences in the 5'-flanking region of the chitobiase genes from human and bovine could account for the widely varied expression levels of the hydrolase within these two species.

Amino Acid Sequence↗
PubMed1999

Engineering a soluble extracellular erythropoietin receptor (EPObp) in Pichia pastoris to eliminate microheterogeneity, and its complex with erythropoietin.

The extracellular ligand-binding domain (EPObp) of the human EPO receptor (EPOR) was expressed both in CHO (Chinese Hamster Ovary) cells and in Pichia pastoris. The CHO and yeast expressed receptors showed identical affinity for EPO binding. Expression levels in P. pastoris were significantly higher, favoring its use as an expression and scale-up production system. Incubation of EPO with a fourfold molar excess of receptor at high protein concentrations yielded stable EPO-EPObp complexes. Quantification of EPO and EPObp in the complex yielded a molar ratio of one EPO molecule to two receptor molecules. Residues that are responsible for EPOR glycosylation and isomerization in Pichia were identified and eliminated by site-specific mutagenesis. A thiol modification was identified and a method was developed to remove the modified species from EPObp. EPObp was complexed with erythropoietin (EPO) and purified. The complex crystallized in two crystal forms that diffracted to 2.8 and 1.9 A respectively. (Form 1 and form 2 crystals were independently obtained at AxyS Pharmaceuticals, Inc. and Amgen, Inc. respectively.) Both contained one complex per asymmetric unit with a stoichiometry of two EPObps to one EPO.

Animals↗
PubMed1999

Colorectal cancer and the Muir-Torre syndrome in a Gypsy family: a review.

OBJECTIVES: The Muir-Torre syndrome (MTS) is characterized by an autosomal dominant predilection to sebaceous adenomas, sebaceous carcinomas, and multiple keratoacanthomas, in concert with the cancer phenotype of hereditary nonpolyposis colorectal cancer (HNPCC). Proof that patients showing a familial aggregation of MTS's cutaneous signs in combination with a specific pattern of visceral cancers which are consonant with an HNPCC diagnosis has been buttressed by the discovery of hMSH2 and hMLH1 germ-line mutations in such families. Our purpose in this investigation was to determine the germ-line mutation in a Gypsy family with MTS in concert with HNPCC cancer features, and to provide genetic counseling. An added objective for this paper is to review the literature on MTS. METHODS: We describe a Gypsy family with MTS in concert with HNPCC cancer features, as well as the molecular genetic and genetic counseling procedures used in the interest of improved compliance with cancer control recommendations. We review the clinical phenotype, natural history, and molecular genetics involved in the MTS variant HNPCC. RESULTS: An hMSH2 germ-line mutation was identified as the culprit germ-line mutation in this family. CONCLUSIONS: The presence of the hMSH2 germ-line mutation in this family provides powerful predictability of colorectal and other HNPCC integral cancers. The gastroenterologist must assume an important role in the diagnosis and management of MTS.

Adult↗
PubMed1999

Interaction of the human immunodeficiency virus type 1 nucleocapsid with actin.

The nucleocapsid (NC) domain of the retrovirus Gag protein plays several important roles in the viral life cycle, including virus assembly, viral genomic RNA encapsidation, primer tRNA placement, and enhancement of viral reverse transcription. In this study, deletion of NC domain of human immunodeficiency virus type 1 (HIV-1) Gag was found to drastically reduce virus particle production in CD4(+) T cells. Cellular fractionation experiments showed that although most of the uncleaved wild-type HIV-1 Gag, unmyristylated Gag, and p6(Gag) domain-truncated Gag molecules copurified with the host cell cytoskeleton, most of the mutant Gag molecules lacking both the NC and p6(Gag) domains failed to cofractionate with cytoskeleton. In wild-type virus-infected cells, in which the viral protease was active, the cleaved NCp7 copurified with the cytoskeleton, whereas most of the MAp17 and CAp24 did not. Monoclonal antibody against actin coimmunoprecipitated full-length Gag and p6(Gag) domain-truncated Gag molecules from cell lysates but failed to precipitate the truncated mutant Gag molecules lacking NC plus p6(Gag). Purified recombinant NCp7, but not CAp24, was able to bind F-actin in cosedimentation experiments. Furthermore, wild-type NCp7 and a zinc finger mutant NCp7(F16A), like a cellular actin-binding protein (the villin headpiece), bound F-actin in a dose-dependent fashion in vitro. Taken together, these results suggest that HIV-1 NCp7 can bind F-actin directly and that interaction between HIV-1 Gag and the actin cytoskeleton through the NC domain may play an important role in HIV-1 assembly and/or other steps of the viral life cycle.

Actins↗
PubMed1999

Formation of virus assembly intermediate complexes in the cytoplasm by wild-type and assembly-defective mutant human immunodeficiency virus type 1 and their association with membranes.

We have previously identified two distinct forms of putative viral assembly intermediate complexes, a detergent-resistant complex (DRC) and a detergent-sensitive complex (DSC), in human immunodeficiency virus type 1 (HIV-1)-infected CD4(+) T cells (Y. M. Lee and X. F. Yu, Virology 243:78-93, 1998). In the present study, the intracellular localization of these two viral assembly intermediate complexes was investigated by use of a newly developed method of subcellular fractionation. In wild-type HIV-1-infected H9 cells, the DRC fractionated with the soluble cytoplasmic fraction, whereas the DSC was associated with the membrane fraction. The DRC was also detected in the cytoplasmic fraction in H9 cells expressing HIV-1 Myr- mutant Gag. However, little of the unmyristylated Gag and Gag-Pol proteins was found in the membrane fraction. Furthermore, HIV-1 Gag proteins synthesized in vitro in a rabbit reticulocyte lysate system in the absence of exogenous lipid membrane were able to assemble into a viral Gag complex similar to that of the DRC identified in infected H9 cells. The density of the viral Gag complex was not altered by treatment with the nonionic detergent Triton X-100, suggesting a lack of association of this complex with endogenous lipid. Formation of the DRC was not significantly affected by mutations in assembly domains M and L of the Gag protein but was drastically inhibited by a mutation in the assembly I domain. Purified DRC could be disrupted by high-salt treatment, suggesting electrostatic interactions are important for stabilizing the DRC. The Gag precursor proteins in the DRC were more sensitive to trypsin digestion than those in the DSC. These findings suggest that HIV-1 Gag and Gag-Pol precursors assemble into DRC in the cytoplasm, a process which requires the protein-protein interaction domain (I) in NCp7; subsequently, the DRC is transported to the plasma membrane through a process mediated by the M domain of the matrix protein. It appears that during this process, a conformational change might occur in the DRC either before or after its association with the plasma membrane, and this change is followed by the detection of virus budding structure at the plasma membrane.

Animals↗
PubMed1999

FAST-2 is a mammalian winged-helix protein which mediates transforming growth factor beta signals.

The mechanisms by which transforming growth factor beta (TGF-beta) and related ligands regulate transcription remain poorly understood. The winged-helix (WH) transcription factor fork head activin signal transducer 1 (FAST-1) was identified as a mediator of activin signaling in Xenopus embryos (X. Chen, M. J. Rubock, and M. Whitman, Nature 383:691-696, 1996). We have cloned a novel WH gene from the mouse which shares many properties with FAST-1. We find that this gene, which we call FAST-2, is able to mediate transcriptional activation by TGF-beta. FAST-2 also interacts directly with Smad2, a cytoplasmic protein which is translocated to the nucleus in response to TGF-beta, and forms a multimeric complex with Smad2 and Smad4 on the activin response element, a high-affinity binding site for FAST-1. Analysis of the sequences of FAST-1 and FAST-2 reveals substantial protein sequence divergence compared to known vertebrate orthologs in the WH family. This suggests that FAST-2 represents a new WH gene related to FAST-1, which functions to mediate TGF-beta signals in mammals. We have also examined the structure of the FAST-2 gene and find that it overlaps with a kinesin motor protein gene. The genes are transcribed in opposite orientations, and their transcripts overlap in the 3' untranslated region.

Activins↗
PubMed1999

Identification of a novel dexamethasone-sensitive RNA-destabilizing region on rat monocyte chemoattractant protein 1 mRNA.

Glucocorticoids are potent anti-inflammatory agents widely used in the treatment of human disease. We have previously shown that the inflammatory cytokine monocyte chemoattractant protein 1 (MCP-1) is regulated posttranscriptionally by glucocorticoids in arterial smooth muscle cells (SMC). To elucidate the mechanism mediating this effect, in vitro-transcribed radiolabeled MCP-1 mRNA was incubated with cytoplasmic extracts from SMC and analyzed by gel electrophoresis. Extracts from SMC treated with platelet-derived growth factor (PDGF) did not degrade the transcripts for up to 3 h. In contrast, extracts from cells treated with 1 microM dexamethasone (Dex) alone or in combination with PDGF degraded the probe with a half-life of approximately 15 min. Dex had maximal effect at concentrations above 0.01 microM and was effective on both rat and human MCP-1 transcripts. By deletion analysis, the Dex-sensitive region of the MCP-1 mRNA was localized to the initial 224 nucleotides (nt) at the 5' end and did not involve an AU-rich sequence in the 3' untranslated end. The 224-nt region conferred Dex sensitivity to heterologous mRNA. These studies provide new insights into the molecular mechanisms underlying the effect of glucocorticoids on gene expression.

Animals↗
PubMed1999

TGF-beta1-mediated hypertrophy involves inhibiting pRB phosphorylation by blocking activation of cyclin E kinase.

When renal epithelial cells are exposed to epidermal growth factor-transforming growth factor-beta1 (EGF-TGF-beta1) the typical EGF-mediated hyperplastic growth response is converted to a hypertrophic growth response. Hypertrophy in this setting involves cell entrance into G(1), but arrest of cell cycle progression at the G(1)/S interface. Late G(1) arrest is mediated by retaining retinoblastoma protein (pRB) in its active, hypophosphorylated state. The present studies examine the mechanism by which pRB is retained in its active state. The results demonstrate that TGF-beta1-mediated conversion of hyperplasia to hypertrophy involves preventing activation of cdk2/cyclin E kinase but has no effect on cdk4(6)/cyclin D kinase activity. Preventing activation of cyclin E kinase is associated with 1) decreased abundance of cdk2/cyclin E complexes and 2) retention of p57(Kip2) in formed cdk2/cyclin E complexes. The development of hypertrophy does not involve regulation of either cdk2, cyclin E, or cdc25A protein abundances, or the abundance of p27(Kip1) or p21 in formed complexes.

Animals↗
PubMed1999

Uterine natural killer cells are targets for a trophoblast cell-specific cytokine, prolactin-like protein A.

PRL-like protein A (PLP-A) is a member of the PRL family expressed in trophoblast cells coincident with establishment of the chorioallantoic placenta. The purpose of this investigation was to identify targets for PLP-A. Using an alkaline phosphatase-tagging strategy, we show that PLP-A specifically interacts with a population of natural killer (NK) lymphocytes within the mesometrial compartment of decidua from pregnant and pseudopregnant rats. These observations are supported by the codistribution of PLP-A targets with cells expressing the rat NK cell surface marker, gp42, the absence of PLP-A binding in conceptuses from NK cell-deficient tg epsilon26 mice, and the specific interaction of PLP-A with a rat NK cell line, RNK-16. We have further demonstrated that PLP-A effectively suppresses RNK-16 cell cytolytic activities. Our results provide evidence for a new paradigm of embryonic-maternal communication involving a PLP-A signaling pathway between trophoblast cells and uterine NK lymphocytes.

Alkaline Phosphatase↗
PubMed1999

Clinical rejection is distinguished from subclinical rejection by increased infiltration by a population of activated macrophages.

It has been reported previously that one-third of protocol renal biopsies in asymptomatic, biochemically stable renal transplant recipients in the first 6 mo show unsuspected subclinical graft rejection (both infiltrate and tubulitis) and that subclinical rejection is a risk factor for chronic renal dysfunction. This study was performed to determine whether differences in phenotype or activation status of graft-infiltrating cells underlie these different manifestations of acute rejection. Biopsies with normal histology (n = 10), subclinical rejection (n = 13), and clinical rejection (n = 9) were studied using immunohistochemistry and computerized image analysis. Subclinical and clinical rejections had similar histologic Banff scores. Univariate analysis showed a trend for a higher infiltration with CD8+ (P = 0.053) and CD68+(P = 0.06) cells in clinical rejection. Of the activation markers studied (CD25, perforin, tumor necrosis factor-alpha), only allograft inflammatory factor-1+-activated macrophages were significantly (P = 0.014) increased in the infiltrate of clinical rejection biopsies. These data suggest that activated macrophages or their products are responsible for acute renal dysfunction associated with clinical rejection episodes.

Acute Disease↗
PubMed1999

The seroepidemiology of genogroup 1 and genogroup 2 Norwalk-like viruses in Italy.

Southampton virus (SV) and Lordsdale viruses (LV) are small round structured viruses characterised recently and belong to two separate genogroups. The capsid genes of these viruses were expressed in insect cells using recombinant baculoviruses. Both SV (genogroup 1) and LV (genogroup 2) capsid proteins self-assembled to form virus-like particles (VLPs). The VLPs were used in a standard enzyme-linked immunosorbent assay (ELISA) to screen for antibodies to SV and LV in 1,729 age-stratified human sera collected in Verona, Italy between January and November 1996. SV VLPs were labile compared with LV VLPs. There was a large difference in the prevalence of SV (28.7%) compared with LV (91.2%). However, presentation of SV VLPs using chicken egg yolk antibody-coated wells (IgY capture ELISA) with a subset of serum samples from patients (0-19 years) increased the number of positive sera significantly (50.5%), indicating that SV antigen integrity is an important factor in the assay. Recent reverse transcription-polymerase chain reaction (RT-PCR) studies have shown that LV is circulating currently and analysis of IgY capture ELISA data showed greater reactivity for LV than SV, reflecting a genuinely lower rate of recent infection by this genogroup 1 virus.

Adolescent↗
PubMed1999

[Study on telomerase inhibition by ribozyme targeted to telomerase RNA component].

OBJECTIVE: To evaluate the possibility of using ribozyme technology for telomerase inhibition and cancer therapy. METHODS: A hammer head ribozyme (telomerase ribozyme, teloRZ) directed against the RNA component of human telomerase (hTR) was designed and synthesized to serve as a telomerase inhibitor. An in vitro transcription plasmid and a eukaryotic expression plasmid containing teloRZ gene were constructed. In vitro cleavage reaction was carried out by mixing the ribozyme RNA with DIG-labeled-hTR in different reaction conditions. Cleavage bands were detected by digoxin chemilumines- cent assay. The eukaryotic expression plasmid was inducted into HeLa cells by lipofectamine; the telomerase activities and bio-characteristics of HeLa cells were detected continuously. RESULTS: teloRZ showed a specific cleavage activity against the telomerase RNA component used as template. The in vitro cleavage ratio reached about 60%. The telomerase activities of cells expressing teloRZ dropped to eight times; the doubling times became longer and apoptosis ratios became higher with increasing population doublings (PDS); at 19-20 PDS 95% cells showed apoptosis. CONCLUSION: These findings support the potential use of this ribozyme against immortalized cancer cells.

Apoptosis↗
PubMed1999

[Expression of MAGE-1 gene in hepatocellular carcinoma].

OBJECTIVE: To explore the possibility of tumor associated antigen encoded by MAGE-1 gene used as a target for immunotherapy in HCC patients. METHOD: The expression of MAGE-1 gene in 39 HCCs and 3 HCC cell lines and 5 non-HCC patients liver tissues was examined by RT-PCR method. RESULTS: The expression rate of MAGE-1 mRNA was significantly higher in HCC patients than in paratumor tissues (P < 0.01, 58.9% vs 10.3%). All the 3 HCC cell lines expressed MAGE-1 gene positively. All 5 non-HCC patients' liver tissues did not express MAGE-1 gene. CONCLUSION: The high frequency of expression of MAGE-1 gene in HCCs suggests the possibility of MAGE-1 used as a target for immunotherapy in HCC patients.

Adult↗