Electrochemical AFM "dip-pen" nanolithography.
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Letter↗
Biomedical subjects
J Liu
Publications and source records attributed to J Liu.
Structural and functional analysis of the HIV gp41 core containing an Ile573 to Thr substitution: implications for membrane fusion.
The envelope glycoprotein of HIV-1 consists of the surface subunit gp120 and the transmembrane subunit gp41. Binding of gp120 to target cell receptors induces a conformational change in gp41, which then mediates the fusion of viral and cellular membranes. A buried isoleucine (Ile573) in a central trimeric coiled coil within the fusion-active gp41 ectodomain core is thought to favor this conformational activation. The role of Ile573 in determining the structure and function of the gp120-gp41 complex was investigated by mutating this residue to threonine, a nonconservative substitution in HIV-1 that occurs naturally in SIV. While the introduction of Thr573 markedly destabilized the gp41 core, the three-dimensional structure of the mutant trimer of hairpins was very similar to that of the wild-type molecule. A new hydrogen-bonding interaction between the buried Thr573 and Thr569 residues appears to allow formation of the trimer-of-hairpins structure at physiological temperature. The mutant envelope glycoprotein expressed in 293T cells and incorporated within pseudotyped virions displayed only a moderate reduction in syncytium-inducing capacity and virus infectivity, respectively. Our results demonstrate that the proper folding of the gp41 core underlies the membrane fusion properties of the gp120-gp41 complex. An understanding of the gp41 activation process may suggest novel strategies for vaccine and antiviral drug development.
Amino Acid Sequence↗
X-ray absorption fine structure spectroscopy determination of the binding mechanism of tetrahedral anions to self assembled monolayers on mesoporous support.
X-ray absorption fine structure (XAFS) spectroscopy is used to investigate the chemical interaction between the end member [Cu(NH2)6] of self-assembled monolayers on mesoporous supports (SAMMS) and the tetrahedral anion SO4. The local structure about Cu indicates monodentate bonding between the SO4 anion and the SAMMS.
Journal Article↗
Up-Regulation of Bcl-2 in microvascular endothelial cells enhances intratumoral angiogenesis and accelerates tumor growth.
Vascular endothelial growth factor (VEGF) has been shown to be a potent mediator of angiogenesis that functions as a survival factor for endothelial cells by up-regulating Bcl-2 expression. We have recently reported that human dermal microvascular endothelial cells (HDMECs) seeded in biodegradable sponges and implanted into severe combined immunodeficient (SCID) mice organize into functional human microvessels that transport mouse blood cells. In this study, we implanted sponges seeded with OSCC-3 (oral squamous cell carcinoma) or SLK (Kaposi's sarcoma) together with endothelial cells into SCID mice to generate human tumors vascularized with human microvessels. This model system was used to examine the role of both endothelial cell Bcl-2 and the proangiogenic chemokine interleukin-8 (IL-8) on tumor growth and intratumoral microvascular density. Coimplantation of HDMECs overexpressing Bcl-2 (HDMEC-Bcl-2) and tumor cells resulted in a 3-fold enhancement of tumor growth when compared with the coimplantation of control HDMECs and tumor cells. This was associated with increased intratumoral microvascular density and enhanced endothelial cell survival. To determine whether the enhanced neovascularization mediated by Bcl-2 overexpression in endothelial cells was influenced by the synthesis of endogenous mediators of angiogenesis, we screened these cells for expression of VEGF, basic fibroblast growth factor (bFGF), and IL-8 by ELISA. HDMEC-Bcl-2 cells and VEGF-treated HDMECs exhibited a 15-fold and 4-fold increase, respectively, in the expression of the proangiogenic chemokine IL-8 in vitro, whereas the expression of VEGF and bFGF remained unchanged. Transfection of antisense Bcl-2 into HDMECs blocked VEGF-mediated induction of IL-8. Conditioned media from HDMEC-Bcl-2 induced proliferation and sprouting of endothelial cells in vitro and neovascularization in rat corneas. Anti-IL-8 antibody added to HDMEC-Bcl-2 conditioned media markedly reduced the potency of these responses. SCID mice bearing VEGF-producing tumor implants that were treated with anti-lL-8 antibody exhibited a 43% reduction in microvessel density and a 50% reduction in tumor weight compared with treatment with a nonspecific antibody. These results demonstrate that the up-regulation of Bcl-2 expression in endothelial cells that constitute tumor microvessels enhances intratumoral microvascular survival and density and accelerates tumor growth. Furthermore, endothelial cells that overexpress Bcl-2 have more angiogenic potential than control cells, and IL-8-neutralizing antibodies attenuate their angiogenic activity in vitro and in vivo.
Animals↗
Developmental changes of (3)H-labelled mu-opioid receptors in brainstems of intra-uterine growth-restricted rats.
The opioid mu-system is involved in brainstem-mediated respiratory control. Infants with intra-uterine growth restriction (IUGR) have more respiratory disorders in the early postnatal period. Using [(3)H]DAGO, a mu-selective ligand, and a computer-based image analysis of autoradiography, we compared the ontogeny and distribution of mu-opioid binding sites in the brainstem of IUGR and control rats in utero (E21), at birth (P0) and on postnatal days 1 (P1), P7, P10, P14 and P21. The ontogeny pattern was found to be similar in both groups. The density of the binding sites, which was low in E21, increased at P0, slightly declined at P1 and remained relatively constant thereafter. The distribution of DAGO-binding sites, also similar in both groups, was heterogeneous and was much denser in the dorsal areas of medulla and pons. In particular, binding sites were highly concentrated in nuclei involved in the cardio-respiratory function. However, DAGO-binding density was higher at all ages (except for P0 and P1) in IUGR than in control rats. Taken together, these results give at least a partial explanation for the effects of IUGR which lowers the Apgar score at birth and raises the incidence of respiratory disorders in infants.
Aging↗
Nitric oxide induces metallothionein (MT) gene expression apparently by displacing zinc bound to MT.
The metal binding protein metallothionein (MT) is involved in zinc homeostasis since it typically binds large amounts of zinc. Free zinc can control MT gene expression by interacting with metal-sensitive transcription factors. However, the precise factors governing intracellular release of metal ions from MT remain unknown. Aerobic nitric oxide (NO) can nitrosate thiol groups in proteins, and MT-bound cadmium is released by NO exposure. Thus, we hypothesized that NO may also be effective at displacing zinc from MT in cultured cells and that this could be an important physiological control mechanism in zinc homeostasis and utilization. In this study, DETA/NO, an agent that spontaneously generates NO with a 20-h half life in physiological media, was used to study the release of zinc from MT and the induction of MT in TRL1215 cells (a normal rat liver cell line). Zinc or cadmium was given at levels inducing MT production, followed by DETA/NO (20-200 microM) to produce controlled NO exposure in both cell lines. Although both metals activated MT gene expression, MT-I mRNA and MT protein were further increased when DETA/NO was given after zinc or cadmium treatment. Additionally, NO from DETA/NO clearly displaced MT-bound zinc, as evidenced by G-75 gel-filtration chromatography. The released zinc or cadmium probably then stimulates further MT gene expression. These results suggest that NO may play an important role in regulation of cellular zinc homeostasis by providing a controlled release mechanism for metal ions stored in MT, and NO-mediated release of MT-bound zinc could in turn activate gene expression, such as with the MT gene.
Animals↗
Analysis of a parametrically driven pendulum.
We study in this paper the behavior of a periodically driven nonlinear mechanical system. Bifurcation diagrams are found which locate regions of quasiperiodic, periodic, and chaotic behavior within the parameter space of the system. We also conduct a symbolic analysis of the model which demonstrates that the symbolic dynamics of two-dimensional maps can be applied effectively to the study of ordinary differential equations in order to gain global knowledge about them.
Journal Article↗
Effects of nitric oxide in the dorsal motor nucleus of the vagus on the extrahepatic biliary system in rabbits.
To investigate whether nitric oxide (NO) in the dorsal motor nucleus of the vagus (DMV) mediated an influence on the extrahepatic biliary system, we studied the effects of microinjection of NO-producing drugs into DMV on the motilities of the gallbladder (GB) and the sphincter of Oddi (SO) in anesthetized rabbits. Microinjection of the NO precursor L-arginine into the rostral DMV produced an increase in the GB and SO motilities, which can be counteracted by both NG-nitro-L-arginine-methyl (L-NAME), an inhibitor of NOS, and reduced hemoglobin (rHb), a scavenger of NO, and were eliminated by bilateral cervical vagotomy. On the other hand, the NO donor sodium nitroprusside (SNP) was able to mimic the excitatory effect of L-arginine. This effect can be antagonized by rHb, but not by L-NAME, for SNP supplied exogenous NO without activating NOS. These results indicate that NO in the DMV mediates an excitatory effect on the extrahepatic biliary system.
Animals↗
A spontaneous recurrent seizure-related Rattus NSF gene identified by linker capture subtraction.
Spontaneous recurrent seizures (SRS) are the major clinical characteristic of epilepsy. In this study, using a SRS-behavior test combined with linker capture subtraction (LCS) to identify genes altered in their expression in response to a single kainic acid (KA)-induced SRS at 3 weeks in the rat hippocampal formation. Dot blot analysis of the differentially expressed cDNA fragments with LCS showed the down-regulation of one cDNA related to SRS, which was designated epilepsy-related gene 1 (ERG1). Northern blot analysis showed that ERG1 mRNA was reduced by KA administration with and without SRS, but more so with SRS. This differential expression had also been confirmed by in situ hybridization, which showed that ERG1 mRNA was down-regulated in the dorsal dentate granule cells (dDGCs) of the hippocampal formation, but remarkable up-regulated in the amygdalohippocampal area (AHi), posteromedial cortical amygdaloid nucleus (PMCo) and perirhinal cortex (PRh). The complete cDNA of ERG1 was cloned, sequenced (AF142097). It encodes a Rattus homologue of N-ethylmaleimide-sensitive fusion protein (NSF), which is an ATPase that plays a key role in mediating docking and/or fusion of transport vesicles in the multi-step pathways of vesicular transport. Sequence analysis revealed that ERG1 has high sequence similarity with the cDNA of the Mus musculus suppressor of K(+) transport growth defect (SKD2), N-ethylmaleimide(NEM)-sensitive fusion protein of Chinese hamster and human NEM-sensitive factor (HSU03985).
Animals↗
A scintillation proximity assay for rna detection.
A homogeneous scintillation proximity assay (SPA) for detection of RNA transcripts is described. 3H-labeled RNA transcripts are hybridized in solution to biotinylated oligodeoxynucleotides (ODNs), which are then bound by streptavidin-coated, scintillant-embedded beads. Only bound 3H-labeled RNA transcripts are brought in close enough proximity to stimulate light emission from the beads. The results from this novel homogeneous assay correlated well with those obtained using the traditional filter-binding methods to measure RNA polymerase activity. The assay has been miniaturized to a 384-well format compatible with automated high-throughput screening. This SPA method has also been successfully used to probe RNA-accessible sites to hybridization, and thus should provide a useful tool for selecting effective antisense ODNs in antisense research.
DNA-Directed RNA Polymerases↗
High-resolution mapping of the blood pressure QTL on chromosome 7 using Dahl rat congenic strains.
It was previously shown using Dahl salt-sensitive (S) and salt-resistant (R) rats that a blood pressure quantitative trait locus (QTL) was present on rat chromosome 7. In the present work, this QTL was localized to a region less than 0.54 cM in size on the linkage map using a series of congenic strains. This region was contained in a single yeast artificial chromosome that was 220 kb long. This small segment still contained the primary candidate locus Cyp11b1 (11beta-hydroxylase), but the adjacent candidate genes Cyp11b2 (aldosterone synthase) and Cyp11b3 were ruled out. It is concluded that 11beta-hydroxylase, through its known genetic variants altering the production of 18-hydroxy-11-deoxy corticosterone, is very likely to account for the blood pressure QTL on chromosome 7 in the Dahl rat model of hypertension. This QTL accounts for about 23 mm Hg under the condition of 2% NaCl diet for 24 days.
Alleles↗
Defective interplay of activators and repressors with TFIH in xeroderma pigmentosum.
Inherited mutations of the TFIIH helicase subunits xeroderma pigmentosum (XP) B or XPD yield overlapping DNA repair and transcription syndromes. The high risk of cancer in these patients is not fully explained by the repair defect. The transcription defect is subtle and has proven more difficult to evaluate. Here, XPB and XPD mutations are shown to block transcription activation by the FUSE Binding Protein (FBP), a regulator of c-myc expression, and repression by the FBP Interacting Repressor (FIR). Through TFIIH, FBP facilitates transcription until promoter escape, whereas after initiation, FIR uses TFIIH to delay promoter escape. Mutations in TFIIH that impair regulation by FBP and FIR affect proper regulation of c-myc expression and have implications in the development of malignancy.
Blotting, Western↗
Erv41p and Erv46p: new components of COPII vesicles involved in transport between the ER and Golgi complex.
Proteins contained on purified COPII vesicles were analyzed by matrix-assisted laser desorption ionization mass spectrometry combined with database searching. We identified four known vesicle proteins (Erv14p, Bet1p, Emp24p, and Erv25p) and an additional nine species (Yip3p, Rer1p, Erp1p, Erp2p, Erv29p, Yif1p, Erv41p, Erv46p, and Emp47p) that had not been localized to ER vesicles. Using antibodies, we demonstrate that these proteins are selectively and efficiently packaged into COPII vesicles. Three of the newly identified vesicle proteins (Erv29p, Erv41p, and Erv46p) represent uncharacterized integral membrane proteins that are conserved across species. Erv41p and Erv46p were further characterized. These proteins colocalized to ER and Golgi membranes and exist in a detergent-soluble complex that was isolated by immunoprecipitation. Yeast strains lacking Erv41p and/or Erv46p are viable but display cold sensitivity. The expression levels of Erv41p and Erv46p are interdependent such that Erv46p was reduced in an erv41Delta strain, and Erv41p was not detected in an erv46Delta strain. When the erv41Delta or ev46Delta alleles were combined with other mutations in the early secretory pathway, altered growth phenotypes were observed in some of the double mutant strains. A cell-free assay that reproduces transport between the ER and Golgi indicates that deletion of the Erv41p-Erv46p complex influences the membrane fusion stage of transport.
COP-Coated Vesicles↗
Trehalose accumulation from soluble starch by Saccharomycopsis fibuligera sdu.
Trehalose accumulation from starch by Saccharomycopsis fibuligera sdu was examined in 300-ml shaken flask culture and Biostat B(2) 2-1 fermentation. In the 300-ml flask, 16.5% (w/w) trehalose accumulated in the yeast cells (cell dry weight) was observed with 100-ml medium shaken at 200 rpm for 50 h at 30 degrees C. We found that 1.0% soluble starch in the medium was most suitable for trehalose accumulation by this yeast strain. In the Biostat B(2) 2-1 fermentor, 18.0% (w/w) trehalose accumulated in the yeast cells (cell dry weight) was observed within 48 h of fermentation when agitation speed was 200 rpm. The trehalose obtained from the yeast cells was identical to standard trehalose from Sigma based on the analysis results of High-Performance Exchange Anionic Chromatography (HPEAC).
Journal Article↗
Triplex targeting of human PDGF-B (c-sis, proto-oncogene) promoter specifically inhibits factors binding and PDGF-B transcription.
Human c-sis/PDGF-B proto-oncogene has been shown to be overexpressed in a large percentage of human tumor cells establishing a growth-promoting, autocrine growth circuit. Triplex forming oligonucleotides (TFOs) can recognize and bind sequences in duplex DNA, and have received considerable attention because of their potential for targeting specific genomic sites. The c-sis/PDGF-B promoter contains a unique homopurine/homopyrimidine sequence (SIS proximal element, SPE), which is crucial for binding nuclear factors that provoke transcription. In order to develop specific transcriptional inhibitors of the human c-sis/PDGF-B proto-oncogene, 20 potential TFOs targeting part or all of the SPE were screened by gel mobility analysis. DNase I footprinting shows that the TFOs we designed can form a sequence-specific triplex with the target. Protein binding assays demonstrate that triplex formation inhibits nuclear factors binding the c-sis/PDGF-B promoter. Both transient and stable transfection experiments demonstrate that the transcriptional activity of the promoter is considerably inhibited by the TFOs. We propose that TFOs represent a therapeutic potential to specifically diminish the expression of c-sis/PDGF-B proto-oncogene in various pathologic settings where constitutive expression of this gene has been observed.
Base Sequence↗
Sino-MONICA project: a collaborative study on trends and determinants in cardiovascular diseases in China, Part i: morbidity and mortality monitoring.
BACKGROUND: The Sino-MONICA project is a 7-year study monitoring trends and determinants of cardiovascular disease (CVD) in geographically defined populations in different parts of China. METHODS AND RESULTS: The study is a community-based prospective disease surveillance that uses the methodology and criteria of the World Health Organization's Monitoring Trends and Determinants in Cardiovascular Disease (WHO MONICA) project, with slight modifications for local use. After a pilot study of 2 years (1985 through 1986), data collection started formally on January 1, 1987, and ended on December 31, 1993. The main results were as follows. By international standards, both the incidence and mortality rate of coronary heart disease in Chinese populations were low. The highest incidence was 108.7 of 100,000 (1987 to 1989), and the lowest was 3.3 of 100,000 for men 35 to 64 years of age, a 33-fold difference. Both the incidence and mortality rate of cerebrovascular disease were high. The highest incidence was 553.3 of 100,000 (1987 to 1989), and the lowest was 33.0 of 100,000 for men 35 to 64 years of age, a 17-fold difference. There were significant geographic variations in both CVD incidence and mortality rate, with higher rates in the north and lower rates in the south. During 1987 to 1993, increasing trends were found in CVD rates in some populations, whereas decreasing trends were found in others. The trends were not significant statistically in most cases. CONCLUSIONS: Monitoring CVD with international standardized methods in China is feasible and urgently needed in view of the rapid socioeconomic development and transition of disease patterns taking place in China. The results are of significance in combating CVD both at home and abroad.
Adult↗
Inhibition of breast and brain cancer cell growth by BCCIPalpha, an evolutionarily conserved nuclear protein that interacts with BRCA2.
BRCA2 is a tumor suppressor gene involved in mammary tumorigenesis. Although important functions have been assigned to a few conserved domains of BRCA2, little is known about the longest internal conserved domain encoded by exons 14-24. We identified a novel protein, designated BCCIPalpha, that interacts with part of the internal conserved region of human BRCA2. Human BCCIP represents a family of proteins that are evolutionarily conserved, and contain three distinct domains: an N-terminus acidic domain (NAD) of 30-60 amino acids, an internal conserved domain (ICD) of 180-220 amino acids, and a C-terminus variable domain (CVD) of 30-60 amino acids. The N-terminal half of the human BCCIP ICD shares moderate homology with regions of calmodulin and M-calpain, suggesting that BCCIP may also bind Ca. Human cells express both a longer, BCCIPalpha, and a shorter, BCCIPbeta, form of the protein, which differ in their CVD. BCCIP is a nuclear protein highly expressed in testis. Although BCCIPbeta expression is relatively consistent in cancer cells, the expression of BCCIPalpha varies in cancer cell lines. The BCCIPalpha gene is located at chromosome 10q25.3-26.2, a region frequently altered in brain and other cancers. Furthermore, expression of BCCIPalpha inhibits breast and brain cancer cell growth, but fails to inhibit HT1080 cells and a non-transformed human skin fibroblast. These results suggest that BCCIPalpha is an important cofactor for BRCA2 in tumor suppression.
Amino Acid Sequence↗
Transcript map and comparative analysis of the 1.5-Mb commonly deleted segment of human 5q31 in malignant myeloid diseases with a del(5q).
Loss of a whole chromosome 5, or a del(5q), are recurring abnormalities in malignant myeloid diseases. In previous studies, we defined a commonly deleted segment (CDS) of 1.5 Mb between D5S479 and D5S500 in patients with a del(5q), and we established a P1 artificial chromosome-based contig encompassing this interval. To identify candidate tumor suppressor genes (TSGs), we developed a transcript map of the CDS. The map contains 18 genes and 12 expressed sequence tags/UniGenes. Among the 18 genes are 10 genes that were previously cloned and 8 novel genes. The newly identified genes include CDC23, which encodes a component of the anaphase-promoting complex; RAB6KIFL, which encodes a kinesin-like protein involved in organelle transport; and KLHL3, which encodes a human homologue of the Drosophila ring canal protein, kelch. We determined the intron/exon organization of 14 genes and eliminated each gene as a classical TSG by mutation analysis. In addition, we established a single-nucleotide polymorphism map as well as a map of the mouse genome that is syntenic to the CDS of human 5q31. The development of a transcription map will facilitate the molecular cloning of a myeloid leukemia suppressor gene on 5q.
Acute Disease↗