Biomedical subjects

L Gan

Publications and source records attributed to L Gan.

At least 55 records · Page 3Linked to original sources

PubMed2001

[Inhibition the growth of human leukemia cells by Lycium barbarum polysaccharide].

The effect and the mechanism of Lycium barbarum polysaccharide (LBP-X) on inhibiting the growth of human leukemia HL-60 cells were examined. LBP-X(20, 100, 500, 1000 mg/L) could inhibit the growth of HL-60 cells in dose-dependent manner and decrease the membrane fluidity of the cell. Agarose gel electrophoresis of DNA from the cells treated with LBP-X revealed a "DNA ladder" and positive TUNEL test. The results showed that the apoptosis of HL-60 cells induced by LBP-X maybe its important mechanism on anti-tumorgenesis.

Antineoplastic Agents, Phytogenic↗

PubMed2000

Huntingtin interacting protein 1 induces apoptosis via a novel caspase-dependent death effector domain.

Huntington disease is a devastating neurodegenerative disease caused by the expansion of a polymorphic glutamine tract in huntingtin. The huntingtin interacting protein (HIP-1) was identified by its altered interaction with mutant huntingtin. However, the function of HIP-1 was not known. In this study, we identify HIP-1 as a proapoptotic protein. Overexpression of HIP-1 resulted in rapid caspase 3-dependent cell death. Bioinformatics analyses identified a novel domain in HIP-1 with homology to death effector domains (DEDs) present in proteins involved in apoptosis. Expression of the HIP-1 DED alone resulted in cell death indistinguishable from HIP-1, indicating that the DED is responsible for HIP-1 toxicity. Furthermore, substitution of a conserved hydrophobic phenylalanine residue within the HIP-1 DED at position 398 eliminated HIP-1 toxicity entirely. HIP-1 activity was found to be independent of the DED-containing caspase 8 but was significantly inhibited by the antiapoptotic protein Bcl-x(L), implicating the intrinsic pathway of apoptosis in HIP-1-induced cell death. Co-expression of a normal huntingtin fragment capable of binding HIP-1 significantly reduced cell death. Our data identify HIP-1 as a novel proapoptotic mediator and suggest that HIP-1 may be a molecular accomplice in the pathogenesis of Huntington disease.

Amino Acid Sequence↗

PubMed2000

Sequencing and expression analysis of the serine protease gene cluster located in chromosome 19q13 region.

The human kallikrein gene cluster, located in the chromosome band 19q13, contains several tissue-specific serine protease genes including the prostate-specific KLK2, KLK3 and prostase genes. To further characterize the gene cluster, we have mapped, sequenced, and analyzed the genomic sequence from the region. The results of EST database searches and GENSCAN gene prediction analysis reveal 13 serine protease genes and several pseudogenes in the region. Expression analysis by RT-PCR indicates that most of these protease genes are expressed only in a subset of the 35 different normal tissues that have been examined. Several protease genes expressed in skin show higher expression levels in psoriatic lesion samples than in non-lesional skin samples from the same patient. This suggests that the imbalance of a complex protease cascade in skin may contribute to the pathology of disease. The proteases, excluding the kallikrein genes, share approximately 40% of their sequences suggesting that the serine protease gene cluster on chromosome 19q13 arose from ancient gene duplications.

Amino Acid Sequence↗

PubMed2000

The basic helix-loop-helix transcription factor capsulin controls spleen organogenesis.

Formation of numerous internal organs involves reciprocal epithelial-mesenchymal signaling and subsequent patterning and growth of the organ primordium. Capsulin is a basic helix-loop-helix transcription factor expressed in mesenchymal cells that encapsulate the epithelial primordia of internal organs, including the kidney and lung, as well as the epicardium, which gives rise to the coronary arteries. Capsulin is also expressed in the mesothelium that gives rise to the spleen. We demonstrate that mice homozygous for a capsulin null mutation fail to form a spleen. The homeobox genes Hox11 and Bapx1, shown previously to be essential regulators of spleen organogenesis, and a lacZ reporter introduced into the capsulin locus, were expressed in the early splenic primordium, derived from the splanchnic mesoderm, of homozygous mutant embryos. However, this primordium failed to develop beyond an initial group of precursor cells and underwent rapid apoptosis. The phenotype of capsulin mutant mice demonstrates that capsulin acts within a subpopulation of splanchnic mesodermal cells to control an essential early step in spleen organogenesis that is likely to represent a point of regulatory convergence of the capsulin, Hox11, and Bapx1 genes.

Animals↗

PubMed2000

Differential immediate-early gene responses to shear stress and intraluminal pressure in intact human conduit vessels.

We have previously shown distinct effects of shear stress and pressure on transcription of several potent vascular mediators. In the present study, we tested the hypothesis that c-jun and c-fos are regulated differentially by shear and pressure. Intact human umbilical veins were perfused with various combinations of shear and pressure during 1.5, 3 and 6 h. Protein and gene expressions were assessed by immunofluorescence and real-time reverse transcription PCR, respectively. Shear stress and pressure exert differential temporal effects on c-jun and c-fos gene and protein expression, and these immediate-early gene responses appear to be cell-type specific for endothelial and smooth muscle cells.

Endothelium, Vascular↗

PubMed2000

Distinct regulation of vascular endothelial growth factor in intact human conduit vessels exposed to laminar fluid shear stress and pressure.

VEGF is a potent angiogenic factor. We tested the hypothesis that biomechanical forces may regulate VEGF expression. By using a computerized perfusion system, human umbilical veins were exposed to high/low shear stress or intraluminal pressure (25/4 dyn/cm(2) or 40/20 mmHg) for 1.5, 3, or 6 h. Quantification of VEGF gene expression was performed with real-time RT-PCR. VEGF protein was characterized by quantitative immunohistochemistry. All perfusion experiments were performed under identical pH, PO(2), and temperature. Shear stress induced significant biphasic regulation pattern of VEGF (P = 0.0044) with significant downregulation by 45 and 40% after 1.5 and 6 h perfusion, respectively (P = 0.006 and P = 0.0002). The temporal changes of the gene expression were accompanied by synchronal changes at the protein level. High pressure induced transient 25% downregulation of VEGF gene expression after 1.5 h perfusion (P = 0.031). These data provide the first evidence on modulating effects of biomechanical forces on the vascular angiogenic property.

Blood Pressure↗

PubMed2000

Fullerene-sensitized

[reaction: see text] Fullerene catalyzes the cycloaddition of dimethyliminodiacetate to maleimides under photolysis to form 2,5-dimethoxycarbonylpyrrolidine derivatives.

Journal Article↗

PubMed2000

Huntingtin is required for normal hematopoiesis.

Huntington's disease (HD) is a neurodegenerative disease associated with polyglutamine expansion in huntingtin, a widely expressed protein. The function of huntingtin is unknown although huntingtin plays a fundamental role in development since gene targeted HD (-) (/-)mouse embryos die shortly after gastrulation. Expression of huntingtin is detected in spleen and thymus but its role in hematopoiesis has not been examined. To determine the function of huntingtin and to provide insight into potential pathologic mechanisms in HD, we analyzed the role of huntingtin in hematopoietic development. Expression of huntingtin was analyzed in a variety of hematopoietic cell types, and in vitro hematopoiesis was assessed using an HD ( +/-)and several HD( -) (/-)embryonic stem (ES) cell lines. Although wild-type, HD ( +/-)and HD( -) (/-)ES cell lines formed primary embryoid bodies (EBs) with similar efficiency, the numbers of hematopoietic progenitors detected at various stages of the in vitro differentiation were reduced in HD ( +/-)and HD( -/-)() ()ES cell lines examined. Expression analyses of hematopoietic markers within the EBs revealed that primitive and definitive hematopoiesis occurs in the absence of huntingtin. However, further analysis using a suspension culture in the presence of hematopoietic cytokines demonstrated a highly significant gene dosage-dependent decrease in proliferation and/or survival of HD ( +/-)and HD( -) (/-)cells. Enrichment for the CD34(+)cells within the EB confirmed that the impairment is intrinsic to the hematopoietic cells. These obser- vations suggest that huntingtin expression is required for the generation and expansion of hematopoietic cells and provides an alternative system in which to assess the function of huntingtin.

Animals↗

PubMed2000

Abnormal polarization and axon outgrowth in retinal ganglion cells lacking the POU-domain transcription factor Brn-3b.

The POU domain transcription factor Brn-3b (also called Brn-3.2) is essential for the normal development of retinal ganglion cells (RGCs) in the mouse. Without Brn-3b, RGCs commit to their fate and migrate to the ganglion cell layer, but most cells die during fetal development. An earlier report (L. Gan et al., 1999, Dev. Biol. 210, 469-480) suggested that cell death was caused by abnormal axon formation. Here, we use retinal explants from wild-type and mutant embryos to show that brn-3b-deficient RGCs are not properly polarized and tend to form dendrites rather than axons. Compared with wild-type explants, neurites of RGCs from brn-3b-deficient retinal explants grew slower, were shorter, and did not fasciculate properly. Mutant neurites had more microtubules than wild-type controls, and the arrangement of microtubules and neurofilaments was characteristic of dendrites rather than axons. Neurites from individual mutant RGCs displayed abnormal polarity and had dendrite-like branches extending outward from their main axis. Most mutant RGCs exhibited abnormal migratory behavior, and their neurites labeled intensely with the dendrite marker MAP-2. A small number of mutant RGCs were not migratory, and their neurites were longer and labeled positively for the axon marker tau-1, suggesting that some RGCs were not as severely affected by the absence of Brn-3b as others. Although tau-1 was not observed in most mutant neurites, it did accumulate in mutant cell bodies, implying that the absence of Brn-3b caused a defect in axon transport. Thus, Brn-3b appears to control the activity of genes that function in establishing RGC polarity, and without Brn-3b, RGCs cannot extend normal axons.

Animals↗

PubMed2000

HIP12 is a non-proapoptotic member of a gene family including HIP1, an interacting protein with huntingtin.

Huntingtin-interacting protein I (HIP1) is a membrane-associated protein that interacts with huntingtin, the protein altered in Huntington disease. HIP1 shows homology to Sla2p, a protein essential for the assembly and function of the cytoskeleton and endocytosis in Saccharomyces cerevisiae. We have determined that the HIP1 gene comprises 32 exons spanning approximately 215 kb of genomic DNA and gives rise to two alternate splice forms termed HIP1-1 and HIP1-2. Additionally, we have identified a novel protein termed HIP12 with significant sequence and biochemical similarities to HIP1 and high sequence similarity to Sla2p. HIP12 differs from HIP1 in its pattern of expression both at the mRNA and protein level. However, HIP1 and HIP12 are both found within the brain and show a similar subcellular distribution pattern. In contrast to HIP1, which is toxic in cell culture, HIP12 does not confer toxicity in the same assay systems. Interestingly, HIP12 does not interact with huntingtin but can interact with HIP1. suggesting a potential interaction in vivo that may influence the function of each respective protein.

Alternative Splicing↗

PubMed2000

Mass spectrometric and NMR characterization of metabolites of roxifiban, a potent and selective antagonist of the platelet glycoprotein IIb/IIIa receptor.

1. The methyl ester prodrug roxifiban is an orally active, potent and selective antagonist of the platelet glycoprotein GPIIb/IIIa receptor and is being developed for the prevention and treatment of arterial thrombosis. 2. Roxifiban was rapidly hydrolyzed to the zwitterion XV459 in vivo and by liver slices from the rat, mouse and human and by intestinal cores from dog. XV459 was metabolized to only a small extent in vitro and in vivo. 3. Studies with rat and dog given radiolabelled roxifiban showed limited oral absorption with the majority of the radiolabel being excreted in faeces. After i.v. doses of 14C-roxifiban, most of the radioactivity was recovered in the urine of rat whereas the dog excreted significant amounts of radioactivity in bile and urine. 4. XV459 could be metabolized extrahepatically by dog gut flora to produce an isoxazoline ring-opened metabolite. In vitro hepatic metabolism of XV459 was mainly by hydroxylation at the prochiral and chiral centres of the isoxazoline ring. These hydroxylated metabolites were not detected in the urine and plasma of human volunteers administered roxifiban. 5. Initial LC/MS identification of metabolites was achieved by dosing the rat with an equimolar mixture of d0:d4 roxifiban and detecting isotopic clusters of pseudomolecular ions. Unequivocal characterization of these metabolites was achieved by LC/MS, LC/NMR and high-field NMR techniques using synthetic standards of the metabolites. 6. The synthesis of one hydroxylated metabolite enabled the assignment of the correct stereochemistry of the substituted hydroxyl group on the isoxazoline ring.

Amidines↗

PubMed2000

Apoptosis in UV-exposed rabbit corneas.

PURPOSE: Apoptosis was studied in rabbit corneas as a possible mechanism of cell death after photokeratitis induced by different UV wavelengths. METHOD: Fourteen albino rabbit corneas were exposed to 280- and 310-nm UV radiation (UVR) in 10-nm full wavebands at doses that cause biomicroscopically significant keratitis (0.12 J/cm2 for 280 nm and 0.47 J/cm2 for 310 nm). Animals were killed 24 and 76 h after exposure. Corneas were processed for light and transmission electron microscopy and in situ end labeling of fragmented DNA by using a modification of the TUNEL technique. RESULTS: Corneas exposed to 280-nm UVR showed TUNEL-positive staining only in epithelial cells and superficial keratocytes at 24 and 76 h after irradiation. Twenty-four hours after 310-nm UVR exposure, TUNEL-positive staining was present in the epithelial cells, keratocytes throughout the entire thickness of the central stroma, and in endothelial cells. Seventy-six hours after exposure to 310-nm UVR, keratocytes disappeared throughout the whole thickness of the damaged stroma. Only a few epithelial cells were TUNEL positive at that time. Transmission electron microscopy (TEM) verified the occurrence of apoptotic nuclei and cells. CONCLUSION: Apoptosis appears to be a mechanism of corneal cell death after UVR. The 310-nm UVR caused more extensive damage to the corneal stroma and endothelium than did the 280-nm UVR.

Animals↗

PubMed2000

Fluid shear stress increases the intra-cellular storage pool of tissue-type plasminogen activator in intact human conduit vessels.

We investigated the effect of shear stress on the expression of tissue-type plasminogen activator (t-PA) in intact human conduit vessels. Human umbilical veins were exposed to high or low shear stress (25 vs < 4 dyn/cm2) at identical intraluminal pressure (20 mmHg) for 1.5, 3, and 6 h in a new computerized biomechanical perfusion system. High shear perfusion induced a marked, time-dependent increase in t-PA immunostaining in both the endothelium and the media. t-PA relative to GAPDH gene expression increased by 54 +/- 14% in high- compared to low-sheared vessels (p = 0.002). By contrast, t-PA release into the perfusion medium was similar in vessels perfused under high or low shear stress conditions. The results show that shear stress independently of pressure is a potent fluid mechanical stimulus for up-regulation of the intracellular storage pool of t-PA in the vascular wall of fresh human conduit vessels. The shear effect is associated with an increased t-PA gene expression.

DNA, Complementary↗

PubMed2000

[Application of factor analysis to identification of overlapped spectrum in XRF].

EFA method has been used in the processing of qualitative XRF data for metal alloys. With mathematically selected scanning steps and a mathematical model based on suitable training set selection, the method can correctly identify spectrum overlaps. Studies reveal that identification ability can be affected by the number of data points and the concentrations of the overlapping elements.

Alloys↗

PubMed1999

Identification of a differentially expressed RNA helicase by gene trapping.

A mouse line was generated that expressed a gene trap reporter construct, betageo, in a dynamic pattern during embryonic development. Differential expression was seen within the developing eyes, limbs, heart, neural tube, and skeleton. Two transcripts were cloned that contained endogenous sequences fused to the gene trap vector sequence. Analysis of the endogenous sequences revealed that the reporter integrated within a gene belonging to a small group of eukaryotic superfamily I helicases. Unexpectedly, the majority of transcripts produced from the trapped locus were not affected by the insertion of the reporter. Although the function of the trapped helicase gene is unknown, its complex transcription patterns and widespread spatial-temporal distribution suggest that the gene product plays a role in RNA metabolism in multiple tissues and organs within the developing embryo.

Amino Acid Sequence↗

PubMed1999

Cell cycle arrest and morphological alterations following microinjection of NIH3T3 cells with Pur alpha.

Levels of Pur alpha, a protein implicated in control of both DNA replication and gene transcription, fluctuate during the cell cycle, being lowest in early S phase and highest just after mitosis. Here we have employed a new video time-lapse technique enabling us to determine the cell cycle position of each cell in an asynchronous culture at a given time and to ask whether introduction of Pur alpha protein at specific times can affect cell cycle progression. Approximately 80% of all NIH3T3 cells injected with Pur alpha were inhibited from passing through mitosis. Cells injected with Pur alpha during S or G2 phases were efficiently blocked with a 4N (G2 phase) DNA level, as determined by quantitative DNA photometry of individual cells. Of the cells injected with Pur alpha during G1 phase, 40% experienced a rapid cell death characterized by extreme cellular fragmentation. Of those G1 injected cells which remained viable, approximately equal numbers were arrested with either 2N or 4N DNA levels. Cells arrested by Pur alpha in G2 phase grew to cover a large surface area. These results link fluctuations in Pur alpha levels to aspects of cell cycle control.

3T3 Cells↗

PubMed1999

Evidence for both the nucleus and cytoplasm as subcellular sites of pathogenesis in Huntington's disease in cell culture and in transgenic mice expressing mutant huntingtin.

A unifying feature of the CAG expansion diseases is the formation of intracellular aggregates composed of the mutant polyglutamine-expanded protein. Despite the presence of aggregates in affected patients, the precise relationship between aggregates and disease pathogenesis is unresolved. Results from in vivo and in vitro studies of mutant huntingtin have led to the hypothesis that nuclear localization of aggregates is critical for the pathology of Huntington's disease (HD). We tested this hypothesis using a 293T cell culture model system by comparing the frequency and toxicity of cytoplasmic and nuclear huntingtin aggregates. Insertion of nuclear import or export sequences into huntingtin fragments containing 548 or 151 amino acids was used to reverse the normal localization of these proteins. Changing the subcellular localization of the fragments did not influence their total aggregate frequency. There were also no significant differences in toxicity associated with the presence of nuclear compared with cytoplasmic aggregates. These studies, together with findings in transgenic mice, suggest two phases for the pathogenesis of HD, with the initial toxicity in the cytoplasm followed by proteolytic processing of huntingtin, nuclear translocation with increased nuclear concentration of N-terminal fragments, seeding of aggregates and resultant apoptotic death. These findings support the nucleus and cytosol as subcellular sites for pathogenesis in HD.

Animals↗

PubMed1999

Wild-type but not Alzheimer-mutant amyloid precursor protein confers resistance against p53-mediated apoptosis.

Amyloid precursor proteins (APPs) are expressed in multiple organs and cell types in diverse species. Their conservation across species and high abundance in brain and the association of various APP missense mutations with autosomal dominant forms of familial Alzheimer's disease (FAD) suggest important roles for APP in the central nervous system. However, the basic functions of APP in the central nervous system remain largely unknown. To assess potential effects of APP on neuronal death and survival, we transfected APP-deficient rat neuroblastoma cells (B103) with DNA constructs encoding wild-type or FAD-mutant human APP. Wild-type, but not FAD-mutant, APP effectively protected cells against apoptosis induced by ultraviolet irradiation, staurosporine, or p53. Wild-type APP also strongly inhibited p53 DNA-binding activity and p53-mediated gene transactivation, whereas FAD-mutant APP did not. We conclude that APP protects neuronal cells against apoptosis by controlling p53 activation at the post-translational level. Disruption of this function by mutations or alterations in APP processing could enhance neuronal vulnerability to secondary insults and contribute to neuronal degeneration.

Alzheimer Disease↗