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Biomedical subjects

Y Guo

Publications and source records attributed to Y Guo.

At least 19 recordsLinked to original sources
PubMed2003

Hsp90 binds CpG oligonucleotides directly: implications for hsp90 as a missing link in CpG signaling and recognition.

CpG motifs originating from bacterial DNA (CpG DNA) can act as danger signals for the mammalian immune system. These CpG DNA motifs like many other pathogen-associated molecular patterns are believed to be recognized by a member of the toll-like receptor family, TLR-9. Here we show results suggesting that heat shock protein 90 (hsp90) is also implicated in the recognition of CpG DNA. Hsp90 was characterized as a binder to oligodeoxynucleotides (ODNs) containing CpG motifs (CpG ODNs) after several purification steps from crude protein extracts of peripheral blood mononuclear cells. This finding was further supported by direct binding of CpG ODNs to commercially available human hsp90. Additionally, immunohistochemistry studies showed redistribution of hsp90 upon CpG ODN uptake. Thus, we propose that hsp90 can act as a ligand transfer molecule and/or play a central role in the signaling cascade induced by CpG DNA.

Cell Line↗
PubMed2002

Ferritin induction protects cortical astrocytes from heme-mediated oxidative injury.

Hemin is released from hemoglobin after CNS hemorrhage and may contribute to its cytotoxic effect. In a prior study, we demonstrated that heme oxygenase-1 induction protected murine cortical astrocytes from hemoglobin toxicity. Since heme metabolism releases iron, this observation suggested that these cells are able to effectively sequester and detoxify free iron. In this study, we tested the hypotheses that astrocytes increased ferritin synthesis after exposure to heme-bound iron, and that this induction protected cells from subsequent exposure to toxic concentrations of hemin. Incubation with low micromolar concentrations of hemin, hemoglobin, or ferrous sulfate increased ferritin expression, as detected on immunoblots stained with a polyclonal antibody that was raised against horse spleen ferritin. Time course studies demonstrated an increase in ferritin levels within 2 h. Weak and scattered cellular staining was detected by immunohistochemistry in control, untreated cultures, while diffuse immunoreactivity was observed in cultures exposed to heme-bound iron. An enhanced ferritin band was detected on immunoblots from cultures that were treated with purified apoferritin, consistent with astrocytic ferritin uptake. Immunoreactivity after apoferritin treatment was not altered by concomitant treatment with cycloheximide. Pretreatment with apoferritin protected astrocytes from hemin toxicity in a concentration-dependent fashion between 1 and 4 mg/ml. At the highest concentration, cell death due to a 6-h exposure to 30 microM hemin was decreased by about 85%. A protective effect was also produced by induction of endogenous ferritin with nontoxic concentrations of ferrous sulfate, hemoglobin, or hemin. These results suggest that cortical astrocytes respond to exogenous heme-bound or free iron by rapidly increasing ferritin synthesis. The combined action of heme oxygenase-1 and ferritin may be a primary astrocytic defense against heme-mediated injury.

Animals↗
PubMed2002

CD34(+) or CD34(-): which is the more primitive?

Remarkable progress has been achieved in the characterization and isolation of primitive hematopoietic stem cells (HSC). HSC represent a very small subset of hematopoietic cells and provide self-renewal, possess differentiation capacity and allow a constant supply of the entire hematopoietic cell spectrum. Until recently, CD34 has been used as a convenient marker for HSC, since CD34(+) cells have been shown to possess colony-forming potential in short-term assays, maintain long-term colony-forming potential in in vitro cultures and allow the expression and differentiation of blood cells from different hematopoietic lineages in in vivo models. Clinical and experimental protocols have targeted CD34(+) cells enriched by a variety of selection models and have readily used these for transplantation, purging and gene therapies and targets for future organ replacement. Recent studies in murine and human models, however, have indicated that CD34(-) HSC exist as well, which possess engraftment potential and distinct HSC characteristics. These studies challenge the dogma that HSC are uniformly found in the CD34(+) subset, and question whether primitive HSC are CD34(+) or CD34(-). In this review, results on murine and human CD34(+) and CD34(-) HSC, differences between them and their possible interactions are examined.

Animals↗
PubMed2002

Automated fragment analysis method for determining androgen receptor CAG repeat length.

Several studies have associated polymorphisms in the androgen receptor gene with the risk of developing hormone-dependent cancers. A highly polymorphic (CAG)n repeat in exon 1 encodes a polyglutamine tract of varying length. The determination of the number of CAG repeats in the androgen receptor has typically been performed on denaturing polyacrylamide gels with autoradiographic orfluorescent detection of differently sized alleles. Samples run on a capillary electrophoresis-based ABI Prism 310 Genetic Analyzer gave anomalous results when internal standards supplied by the manufacturer were used Here we report a modified procedure for androgen receptor allele size determination that can be used on an automated capillary electrophoresis-based DNA sequencer equipped with the appropriate software. The assay is very precise, comparable to DNA sequencing, and is compatible with the latest generation of automated DNA sequencers.

Electrophoresis, Capillary↗
PubMed2001

Thermolysis of alkyl sulfoxides and derivatives: a comparison of experiment and theory.

Gas-phase activation data were obtained for model sulfoxide elimination reactions. The activation enthalpy for methyl 3-phenylpropyl sulfoxide is 32.9 +/- 0.9 kcal/mol. Elimination by methyl vinyl sulfoxide to form acetylene has an enthalpic barrier of 41.6 +/- 0.8 kcal/mol and that of 3-phenylpropyl methanesulfinate to form hydrocinnamaldehyde is 34.6 +/- 0.6 kcal/mol. Calculations at the MP2/6-311+G(3df,2p)//MP2/6-31G(d,p) level for simplified models of these reactions provide barriers of 32.3, 40.3, and 32.7 kcal/mol, respectively. A series of other compounds are examined computationally, and it is shown that the substituent effects on the sulfoxide elimination reaction are much more straightforward to interpret if DeltaH data are available in addition to the usually determined DeltaH++. The activation enthalpy of the reverse addition reaction is also subject to structural variation and can usually be rationalized on the basis of nucleophilicity of the sulfur or polarity matching between the sulfenic acid and olefin derivative.

Journal Article↗
PubMed2001

Identification of a novel family of putative methyltransferases that interact with human and Drosophila presenilins.

Mutations in the presenilin genes have been shown to cause the majority of cases of early-onset familial Alzheimer's disease (AD). In addition to their role in AD, presenilins are also known to function during development by interacting with the Notch pathway. To determine if presenilins have additional functions during development and AD we have used a yeast two-hybrid approach to search for proteins that can bind to presenilins. Here, we show the identification and characterization of a novel putative methyltransferase (Metl) that interacts with the loop region of Drosophila presenilin as well as human presenilin-1 and presenilin-2, suggesting that this interaction is evolutionarily conserved and functionally important. Metl appears to be a member of a conserved family of methyltransferases that share homology with, but are distinct from, the UbiE family of methyltransferases involved in ubiquinone and menaquinone biosynthesis. In Drosophila, the metl gene gives rise to two major isoforms by alternative splicing that are broadly expressed throughout development and found in the central nervous system in an overlapping pattern with Drosophila presenilin. Finally, we show that two independent dominant adult phenotypes produced by overexpression of presenilin can be enhanced by overexpression of metl in the same tissue. Taken together, these results suggest that presenilin and Metl functionally and genetically interact during development.

Alternative Splicing↗
PubMed2001

An ERCC1 splicing variant involving the 5'-UTR of the mRNA may have a transcriptional modulatory function.

Human ovarian cancer cells and tissues were examined for the presence or absence of a 42-bp splicing variant of ERCC1 gene, and for a possible functional role of this 42-bp sequence. This specific sequence exists in exon I, the 5'-UTR of the gene. Loss of this 42-bp sequence was associated with increased ERCC1 mRNA expression, in an assessment of 121 ovarian cancer specimens (p2<10(-6)). In cells in tissue culture, the absence of the 42-bp segment was associated with a twofold increased ability to drive transcription in a Luciferase reporter system. Protein can be demonstrated in ovarian cancer cells based on EMSA analysis. Computer analysis shows that this 42-bp sequence contains several binding sites, including a core-binding domain for protein RFX1, transcriptional repressor. These preliminary results lay the groundwork in determination of potential roles for a negative regulatory element in NER repair pathway.

5' Untranslated Regions↗
PubMed2001

Absence of mast cell involvement in active systemic anaphylaxis in rats.

This study investigates the role of mast cells in the hypotension induced by antigen-mediated anaphylaxis, compound 48/80 and dextran in mast cell-deficient white spotting (Ws/Ws) and normal wild type (+/+) rats. Rats were sensitized with 10 microg of intraperitoneal ovalbumin in saline or saline alone (sham-sensitized). Sensitized rats, both Ws/Ws and +/+ but not sham-sensitized rats, challenged intravenously with ovalbumin exhibited hypotensive responses. There was no evidence of mast cell activation in rat mesentery 20 min after intravenous antigen challenge in sensitized +/+ rats. Hypotension induced by intravenous injection of dextran (Dextran-162, 6%, 2 ml kg(-1)) or compound 48/80 (1 mg kg(-1)) occurred in +/+ rats, but not in Ws/Ws rats, and was inhibited by pretreatment with a combination of chlorpheniramine and cimetidine. Taken together, these data indicate that the hypotensive response induced by antigen-mediated anaphylaxis is independent of mast cell activation, whereas mast cell amines play the main role in the hypotensive response induced by dextran or compound 48/80.

Anaphylaxis↗
PubMed2001

Evidence for a mechanism of repression of heat shock factor 1 transcriptional activity by a multichaperone complex.

In the absence of stress, human heat shock factor 1 (hHSF1) is in its unactivated form. hHSF1 polypeptide is in a dynamic heterocomplex with Hsp90 and is incapable of specifically binding DNA. When cells are stressed, heterocomplex assembly is disrupted. Unbound hHSF1 homotrimerizes, acquires DNA binding activity, and concentrates in the nucleus, but remains transcriptionally inactive. A subsequent reaction converts this inactive, trimeric form into the active, hyperphosphorylated transcription factor. Subsequent to the stressful event, hHSF1 is deactivated and eventually returned to its unactivated form. Evidence is presented herein that trimeric hHSF1 has the propensity to dynamically associate with an Hsp90-immunophilin-p23 complex through its regulatory domain. Formation of this heterocomplex results in repression of the transcriptional activity of trimeric hHSF1. Stress-denatured proteins effectively compete with trimeric hHSF1 for Hsp90-immunophilin-p23 complex, counteracting assembly of the heterocomplex and repression of hHSF1 transcriptional activity. This repression mechanism may be required for a proportional transcriptional response to stress. Formation of the heterocomplex may also represent the first step toward returning the hHSF1 to its unactivated form.

Base Sequence↗
PubMed2001

The effect of cholesterol in a liposomal Muc1 vaccine.

A liposomal Muc1 mucin vaccine for treatment of adenocarcinomas was formulated by incorporating a synthetic Muc1 mucin-based lipopeptide and Lipid A into a DPPC/cholesterol bilayer. Vaccination of mice with the liposomal formulation produced a peptide-specific immune response dependent on the cholesterol content. The response occurred at a threshold of 20-23 mol% cholesterol, and was optimal at cholesterol levels of > or =30 mol%. To understand this cholesterol dependency, we studied the effect of cholesterol on the liposomal bilayer and surface properties. Freeze-fracture electron microscopy showed a unique surface texture that was codependent upon cholesterol (> or =20 mol%) and lipopeptide content. Fluorescence anisotropy measurements exhibited a significant decrease in the rotational motion of 1,6-diphenyl-1,3,5-hexatriene in formulations containing >20 mol% cholesterol and only in the presence of the lipopeptide. At 20 mol% cholesterol and with lipopeptide, DSC showed a significant increase in the main phase transition of the DPPC bilayers, while Raman spectroscopy indicated a more ordered arrangement of DPPC molecules compared to control liposomes containing DPPC/cholesterol alone. Taken together, the data suggest the presence of lipopeptide-rich microdomains at and above a threshold of 20 mol% cholesterol that may play a role in the induction of a peptide-specific immunological response.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed2001

Opposing cardioprotective actions and parallel hypertrophic effects of delta PKC and epsilon PKC.

Conflicting roles for protein kinase C (PKC) isozymes in cardiac disease have been reported. Here, deltaPKC-selective activator and inhibitor peptides were designed rationally, based on molecular modeling and structural homology analyses. Together with previously identified activator and inhibitor peptides of epsilonPKC, deltaPKC peptides were used to identify cardiac functions of these isozymes. In isolated cardiomyocytes, perfused hearts, and transgenic mice, deltaPKC and epsilonPKC had opposing actions on protection from ischemia-induced damage. Specifically, activation of epsilonPKC caused cardioprotection whereas activation of deltaPKC increased damage induced by ischemia in vitro and in vivo. In contrast, deltaPKC and epsilonPKC caused identical nonpathological cardiac hypertrophy; activation of either isozyme caused nonpathological hypertrophy of the heart. These results demonstrate that two related PKC isozymes have both parallel and opposing effects in the heart, indicating the danger in the use of therapeutics with nonselective isozyme inhibitors and activators. Moreover, reduction in cardiac damage caused by ischemia by perfusion of selective regulator peptides of PKC through the coronary arteries constitutes a major step toward developing a therapeutic agent for acute cardiac ischemia.

Amino Acid Sequence↗
PubMed2001

Transient suppression of X-ray-induced apoptosis by exposure to power frequency magnetic fields in MCF-7 cells.

Epidemiological studies suggest that exposure to power frequency magnetic fields may be a risk factor for breast cancer in humans. To study the relationship between exposure to 60-Hz magnetic fields (MFs) and breast cancer, cell cycle distribution, apoptosis, and the expression of related proteins (p21, Bax, and Bcl-2) were determined in MCF-7 cells following exposure to magnetic fields (60 Hz, 5 mT) alone or in combination with X rays. It was found that exposure of MCF-7 cells to 60-Hz MFs for 4, 8, and 24 h had no effect on cell cycle distribution. Furthermore, 60-Hz MFs failed to affect cell growth arrest and p21 expression induced by X rays (4 Gy). Similarly, 60-Hz MFs did not induce apoptosis or the expression of Bax and Bcl-2, two proteins related to apoptosis. However, exposure of cells to 60-Hz MFs for 24 h after irradiation by X rays (12 Gy) significantly decreased apoptosis and Bax expression but increased Bcl-2 expression. The effects of exposure to 60-Hz MFs on X-ray-induced apoptosis and Bax and Bcl-2 expressions were not observed at 72 h. These data suggest that exposure to 60-Hz MFs has no effects on the growth of MCF-7 cells, but it might transiently suppress X-ray-induced apoptosis through increasing the Bcl-2/Bax ratio.

Apoptosis↗
PubMed2001

Interleukin-2-induced small unilamellar vesicle coalescence.

Recombinant human interleukin-2 (rhIL-2) was incorporated in liposomes for potential therapeutic applications using a novel process. In this process, rhIL-2 caused the formation of large, unique multilamellar vesicles (MLVs) from small unilamellar vesicles (SUVs) of dimyristoylphosphatidylcholine (DMPC). Vesicle coalescence occurred most rapidly at 19 degrees C, between the pre- and main phase transition temperatures of DMPC, and showed a dependence upon pH (pH <5.5), ionic strength (>50 mM) and the initial size of the unilamellar vesicles (<or=25 nm). Intermediates (partially coalesced vesicles) within the forming multilamellar structures were identified by freeze-fracture electron microscopy and their presence was corroborated by differential scanning calorimetry. Several distinct steps were identified in the coalescence process. In the initial step, rhIL-2 rapidly bound to the DMPC SUVs. This was followed by a pH-dependent conformational change in the protein, as evidenced by an increase in tryptophan fluorescence intensity. The SUVs then aggregated in large clusters that eventually annealed to form closed MLVs. In this process over 90% of the rhIL-2 was bound to and incorporated within the multilamellar structures.

Calorimetry, Differential Scanning↗
PubMed2001

Identification and characterization of a novel androgen receptor coregulator ARA267-alpha in prostate cancer cells.

The androgen receptor (AR) is a member of the steroid receptor superfamily that binds to the androgen response element to regulate target gene transcription. AR may need to interact with some selected coregulators for maximal or proper androgen function. Here we report the isolation of a new AR coregulator with a calculated molecular mass of 267 kDa named the androgen receptor-associated protein 267-alpha (ARA267-alpha). ARA267-alpha contains 2427 amino acids, including one Su(var)3-9, Enhancer-of-zeste, and Trithorax (SET) domain, two LXXLL motifs, three nuclear translocation signal (NLS) sequences, and four plant homeodomain (PHD) finger domains. Northern blot analyses reveal that ARA267-alpha is expressed predominantly in the lymph node as 13- and 10-kilobase transcripts. HepG2 is the only cell line tested that does not express ARA267-alpha. Yeast two-hybrid and glutathione S-transferase pull-down assays show that both the N and C terminus of ARA267-alpha interact with the AR DNA- and ligand-binding domains. Unlike other coregulators, such as CBP, which enhance the interaction between the N and C terminus of AR, we found that ARA267-alpha had little influence on the interaction between the N and C terminus of AR. Luciferase and chloramphenicol acetyltransferase assays show that ARA267-alpha can enhance AR transactivation in a dihydrotestosterone-dependent manner in PC-3 and H1299 cells. ARA267-alpha can also enhance AR transactivation with other coregulators, such as ARA24 or PCAF, a histone acetylase, in an additive manner. Together, our data demonstrate that ARA267-alpha is a new AR coregulator containing the SET domain with an exceptionally large molecular mass that can enhance AR transactivation in prostate cancer cells.

Amino Acid Sequence↗
PubMed2001

[Experimental observation of antagonism of polyaspartic acid against gentamicin-induced phospholipidosis in cochlea of guinea pig].

OBJECTIVE: To observe the effect of polyaspartic acid (PAA) on gentamicin-induced disturbance of phospholipid metabolism in cochlea of Guinea pig. METHODS: Ninety-four Guinea pigs were divided into 4 groups administered with PAA, PAA + GM, GM, or normal saline respectively. Auditory brain-stem response (ABR) was recorded before and one, five and ten days after the experiment. High performance liquid chromatography (HPLC) was used to determinate the amount of phospholipid in cochlear tissue, and ultrastructural change of lysosomes in the cochlear hair cells was observed by transmission electron microscopy one, five, and ten days after the experiment. RESULTS: No difference in any indicator was found one day after the beginning of experiment in all groups. The amounts of phosphatidylinositol (PI) and phosphatidylinositol-phosphate (PIP) increased significantly five days after administration of GM (19.7 micrograms +/- 6.6 micrograms and 121 micrograms +/- 21 micrograms respectively). The amount of PIP increased significantly 10 days after administration of GM (126 micrograms +/- 8 micrograms). Transmission electron microscopy showed an increase of number and volume of lysosome in cochlear outer hair cells and appearance of Hensen's body five days after administration of GM. Such changes became more significant and some lysosomes broke in the 10th day. The ABR threshold began to increase 5 days after the administration of GM, and continued to increase with time. CONCLUSION: PAA has inhibitory effect on gentamicin-induced lysosomal phospholipidosis in cochlea of Guinea pig, thus inhibiting gentamicin-induce hearing loss.

Animals↗
PubMed2001

An essential role of the JAK-STAT pathway in ischemic preconditioning.

The goal of this study was to determine the role of the Janus tyrosine kinase (JAK)-signal transducers and activators of transcription (STAT) pathway in the late phase of ischemic preconditioning (PC). A total of 230 mice were used. At 5 min after ischemic PC (induced with six cycles of 4-min coronary occlusion/4-min reperfusion), immunoprecipitation with anti-phosphotyrosine (anti-pTyr) antibodies followed by immunoblotting with anti-JAK antibodies revealed increased tyrosine phosphorylation of JAK1 (+257 +/- 53%) and JAK2 (+238 +/- 35%), indicating rapid activation of these two kinases. Similar results were obtained by immunoblotting with anti-pTyr-JAK1 and anti-pTyr-JAK2 antibodies. Western analysis with anti-pTyr-STAT antibodies demonstrated a marked increase in nuclear pTyr-STAT1 (+301 +/- 61%) and pTyr-STAT3 (+253 +/- 60%) 30 min after ischemic PC, which was associated with redistribution of STAT1 and STAT3 from the cytosolic to the nuclear fraction and with an increase in STAT1 and STAT3 gamma-IFN activation site DNA-binding activity (+606 +/- 64%), indicating activation of STAT1 and STAT3. No nuclear translocation or tyrosine phosphorylation of STAT2, STAT4, STAT5A, STAT5B, or STAT6 was observed. Pretreatment with the JAK inhibitor AG-490 20 min before the six occlusion/reperfusion cycles blocked the enhanced tyrosine phosphorylation of JAK1 and JAK2 and the increased tyrosine phosphorylation, nuclear translocation, and enhanced DNA-binding activity of STAT1 and STAT3. The same dose of AG-490 abrogated the protection against myocardial infarction and the concomitant up-regulation of inducible NO synthase (iNOS) protein and activity observed 24 h after ischemic PC. Taken together, these results demonstrate that ischemic PC induces isoform-selective activation of JAK1, JAK2, STAT1, and STAT3, and that ablation of this response impedes the up-regulation of iNOS and the concurrent acquisition of ischemic tolerance. This study demonstrates that the JAK-STAT pathway plays an essential role in the development of late PC. The results reveal a signaling mechanism that underlies the transcriptional up-regulation of the cardiac iNOS gene and the adaptation of the heart to ischemic stress.

Animals↗
PubMed2001

Isotopic homogeneity of iron in the early solar nebula.

The chemical and isotopic homogeneity of the early solar nebula, and the processes producing fractionation during its evolution, are central issues of cosmochemistry. Studies of the relative abundance variations of three or more isotopes of an element can in principle determine if the initial reservoir of material was a homogeneous mixture or if it contained several distinct sources of precursor material. For example, widespread anomalies observed in the oxygen isotopes of meteorites have been interpreted as resulting from the mixing of a solid phase that was enriched in 16O with a gas phase in which 16O was depleted, or as an isotopic 'memory' of Galactic evolution. In either case, these anomalies are regarded as strong evidence that the early solar nebula was not initially homogeneous. Here we present measurements of the relative abundances of three iron isotopes in meteoritic and terrestrial samples. We show that significant variations of iron isotopes exist in both terrestrial and extraterrestrial materials. But when plotted in a three-isotope diagram, all of the data for these Solar System materials fall on a single mass-fractionation line, showing that homogenization of iron isotopes occurred in the solar nebula before both planetesimal accretion and chondrule formation.

Journal Article↗