Biomedical subjects

Y Hu

Publications and source records attributed to Y Hu.

At least 19 recordsLinked to original sources

PubMed2000

Establishment and characterization of HKESC-1, a new cancer cell line from human esophageal squamous cell carcinoma.

The establishment of an esophageal cancer cell line can facilitate the search for molecular mechanisms involved in esophageal carcinogenesis. A new human cancer cell line, HKESC-1, was established from a primary moderately-differentiated squamous cell carcinoma of the esophagus from a 47-year-old Hong Kong Chinese man. The pathological characteristics (morphology, immunohistochemical, and electron microscopic studies), the tumorigenecity in nude mice, the cytogenetic features, the DNA ploidy, and telomerase activity of the cell line were investigated. The HKESC-1 cells have been maintained continuously in vitro for more than 16 months and passaged over 96 times. HKESC-1 cells grow as a monolayer, with a doubling time of 46 hours. The HKESC-1 cells are of a squamous epithelial origin, as shown by their immunopositivity with the anti-cytokeratin antibodies and ultrastructural demonstration of tonofilaments and desmosomes. The HKESC-1 cells possess characteristics of malignancy because they are highly tumorigenic in nude mice and have strong telomerase activity. The HKESC-1 cells had an aneuploid DNA content, as demonstrated by flow cytometric analysis. Cytogenetic analysis revealed hyperdiploidy of greater than 50 in 80% of analyzable metaphases. Chromosome gains and losses were common, and loss of the Y chromosome was a consistent numerical aberration. Additionally, many structural chromosomal abnormalities were encountered, with frequent breakpoints at 1p32, 7p22, 7q34, and 20q13. This newly established cell line serves as a useful model for studying the molecular pathogenesis, and testing new therapeutic reagents for esophageal squamous cell carcinoma.

Animals↗

PubMed2000

Neutrophil tethering on E-selectin activates beta 2 integrin binding to ICAM-1 through a mitogen-activated protein kinase signal transduction pathway.

On inflamed endothelium selectins support neutrophil capture and rolling that leads to firm adhesion through the activation and binding of beta 2 integrin. The primary mechanism of cell activation involves ligation of chemotactic agonists presented on the endothelium. We have pursued a second mechanism involving signal transduction through binding of selectins while neutrophils tether in shear flow. We assessed whether neutrophil rolling on E-selectin led to cell activation and arrest via beta 2integrins. Neutrophils were introduced into a parallel plate flow chamber having as a substrate an L cell monolayer coexpressing E-selectin and ICAM-1 (E/I). At shears >/=0.1 dyne/cm2, neutrophils rolled on the E/I. A step increase to 4.0 dynes/cm2 revealed that approximately 60% of the interacting cells remained firmly adherent, as compared with approximately 10% on L cells expressing E-selectin or ICAM-1 alone. Cell arrest was dependent on application of shear and activation of Mac-1 and LFA-1 to bind ICAM-1. Firm adhesion was inhibited by blocking E-selectin, L-selectin, or PSGL-1 with Abs and by inhibitors to the mitogen-activated protein kinases. A chimeric soluble E-selectin-IgG molecule specifically bound sialylated ligands on neutrophils and activated adhesion that was also inhibited by blocking the mitogen-activated protein kinases. We conclude that neutrophils rolling on E-selectin undergo signal transduction leading to activation of cell arrest through beta 2 integrins binding to ICAM-1.

Adult↗

PubMed2000

Histidine 295 and histidine 510 are crucial for the enzymatic degradation of heparan sulfate by heparinase III.

The heparinases from Flavobacterium heparinum are powerful tools in understanding how heparin-like glycosaminoglycans function biologically. Heparinase III is the unique member of the heparinase family of heparin-degrading lyases that recognizes the ubiquitous cell-surface heparan sulfate proteoglycans as its primary substrate. Given that both heparinase I and heparinase II contain catalytically critical histidines, we examined the role of histidine in heparinase III. Through a series of diethyl pyrocarbonate modification experiments, it was found that surface-exposed histidines are modified in a concentration-dependent fashion and that this modification results in inactivation of the enzyme (k(inact) = 0.20 +/- 0.04 min(-)(1) mM(-)(1)). The DEPC modification was pH dependent and reversible by hydroxylamine, indicating that histidines are the sole residue being modified. As previously observed for heparinases I and II, substrate protection experiments slowed the inactivation kinetics, suggesting that the modified residue(s) was (were) in or proximal to the active site of the enzyme. Proteolytic mapping experiments, taken together with site-directed mutagenesis studies, confirm the chemical modification experiments and point to two histidines, histidine 295 and histidine 510, as being essential for heparinase III enzymatic activity.

Animals↗

PubMed2000

Anti-stress effects of dehydroepiandrosterone: protection of rats against repeated immobilization stress-induced weight loss, glucocorticoid receptor production, and lipid peroxidation.

In the present study, we have (i) examined the biological effects of repeated immobilization stress, and (ii) tested the hypothesis that the adrenal steroid hormone dehydroepiandrosterone (DHEA) is an anti-stress hormone, using male Sprague-Dawley rats. Rats (N = 6) were divided into the following four groups: (i) control, (ii) repeated immobilization stress (2 hr daily, for 60 days), (iii) repeated immobilization stress (2 hr daily, for 60 days) plus daily i.p. administration of 5 mg DHEA/0.1 mL DMSO, and (i.v.) daily i.p. administration of 5 mg DHEA/0.1 mL DMSO alone. Results obtained showed that repeated immobilization stress resulted in a significant (25%) inhibition in body weight gain, a significant increase in adrenal weight, an increase in glucocorticoid receptor (GR) in the liver, thymus, and spleen, decreased plasma triglyceride levels, and increased lipid peroxidation in the liver and heart as compared with control unstressed animals. Interestingly, DHEA administration resulted in a significant reversal in stress-induced inhibition in body weight gain, adrenal weight, GR levels in liver, thymus, and spleen, and lipid peroxidation levels in the liver and heart. In addition, animals treated with DHEA alone without stress showed a significant (15%) inhibition in body weight gain and an almost 60% decrease in plasma triglyceride levels as compared with control unstressed animals. It is concluded that DHEA acts as an anti-stress hormone in rats, as shown in its antagonizing the effects of repeated immobilization stress on total body weight, adrenal weight, GR levels, and free radical generation.

Animals↗

PubMed2000

Expression and functional analysis of Apaf-1 isoforms. Extra Wd-40 repeat is required for cytochrome c binding and regulated activation of procaspase-9.

Apaf-1 is an important apoptotic signaling molecule that can activate procaspase-9 in a cytochrome c/dATP-dependent fashion. Alternative splicing can create an NH(2)-terminal 11-amino acid insert between the caspase recruitment domain and ATPase domains or an additional COOH-terminal WD-40 repeat. Recently, several Apaf-1 isoforms have been identified in tumor cell lines, but their expression in tissues and ability to activate procaspase-9 remain poorly characterized. We performed analysis of normal tissue mRNAs to examine the relative expression of the Apaf-1 forms and identified Apaf-1XL, containing both the NH(2)-terminal and COOH-terminal inserts, as the major RNA form expressed in all tissues tested. We also identified another expressed isoform, Apaf-1LN, containing the NH(2)-terminal insert, but lacking the additional WD-40 repeat. Functional analysis of all identified Apaf-1 isoforms demonstrated that only those with the additional WD-40 repeat activated procaspase 9 in vitro in response to cytochrome c and dATP, while the NH(2)-terminal insert was not required for this activity. Consistent with this result, in vitro binding assays demonstrated that the additional WD-40 repeat was also required for binding of cytochrome c, subsequent Apaf-1 self-association, binding to procaspase-9, and formation of active Apaf-1 oligomers. These experiments demonstrate the expression of multiple Apaf-1 isoforms and show that only those containing the additional WD-40 repeat bind and activate procaspase-9 in response to cytochrome c and dATP.

Adenosine Triphosphate↗

PubMed2000

Structures of the superoxide reductase from Pyrococcus furiosus in the oxidized and reduced states.

Superoxide reductase (SOR) is a blue non-heme iron protein that functions in anaerobic microbes as a defense mechanism against reactive oxygen species by catalyzing the reduction of superoxide to hydrogen peroxide [Jenney, F. E., Jr., Verhagen, M. F. J. M., Cui, X. , and Adams, M. W. W. (1999) Science 286, 306-309]. Crystal structures of SOR from the hyperthermophilic archaeon Pyrococcus furiosus have been determined in the oxidized and reduced forms to resolutions of 1.7 and 2.0 A, respectively. SOR forms a homotetramer, with each subunit adopting an immunoglobulin-like beta-barrel fold that coordinates a mononuclear, non-heme iron center. The protein fold and metal center are similar to those observed previously for the homologous protein desulfoferrodoxin from Desulfovibrio desulfuricans [Coelho, A. V., Matias, P., Fülöp, V., Thompson, A., Gonzalez, A., and Carrondo, M. A. (1997) J. Bioinorg. Chem. 2, 680-689]. Each iron is coordinated to imidazole nitrogens of four histidines in a planar arrangement, with a cysteine ligand occupying an axial position normal to this plane. In two of the subunits of the oxidized structure, a glutamate carboxylate serves as the sixth ligand to form an overall six-coordinate, octahedral coordinate environment. In the remaining two subunits, the sixth coordination site is either vacant or occupied by solvent molecules. The iron centers in all four subunits of the reduced structure exhibit pentacoordination. The structures of the oxidized and reduced forms of SOR suggest a mechanism by which superoxide accessibility may be controlled and define a possible binding site for rubredoxin, the likely physiological electron donor to SOR.

Crystallography, X-Ray↗

PubMed2000

Reduced neointima hyperplasia of vein bypass grafts in intercellular adhesion molecule-1-deficient mice.

Recently, we established a new mouse model of vein graft arteriosclerosis through the grafting of vena cava to carotid arteries. In many respects, the morphological features of this murine vascular graft model resemble those of human venous bypass graft disease. With this model, we studied the role of intercellular adhesion molecule-1 (ICAM-1) in the development of vein graft arteriosclerosis in ICAM-1-deficient mice. Neointimal hyperplasia of vein grafts in ICAM-1 -/- mice was reduced 30% to 50% compared with that of wild-type control animals. Immmunofluorescent analysis revealed that increased ICAM-1 expression was observed on the endothelium and smooth muscle cells (SMCs) of the grafted veins in wild-type, but not ICAM-1 -/-, mice. MAC-1 (CD11b/18)-positive cells that adhered to the surface of vein grafts in ICAM-1 -/- mice were significantly less as identified with en face immunofluorescence, and these positive cells were more abundant in the intimal lesions of vein grafts in wild-type mice. Furthermore, aortic SMCs cultivated from wild-type mice exhibited high ICAM-1 expression in response to tumor necrosis factor-alpha. When tumor necrosis factor-alpha-stimulated SMCs were incubated with mouse spleen leukocytes, the number of cells that adhered to ICAM-1 -/- SMCs was significantly lower than the number that adhered to ICAM-1 +/+ SMCs, which was markedly blocked through pretreatment of leukocytes with the anti-MAC-1 antibody. Taken together, our findings demonstrate that ICAM-1 is critical in the development of venous bypass graft arteriosclerosis, which provides essential information for therapeutic intervention for vein graft disease in patients undergoing bypass surgery.

Animals↗

PubMed2000

Expression of emmprin by oral squamous cell carcinoma.

A transmembrane glycoprotein recently identified on some tumor cells, extracellular matrix metalloproteinase inducer (EMMPRIN), has been shown to induce metalloproteinase (MMP) production by peritumor fibroblasts (PTF). We examined biopsy specimens of normal human oral mucosa and oral squamous cell carcinoma (SCC) for expression of EMMPRIN. In normal mucosa, EMMPRIN was expressed at the cell membrane throughout the epithelium with a slight enhancement along the basal cell layer. In oral SCC, EMMPRIN was expressed at the cell membrane throughout the entire lesion. Immunofluorescence microscopy localized EMMPRIN to the cell membrane in a highly invasive oral SCC cell line in agreement with our in vivo observations. Function-blocking antibodies to EMMPRIN significantly inhibited oral SCC cell migration on tenascin-C (TN-C) and fibronectin as well as invasion through a reconstituted basement membrane (RBM). We previously showed that soluble factors from SCC cells and PTF are required for deposition of a TN-C matrix. To determine whether EMMPRIN may modulate the release or expression of these soluble factors, we again used function-blocking antibodies. Antibodies to EMMPRIN completely inhibited the organization of TN-C matrices and partially reduced the deposition of FN matrices by oral SCC cell /PTF co-cultures. In addition, antibodies to EMMPRIN perturbed the expression of MMP-2. Moreover, antibodies to MMP-2 perturbed oral SCC cell invasion of an RBM by approx. 75%. Our results demonstrate that EMMPRIN is highly expressed in oral SCC, facilitates tumor cell motility, and mediates TN-C matrix deposition. Taken together, these results suggest that EMMPRIN may help regulate oral squamous cell carcinoma invasion.

Antigens, CD↗

PubMed2000

Antagonistic signals between BMP4 and FGF8 define the expression of Pitx1 and Pitx2 in mouse tooth-forming anlage.

Members of the Pitx/RIEG family of homeodomain-containing transcription factors have been implicated in vertebrate organogenesis. In this study, we examined the expression and regulation of Pitx1 and Pitx2 during mouse tooth development. Pitx1 expression is detected in early development in a widespread pattern, in both epithelium and mesenchyme, covering the tooth-forming region in the mandible, and is then maintained in the dental epithelium from the bud stage to the late bell stage. Pitx2 expression, on the other hand, is restricted to the dental epithelium throughout odontogenesis. Interestingly, from E9.5 to E10.5, the expression domains of Pitx1 and Pitx2, in the developing mandible, overlap with that of Fgf8 but are exclusive to the zone of Bmp4 expression. Bead implantation experiments demonstrate that ectopic expression of Fgf8 can induce/maintain the expression of both Pitx1 and Pitx2 at E9.5. In contrast, Bmp4-expressing tissues and BMP4-soaked beads were able to repress Pitx1 expression in mandibular mesenchyme and Pitx2 expression in the presumptive dental epithelium, respectively. However, the effects of FGF8 and BMP4 are transient. It thus appears that the early expression patterns of Pitx1 and Pitx2 in the developing mandible are regulated by the antagonistic effects of FGF8 and BMP4 such that the Pitx1 and Pitx2 expression patterns are defined. These results indicate that the epithelial-derived signaling molecules are responsible not only for restricting specific gene expression in the dental mesenchyme, but also for defining gene expression in the dental epithelium.

Animals↗

PubMed2000

Biomechanical stress-induced apoptosis in vein grafts involves p38 mitogen-activated protein kinases.

The present study was designed to investigate whether apoptosis occurs in early-stage vein grafts and to determine the mechanisms by which mechanical stress contributes to apoptosis in vascular smooth muscle cells (SMCs). Apoptosis in vessel walls of mouse vein grafts was confirmed by morphological changes and by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL). TUNEL(+) cells in vein grafts 1, 4, and 8 wk postoperatively was 13%, 29%, and 21%, respectively, and apoptosis occurred mainly in veins grafted to arteries, remaining unchanged in vein-to-vein grafts. When mouse, rat, and human arterial SMCs were cultured on a flexible membrane and subjected to cyclic strain stress, apoptosis was observed in a time- and strength-dependent manner. All three types of SMCs showed apoptotic death as confirmed by TUNEL, propidium iodide, and annexin V staining. To further study the signal pathways leading to apoptosis, activities of p38, a subfamily of mitogen-activated protein kinases (MAPKs), were determined. Mechanical stress resulted in p38 MAPK activation, reaching high levels within 8 min. SB 202190, a specific inhibitor for p38 MAPKs, prevented SMC apoptosis in response to mechanical stress. SMC lines stably transfected with a dominant negative rac, an upstream signal transducer, or overexpressing MAPK phosphatase-1, a negative regulator for MAPKs, completely inhibited mechanical stress stimulated p38 activation and abolished mechanical stress-induced apoptosis. Thus, we provide solid evidence that one of the earliest events in venous bypass grafts is apoptosis, in which mechanical stress-induced p38-MAPK activation is responsible for transducing signals leading to apoptosis.-Mayr, M., Li, C., Zou, Y., Huemer, U., Hu, Y., Xu, Q. Biomechanical stress-induced apoptosis in vein grafts involves p38 mitogen-activated protein kinases.

Animals↗

PubMed2000

Further validation of the IBS-QOL: a disease-specific quality-of-life questionnaire.

OBJECTIVE: There has been growing interest in the investigation of health-related quality of life (HRQOL) among patients with gastrointestinal (GI) disorders. We recently reported on the development and preliminary validation of the IBS-QOL, a specific quality-of-life measure for irritable bowel syndrome (IBS). The aim of this study was to determine the longitudinal construct validity (responsiveness) of the IBS-QOL. METHODS: Female patients enrolled in a multicenter treatment trial for functional bowel disorders were studied pre- and posttreatment with the IBS-QOL and other health status measures. Based on the response to treatment for several variables (pain/14-day score, daily function, and days in bed/3 months), patients were stratified into Responders, Partial Responders, and Nonresponders. Change scores in the IBS-QOL were then statistically compared with changes in the other variables to determine their correlation and whether Responders were significantly different from non- and Partial Responders on the IBS-QOL. RESULTS: There was a significant correlation between change scores on the IBS-QOL and the other measures of treatment effect (Pain/14 days, r = 0.25, p < 0.002; Sickness Impact Profile [SIP] Total Score, r = 0.28, p < 0.0004). In addition, the IBS-QOL scores significantly differentiated Responders from Nonresponders for most of the variables tested (regression trend test for Pain/14 days, p < 0.04; SIP Total, p < 0.0001; SIP Physical, p < 0.0001; SIP Psychosocial, p < 0.002, and SIP Eating, p < 0.04). CONCLUSION: The IBS-QOL is responsive to treatment in a referral-based clinical population of patients with functional bowel disorders.

Adult↗

PubMed2000

Hyperexpression and activation of extracellular signal-regulated kinases (ERK1/2) in atherosclerotic lesions of cholesterol-fed rabbits.

A hallmark of hyperlipidemia-induced atherosclerosis is altered gene expression that initiates cell proliferation and (de)differentiation in the intima of the arterial wall. The molecular signaling that mediates this process in vivo has yet to be identified. Extracellular signal-regulated kinases (ERKs) are thought to play a pivotal role in transmitting transmembrane signals required for cell proliferation in vitro. The present studies were designed to investigate the activity, abundance, and localization of ERK1/2 in atherosclerotic lesions of cholesterol-fed rabbits. Immunofluorescence analysis revealed abundant and heterogeneous distribution of ERK1/2, mainly localized in the cap and basal regions of atheromas. A population of ERK-enriched cells was identified as alpha-actin-positive smooth muscle cells (SMCs). ERK1 and 2 were heavily phosphorylated on tyrosyl residues and coexpressed with proliferating cell nuclear antigen in atherosclerotic lesions. ERK1/2 protein levels in protein extracts from atherosclerotic lesions were 2- to 3-fold higher than the vessels of chow-fed rabbits, and their activities were elevated 3- to 5-fold over those of the normal vessel. SMCs derived from atherosclerotic lesions had increased migratory/proliferative ability and higher ERK activity in response to LDL stimulation compared with cells from the normal vessel. Inhibition of ERK activation by PD98059, a specific inhibitor of mitogen-activated protein kinase kinases (MEK1/2), abrogated LDL-induced SMC proliferation in vitro. Taken together, our findings support the proposition that persistent activation and hyperexpression of ERK1/2 may be a critical element to initiate and perpetuate cell proliferation during the development of atherosclerosis.

Animals↗

PubMed2000

Mouse model of transplant arteriosclerosis: role of intercellular adhesion molecule-1.

Transplant-accelerated arteriosclerosis in coronary arteries is the major limitation to long-term survival of patients with heart transplantation. The pathogenesis of this disease is not fully understood. Herein, we describe a simplified model of artery allografts in the mouse that allows us to take advantage of transgenic, knockout, or mutant animals. Common carotid arteries or aortic vessels were end-to-end allografted into carotid arteries between C57BL/6J and BALB/c mice. Neointimal lesions were observed as early as 2 weeks after surgery and had progressed at 4 and 6 weeks postoperatively. The lumen of grafted arteries was significantly narrowed due to neointima hyperplasia 4 weeks after transplantation. Using this model, we studied the role of intercellular adhesion molecule-1 (ICAM-1) in the development of transplant arteriosclerosis in ICAM-1-deficient mice. Neointimal lesions of artery grafts from ICAM-1 -/- C57BL/6J to BALB/c mice were reduced up to 60% compared with wild-type controls. MAC-1 (CD11b/18)-positive cells adhering to the surface of ICAM-1 -/- artery grafts were significantly less as identified by en face immunofluorescence, and these positive cells were more abundant in intimal lesions of artery grafts in wild-type mice. Furthermore, the major cell component of neointimal lesions 4 weeks after surgery was found to be alpha-actin-positive smooth muscle cells, which were significantly reduced in lesions of ICAM-1 -/- artery grafts. Thus, this model has been proven to be useful for understanding the mechanism of transplant arteriosclerosis. Our findings demonstrate that ICAM-1 is critical in the development of allograft arteriosclerosis via mediation of leukocyte adhesion to, and infiltration into, the vessel wall.

Actins↗

PubMed2000

Ras/Rac-Dependent activation of p38 mitogen-activated protein kinases in smooth muscle cells stimulated by cyclic strain stress

p38, a subfamily of the mitogen-activated protein kinases (MAPKs), is a crucial signal transducer between a variety of extracellular stimuli and gene expression in mammalian cells. This kinase is activated in cultured cells stimulated by heat shock, osmotic stress, and proinflammatory cytokines, but a similar activation of p38 MAPKs in vascular smooth muscle cells (SMCs) stimulated by mechanical stress has yet to be studied. We studied signal pathways leading to time- and strength-dependent p38 activation in rat SMCs in response to cyclic strain stress. p38 phosphorylation in stressed SMCs showed maximal activation at 10 minutes. This activation was significantly inhibited by pretreatment of the SMCs with pertussis toxin, a G-protein antagonist, and enhanced by treatment with suramin, a growth factor receptor antagonist, but opposite effects in the activation of extracellular signal-regulated kinases stimulated by mechanical forces were found. p38 activation was markedly reduced in stressed SMCs after protein kinase C depletion. Interestingly, SMC lines stably expressing dominant-negative ras (ras N17) or rac1 (rac1 N17) almost abolished p38 phosphorylation induced by cyclic strain stress. When p38 activation was inhibited by the specific inhibitor SB 202190, SMC migration, determined in a Boyden chamber in response to stimulation with platelet-derived growth factor-BB, and SMC proliferation, stimulated by cyclic strain stress, were abrogated. Thus, we provide the first evidence that cyclic strain stress rapidly activates p38 MAPKs via activation of protein kinase C ras/rac signal pathways, suggesting that p38 MAPKs are important signal transducers mediating the mechanical stress-induced cell responses essential for SMC migration and proliferation.

Journal Article↗

PubMed2000

A new function of BMP4: dual role for BMP4 in regulation of Sonic hedgehog expression in the mouse tooth germ.

The murine tooth development is governed by sequential and reciprocal epithelial-mesenchymal interactions. Multiple signaling molecules are expressed in the developing tooth germ and interact each other to mediate the inductive tissue interactions. Among them are Sonic hedgehog (SHH), Bone Morphogenetic Protein-2 (BMP2) and Bone Morphogenetic Protein-4 (BMP4). We have investigated the interactions between these signaling molecules during early tooth development. We found that the expression of Shh and Bmp2 is downregulated at E12.5 and E13.5 in the dental epithelium of the Msx1 mutant tooth germ where Bmp4 expression is significantly reduced in the dental mesenchyme. Inhibition of BMP4 activity by noggin resulted in repression of Shh and Bmp2 in wild-type dental epithelium. When implanted into the dental mesenchyme of Msx1 mutants, beads soaked with BMP4 protein were able to restore the expression of both Shh and Bmp2 in the Msx1 mutant epithelium. These results demonstrated that mesenchymal BMP4 represents one component of the signal acting on the epithelium to maintain Shh and Bmp2 expression. In contrast, BMP4-soaked beads repressed Shh and Bmp2 expression in the wild-type dental epithelium. TUNEL assay indicated that this suppression of gene expression by exogenous BMP4 was not the result of an increase in programmed cell death in the tooth germ. Ectopic expression of human Bmp4 to the dental mesenchyme driven by the mouse Msx1 promoter restored Shh expression in the Msx1 mutant dental epithelium but repressed Shh in the wild-type tooth germ in vivo. We further demonstrated that this regulation of Shh expression by BMP4 is conserved in the mouse developing limb bud. In addition, Shh expression was unaffected in the developing limb buds of the transgenic mice in which a constitutively active Bmpr-IB is ectopically expressed in the forelimb posterior mesenchyme and throughout the hindlimb mesenchyme, suggesting that the repression of Shh expression by BMP4 may not be mediated by BMP receptor-IB. These results provide evidence for a new function of BMP4. BMP4 can act upstream to Shh by regulating Shh expression in mouse developing tooth germ and limb bud. Taken together, our data provide insight into a new regulatory mechanism for Shh expression, and suggest that this BMP4-mediated pathway in Shh regulation may have a general implication in vertebrate organogenesis.

Animals↗

PubMed1999

Ascorbate and glutathione regulation in hibernating ground squirrels.

Ground squirrels withstand up to 90% reductions in cerebral blood flow during hibernation as well as rapid reperfusion upon periodic arousals from torpor. Metabolic suppression likely plays a primary adaptive role which allows hibernating species to tolerate such phenomena. However, several other aspects of hibernation physiology are also consistent with tolerance to dramatic fluctuations in cerebral blood flow, suggesting that multiple neuroprotective adaptations may work in concert during hibernation. The purpose of the present work was to study the dynamics of the low molecular weight antioxidants, ascorbate and glutathione (GSH), during hibernation. Alterations in concentrations of ascorbate during hibernation and arousal in two species of hibernating ground squirrels suggest that it could play a protective role during hibernation or arousal. Samples were collected during the hibernation season from arctic ground squirrels (AGS; Spermophilus parryii) and 13-lined ground squirrels (TLS; S. tridecemlineatus) during prolonged torpor and in squirrels that did not hibernate or had not been hibernating for several weeks. We determined antioxidant levels in plasma, cerebrospinal fluid (CSF), and in frontal cortex, hippocampus and cerebellum using high-performance liquid chromatography (HPLC). Plasma ascorbate concentrations increased dramatically (3-4-fold) in both species during hibernation and rapidly returned to prehibernation levels upon arousal. By contrast, plasma GSH concentrations fell slightly or remained stable during hibernation. Ascorbate levels in the CSF doubled in hibernating AGS (not determined in TLS), while brain ascorbate content fell slightly (10-15%) in both species. Substantial increases in plasma and CSF ascorbate concentrations suggest that this antioxidant could play a protective role during hibernation and reperfusion upon arousal from hibernation.

Animals↗

PubMed1999

The Arabidopsis SKP1-LIKE1 gene is essential for male meiosis and may control homologue separation.

The yeast and human SKP1 genes regulate the mitotic cell cycle but are not yet known to be required for meiosis. Nine Arabidopsis SKP1 homologues have been uncovered and are named ASK1 through ASK9. Here, we report the isolation and characterization of a male sterile Arabidopsis mutant and show that the mutant defect was caused by a Ds transposon insertion into the ASK1 gene. In the ask1-1 mutant, abnormal microspores exhibit a range of sizes. Furthermore, during mutant male meiosis, although homologous chromosome pairing appeared normal at metaphase I, chromosome segregation at anaphase I is unequal, and some chromosomes are abnormally extended. Therefore, in ask1-1, at least some homologues remain associated after metaphase I. In addition, immunofluorescence microscopy indicates that the mutant spindle morphology at both metaphase I and early anaphase I is normal; thus, the abnormal chromosome segregation is not likely caused by a spindle defect. Because the yeast Skp1p is required for targeting specific proteins for ubiquitin-mediated proteolysis, we propose that ASK1 controls homologue separation by degrading or otherwise removing a protein that is required directly or indirectly for homologue association before anaphase I.

Amino Acid Sequence↗

PubMed1999

Structural and functional characterization of the 5'-flanking region of the rat and human cytochrome P450 2E1 genes: identification of a polymorphic repeat in the human gene.

Cytochrome P450 2E1 (CYP2E1) is a toxicologically very important enzyme with a high extent of interindividual variability in expression. We sequenced and characterized the 5'-flanking region of the human and rat CYP2E1 genes. The identity between the human and rat sequences (-3.8 kb to +1 kb) was generally between 35 and 60%, and the most similar regions were found in the proximal part of the sequence. Two more distant regions at -1.6 to -2.0 kb and -2.5 to -2. 8 kb in the human sequence were also found to have high identity to the rat sequence. A polymorphic repeat sequence in the human gene was found between -2178 to -1945 bp. The common allele (CYP2E1*1C) contained 6 repeats (each 42-60 bp long) and the rare allele (CYP2E1*1D) had 8 repeats with an allele frequency of 1% among Caucasians and 23% among Chinese. The CYP2E1 5'-flanking regions of the human (-3712 bp to +10 bp) and rat (-3685 bp to +28 bp) genes were ligated in front of a luciferase reporter gene and transfected into rat hepatoma Fao and human hepatoma B16A2 cells. Important species specificity was noted in the control of gene expression and regions of negative and positive cis-acting elements were localized. No difference was seen in the constitutive expression between the two polymorphic forms. The importance of this repeat polymorphism for high and low inducible CYP2E1 phenotypes is discussed.

Alleles↗