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H Li

Publications and source records attributed to H Li.

At least 1,405 records · Page 78Linked to original sources
PubMed1996

Defining the enzyme binding domain of a ribonuclease III processing signal. Ethylation interference and hydroxyl radical footprinting using catalytically inactive RNase III mutants.

Ethylation interference and hydroxyl radical footprinting were used to identify substrate ribose-phosphate backbone sites that interact with the Escherichia coli RNA processing enzyme, ribonuclease III. Two RNase III mutants were employed, which bind substrate in vitro similarly as wild-type enzyme, but lack detectable phosphodiesterase activity. Specifically, altering glutamic acid at position 117 to lysine or alanine uncouples substrate binding from cleavage. The two substrates examined are based on the bacteriophage T7 R1.1 RNase III processing signal. One substrate, R1.1 RNA, undergoes accurate single cleavage at the canonical site, while a close variant, R1.1[WC-L] RNA, undergoes coordinate double cleavage. The interference and footprinting patterns for each substrate (i) overlap, (ii) exhibit symmetry and (iii) extend approximately one helical turn in each direction from the RNase III cleavage sites. Divalent metal ions (Mg2+, Ca2+) significantly enhance substrate binding, and confer stronger protection from hydroxyl radicals, but do not significantly affect the interference pattern. The footprinting and interference patterns indicate that (i) RNase III contacts the sugar-phosphate backbone; (ii) the RNase III-substrate interaction spans two turns of the A-form helix; and (iii) divalent metal ion does not play an essential role in binding specificity. These results rationalize the conserved two-turn helix motif seen in most RNase III processing signals, and which is necessary for optimal processing reactivity. In addition, the specific differences in the footprint and interference patterns of the two substrates suggest why RNase III catalyzes the coordinate double cleavage of R1.1[WC-L] RNA, and dsRNA in general, while catalyzing only single cleavage of R1.1 RNA and related substrates in which the scissle bond is within an asymmetric internal loop.

Base Sequence↗
PubMed1996

Molecular cloning and chromosomal localization of the human cyclin C (CCNC) and cyclin E (CCNE) genes: deletion of the CCNC gene in human tumors.

The human Gi-phase cyclins are important regulators of cell cycle progression that interact with various cyclin-dependent kinases and facilitate entry into S-phase. We have confirmed the localization of the human cyclin C (CCNC) gene to chromosome 6q21 and of human cyclin E (CCNE) to 19q12. The CCNC gene structure was also determined, and we have shown that it is deleted in a subset of acute lymphoblastic leukemias, including a patient sample containing a t(2;6)(p21;q15), with no apparent cytogenetic deletion. Single-strand conformational polymorphism analysis of the remaining CCNC allele from patients with a deletion of one allele established that there were no further mutations within the exons or the flanking intronic sequences. These results suggest either that haploinsufficiency of the cyclin C protein is sufficient to promote tumorigenesis or that the important tumor suppressor gene is linked to the CCNC locus.

Chromosome Mapping↗
PubMed1996

A single MEF2 site governs desmin transcription in both heart and skeletal muscle during mouse embryogenesis.

Desmin, the muscle-specific intermediate filament protein is one of the earliest known myogenic makers both in heart and in somites. We have previously shown that high levels of desmin expression in the skeletal cell line C2C12 are due to a distal enhancer, which contains a muscle-specific factor-(2MEF2) binding site, adjacent to an E box, the binding site of the myogenic Helix-Loop-Helix (mHLH) regulators. We have further shown that MEF2C, a myocyte restricted member of the MEF2 family and all four mHLH factors can bind to their corresponding sites and through a cooperation with a second proximal E box can transactivate the desmin promoter. To study the significance of these regulatory elements in vivo, we have generated transgenic mice with desmin-lacZ reporters, intact or mutated at the MEF2 and E box of the enhancer. We show that the cis-acting DNA sequences within the 1-kb 5' flanking region of the mouse desmin gene are sufficient to direct appropriate temporal transcription both in heart and in skeletal muscle during mouse embryogenesis. Mutation at the MEF2 site completely suppressed transcription of the linked lacZ transgene in both developing heart and somites of the embryos. Mutation of the E box only suppressed activation in skeletal muscle precursors (somites and limb buds) but not in cardiac muscle. These data demonstrate that the MEF2 site is indispensable for the desmin enhancer function both in heart and in skeletal muscle. In addition, MEF2 cooperation with the mHLH regulators is absolutely necessary for proper transcriptional activity during embryonic skeletal muscle development.

Animals↗
PubMed1996

Cyp1a2(-/-) null mutant mice develop normally but show deficient drug metabolism.

Cytochrome P450 1A2 (CYP1A2) is a predominantly hepatic enzyme known to be important in the metabolism of numerous foreign chemicals of pharmacologic, toxicologic, and carcinogenic significance. CYP1A2 substrates include aflatoxin B1, acetaminophen, and a variety of environmental arylamines. To define better the developmental and metabolic functions of this enzyme, we developed a CYP1A2-deficient mouse line by homologous recombination in embryonic stem cells. Mice homozygous for the targeted Cyp1a2 gene, designated Cyp1a2(-/-), are completely viable and fertile; histologic examination of 15-day embryos, newborn pups, and 3-week-old mice revealed no abnormalities. No CYP1A2 mRNA was detected by Northern blot analysis. Moreover, mRNA levels of Cyp1a1, the other gene in the same subfamily, appear unaffected by loss of the Cyp1a2 gene. Because the muscle relaxant zoxazolamine is a known substrate for CYP1A2, we studied the Cyp1a2(-/-) genotype by using the zoxazolamine paralysis test: the Cyp1a2(-/-) mice exhibited dramatically lengthened paralysis times relative to the Cyp1a2(+/+) wild-type animals, and the Cyp1a2(+/-) heterozygotes showed an intermediate effect. Availability of a viable and fertile CYP1A2-deficient mouse line will provide a valuable tool for researchers wishing to define the precise role of CYP1A2 in numerous metabolic and pharmacokinetic processes.

Animals↗
PubMed1996

Gsh-1, an orphan Hox gene, is required for normal pituitary development.

The anterior pituitary regulates the function of multiple organ systems as well as body growth, and in turn is controlled by peptides released by the hypothalamus. We find that mutation of the Gsh-1 homeobox gene results in pleiotropic effects on pituitary development and function. Homozygous mutants exhibit extreme dwarfism, sexual infantilism and significant perinatal mortality. The mutant pituitary is small in size and hypocellular, with severely reduced numbers of growth hormone- and prolactin-producing cells. Moreover, the pituitary content of a subset of pituitary hormones, including growth hormone, prolactin and luteinizing hormone, is significantly decreased. The hypothalamus, although morphologically normal, is also perturbed in mutants. The gsh-1 gene is shown to be essential for growth hormone-releasing hormone (GHRH) gene expression in the arcuate nucleus of the hypothalamus. Further, sequence and electrophoretic mobility shift data suggest the Gsh-1 and GHRH genes as potential targets regulated by the Gsh-1-encoded protein. The mutant phenotype indicates a critical role for Gsh-1 in the genetic hierarchy of the formation and function of the hypothalamic-pituitary axis.

Amino Acid Sequence↗
PubMed1996

Blockage of urokinase receptor reduces in vitro the motility and the deformability of endothelial cells.

The binding of urokinase (u-PA) to its cell surface receptor (u-PAR) is critical for tumor cell invasion. Here, we report that the distribution of this binding by a u-PAR antagonist ATF-HSA inhibits in vitro the motility of endothelial cells in a dose-dependent manner. This inhibition was also observed when the cells were first stimulated with potent angiogenic factors, including bFGF or VEGF. [3H]thymidine incorporation assay demonstrated that ATF-HSA did not affect the cell proliferation. ATF-HSA was more potent than plasmin inhibitors, suggesting that it exerts its effects not solely by inhibiting the remodeling of the extracellular matrix. In fact, analysis of the cell shape change during migration revealed for the first time that its effect is related to a decrease in cell deformability. These results suggest that u-PAR antagonist may be a new approach to control angiogenesis.

Aprotinin↗
PubMed1996

Progesterone inhibits cholesterol biosynthesis in cultured cells. Accumulation of cholesterol precursors.

Cells acquire cholesterol through endogenous synthesis and through receptor-mediated uptake of cholesterol-rich low density lipoprotein (LDL). Esterification of LDL-derived cholesterol is catalyzed by acyl-CoA:cholesterol acyltransferase (ACAT) in the endoplasmic reticulum (ER). Progesterone inhibits esterification, and, although the mechanism of inhibition is not completely understood, this inhibition results from progesterone's ability to inhibit the activity of multiple drug resistance (MDR) P-glycoproteins (P. DeBry and J. E. Metherall, submitted for publication). In the current manuscript, we demonstrate that progesterone inhibits cholesterol biosynthesis resulting in the accumulation of a number of sterol precursors. In Chinese hamster ovary (CHO) cells, high concentrations (100 microM) of progesterone completely blocked cholesterol production, resulting in the accumulation of lanosterol and a lanosterol precursor. Lower concentrations (40 microM) of progesterone cause plasma membrane accumulation of several sterol products. The majority of these sterols are precursors of cholesterol since they were efficiently converted to cholesterol upon removal of progesterone from the culture medium. Although very high concentrations (> 200 microM) of progesterone killed CHO cells, their growth was restored by the addition of cholesterol to the growth medium, indicating that progesterone toxicity resulted from cholesterol auxotrophy. The effect of progesterone was not unique to CHO cells; progesterone also inhibited cholesterol biosynthesis in all human cell lines tested. These observations suggest that a common progesterone-sensitive pathway is involved in both cholesterol biosynthesis and the processing of LDL-derived cholesterol.

Animals↗
PubMed1996

Role of multidrug resistance P-glycoproteins in cholesterol biosynthesis.

Multidrug resistance (MDR) P-glycoproteins were first recognized for their ability to catalyze ATP-dependent efflux of cytotoxic agents from tumor cells when overexpressed. Despite extensive study, little is known about the normal substrate(s) and normal cellular function of these proteins. In the accompanying manuscript (Metherall, J. E., Waugh, K., and Li, H. (1996) J. Biol. Chem. 271, 2627-2633), we demonstrate that progesterone inhibits cholesterol biosynthesis, causing the accumulation of a number of cholesterol precursors. In the current manuscript, we use several criteria to show that the progesterone receptor is not involved in this inhibition. Rather, we demonstrate that progesterone inhibits cholesterol biosynthesis by interfering with MDR activity. We show that a steroid hormone's ability to inhibit cholesterol biosynthesis is correlated with: 1) its general hydrophobicity and 2) its ability to inhibit MDR activity. The only exception to this finding is beta-estradiol, which is a more potent inhibitor of cholesterol biosynthesis than expected based solely on hydrophobicity and MDR inhibition. We further demonstrate that nonsteroidal inhibitors of MDR also inhibit cholesterol biosynthesis. Since MDR activity is required for esterification of LDL-derived cholesterol (P. DeBry and J. E. Metherall, submitted for publication), we investigated the relationship between these phenomena and show that inhibition of cholesterol esterification does not cause inhibition of cholesterol biosynthesis and that inhibition of cholesterol biosynthesis does not cause inhibition of cholesterol esterification. We propose a model in which MDR is required for transport of sterols from the plasma membrane to the endoplasmic reticulum (ER). Inhibiting this transport prevents cholesterol esterification and cholesterol biosynthesis by preventing sterol substrates from reaching ER-resident enzymes.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

Estrogen reduces myointimal proliferation after balloon injury of rat carotid artery.

BACKGROUND: Vascular disease progresses more slowly in females with functional ovaries than in males. The mechanisms of this vasoprotective effect of female sex are incompletely understood. This study tested (1) whether there is a sex difference in the development of myointimal proliferation after balloon injury of the rat carotid artery in vivo, (2) whether this response is estrogen or androgen dependent, and (3) whether there is a sexual dimorphism in expression of the c-myc proto-oncogene in intact and/or damaged rat carotid arteries. METHODS AND RESULTS: Ten-week-old male and female Sprague-Dawley rats were either gonadectomized or studied intact. Gonadectomized rats of both sexes were implanted with estradiol, testosterone, or nothing (control) 3 days before vascular injury. Two weeks later, the rats were killed by overdose of pentobarbital, and the injured right and uninjured control left carotid arteries were fixed and subjected to morphometric analysis for evaluation of the degree of myointimal thickening. Separate groups of intact male and female rats were killed at 1 and 2 hours after vascular injury, and total RNA from injured and uninjured vessels was subjected to Northern blot analysis for assessment of steady state c-myc mRNA levels. Neointimal area and the ratio of neointimal to medial area were significantly less in intact female rats than in intact male rats (P < .05). Gonadectomy of female rats was associated with a greater increase in neointima formation after balloon injury than that observed in intact females (P < .05), but testosterone replacement did not further enhance this response. Estradiol treatment significantly inhibited myointimal proliferation after vascular injury in gonadectomized rats of both sexes (P < .05). Neither gonadectomy nor gonadectomy plus testosterone replacement altered the myointimal proliferative response to balloon injury in male rats. Steady state c-myc mRNA levels were detectable in undamaged carotid arteries in intact rats of both sexes and were significantly greater in males than in females; c-myc mRNA levels were increased in both sexes after carotid injury, but the response was significantly larger in magnitude and more rapid in males than in females. CONCLUSIONS: These data indicate that the sex difference in myointimal proliferation after vascular injury is estrogen dependent. C-myc gene expression is greater in the undamaged carotid artery of the male than in that of the female, and the responsiveness of this gene to balloon injury of the artery is more rapid and more robust in the male than in the female rat. These findings have direct implications for the prevention and treatment of vascular disease in humans.

Animals↗
PubMed1996

Inhibition of IFN-stimulated gene expression and IFN induction of cytolytic resistance to natural killer cell lysis correlate with E1A-p300 binding.

Treatment of target cells with IFN induces resistance to NK cell lysis. This process is blocked by expression of E1A gene products in adenovirus (Ad)-infected and Ad-transformed cells. We compared the ability of adenovirus serotype 5 (Ad5) E1A exon 1 mutants to inhibit the induction of cytolytic resistance by IFN and block IFN-stimulated gene expression with their capacity to bind the cellular proteins p105 (retinoblastoma gene product), p107, and p300. E1A mutants that did not express conserved region 3 (CR3; residues 138-184) or contained deletions in the nonconserved regions between residues 26-35 or 86-120, bound p105, p107, and p300 and were not impaired in their capacity to block IFN-stimulated gene expression or IFN's induction of cytolytic resistance. E1A mutants with deletions in CR2 (residues 121-138) could not bind p105 or p107, but blocked IFN-stimulated gene expression and IFN's induction of cytolytic resistance. In contrast, mutants in CR1 or the N-terminal nonconserved region (residues 2, 4-25, and 48-60), which define E1A's binding site for p300, were unable to block either IFN-stimulated gene expression or IFN's induction of cytolytic resistance. We conclude that E1A's capacity to block both IFN-stimulated gene expression and IFN's induction of cytolytic resistance appears to be transduced through a pathway that involves E1A-p300 binding. The capacity of E1A to block IFN's induction of cytolytic resistance is probably secondary to E1A's more general ability to inhibit IFN-stimulated gene expression.

Cytotoxicity, Immunologic↗
PubMed1996

The DExH box protein Suv3p is a component of a yeast mitochondrial 3'-to-5' exoribonuclease that suppresses group I intron toxicity.

The yeast mitochondrial protein Suv3p is a putative NTP-dependent RNA helicase. Here we report that in cells lacking Suv3p, there is an approximately 50-fold increase in the excised form of the group I intron omega of the mitochondrial 31S rRNA gene. Surprisingly, little mature 21S rRNA accumulates in those cells; instead, unligated 21S rRNA exons appear. Intron overaccumulation could lead to spliced exon reopening via a reaction known to be catalyzed by group I introns in vitro. We also show that Suv3p is a functional component of a novel mitochondrial NTP-dependent 3'-to-5' exoribonuclease activity that can degrade group I intron RNAs. These findings account for group I intron overaccumulation in cells lacking Suv3p and define a novel function for putative RNA helicases in direct RNA degradation.

Base Sequence↗
PubMed1996

Identification of the site in the substance P (NK-1) receptor for modulation of peptide binding by sulfhydryl reagents.

Substance P (SP) is a peptide neurotransmitter that is involved in multiple responses in both the central and the peripheral nervous systems through a G-protein-coupled contains a number of conserved cysteine residues. To localize and identify the cysteine residues that participate in receptor binding, intact Chinese hamster ovary cells expressing the SP receptor were treated with various sulfhydryl reagents and the effect of these reagents on radioiodinated SP binding affinity and dissociation rate was determined. We used a series of amphiphilic maleimide derivatives in which the reactive maleimide group penetrates to different depths within the plane of membrane. Only the maleimide derivatives with intermediate chain lengths modified receptor binding properties, indicating that the reactive sulfhydryl group is located within a transmembrane domain of the receptor close (within 1.7 nm) to the extracellular border. Since peptide binding to a mutant receptor C199S, in which Cys-199 was replaced by a serine, was found to be insensitive to modulation by sulfhydryl reagents, this reactive sulfhydryl group is on Cys-199 of the receptor. Receptor occupancy by SP protects Cys-199 from modification and thus this residue is either located at or conformationally linked to the SP binding site.

Alkylation↗
PubMed1996

Mixed allogeneic chimerism induced by a sublethal approach prevents autoimmune diabetes and reverses insulitis in nonobese diabetic (NOD) mice.

Evidence in experimental models suggests that many autoimmune diseases can be prevented by transplantation of bone marrow from disease-resistant donors. For potential clinical application, it would be important to avoid the morbidity and mortality associated with lethal conditioning and achieve mixed chimerism using less than complete recipient ablation. We report here for the first time that stable chimerism achieved in NOD mice using a sublethal radiation-based conditioning approach is sufficient to prevent beta-cell destruction and abrogate insulitis in prediabetic NOD mice. The percentage of NOD mouse recipients (8 wk of age) that engrafted with donor bone marrow correlated with the dose of irradiation and number of bone marrow cells transplanted. Engraftment of B10.BR bone marrow occurred in > or = 94% of animals receiving > or = 750 cGy of total body irradiation before bone marrow transplantation and > or = 30 x 10(6) bone marrow cells, while reproducible engraftment did not occur at radiation doses of less than 700 cGy and cellular doses of less than 30 x 10(6) bone marrow cells. All chimeric animals remained free of diabetes (n = 38) for 10 mo following bone marrow transplantation. Moreover, in all animals examined, no insulitis was present from 12 to 36 wk following reconstitution. In striking contrast, 61% (22 of 36) of NOD recipients that were conditioned but did not receive bone marrow developed acute diabetes by 12 mo. Insulitis was present in all remaining animals. These results suggest that allogeneic chimerism achieved using a sublethal conditioning approach can prevent the onset of diabetes and even reverse preexisting insulitis in NOD mice.

Animals↗
PubMed1996

Elimination of fat emulsion particles from plasma during glucose infusion.

To determine the optimal rate of infusion of a fat emulsion (long-chain triglycerides; LCTs) for parenteral nutrition, the rate of elimination of triglycerides from plasma was measured on two occasions in three healthy subjects, once while receiving LCTs alone (study I) and once while receiving a combined infusion of glucose and LCTs (study 2). In study 1 a hypertriglyceridaemic clamp was set up by raising the concentration of triglycerides to 2 mmol l(-1) (60-min priming infusion at 0.2 g LCTs per kg body-weight per h) and maintaining this value for 300 min. In study 2 a constant infusion of glucose at a rate of 0.32 g per kg body-weight per h was given throughout the 420-min test, and the hypertriglyceridaemic clamp was set up after 70 min. The infusion of glucose induced a progressive increase in the mean(s.d.) concentration of insulin from 4.3(0.4) microU ml(-1) to a plateau value of 11.0(0.2) microU ml(-1) which was sustained during the last 310 min of the study. During the steady state of the hypertriglyceridaemic clamp, the concentration of triglycerides (2.16(0.17) versus 2.12(0.20) mmol l(-1), P = 0.26), as well as the mean(s.d.) rates of infusion of LCTs (0.12(0.08) versus 0.12(0.04) per kg body-weight per h, P = 0.38), did not differ between studies 1 and 2. Thus, an infusion of glucose (and the associated physiological increase in the concentration of insulin) at doses commonly used in parenteral nutrition does not influence the rate of elimination of triglycerides from plasma in normal subjects.

Adult↗
PubMed1996

Hypergastrinemia increases proliferation of gastroduodenal epithelium during gastric ulcer healing in rats.

We investigated if hypergastrinemia exerted any influence on the proliferation of gastroduodenal epithelium during the healing of ulcers in rats. A mucosal ulcer was induced in the corpus region of the stomach in three groups of rats, which were then given vehicle, omeprazole (400 mumol/kg/day), or gastrin-17 (60 nmol/kg/day) for three or six days. A fourth group of unoperated rats served as controls. One hour before killing, [3H]thymidine was injected. The ulcer margin and corresponding control tissues were excised and processed for light microscopic determination of epithelial labeling index (LI), mitotic index, and apoptotic index. LI was also determined in other parts of the gastroduodenal mucosa. Three and six days after the ulcer operation, the LI in the vehicle-treated ulcer rats was significantly increased in the ulcer margin and in the duodenum, in comparison with the intact controls. In the ulcer margin, the mitotic index was significantly increased, in parallel with the LI; the apoptotic index remained at the control level. The LI in the ulcer margin was increased further after administration of omeprazole or gastrin-17, which elevated the plasma gastrin levels by 5-15 times. It is concluded that hypergastrinemia may increase cell proliferation in the ulcer margin, which may accelerate the rate of healing.

Animals↗