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C Li

Publications and source records attributed to C Li.

At least 181 records · Page 10Linked to original sources
PubMed2001

An extended Escherichia coli "selenocysteine insertion sequence" (SECIS) as a multifunctional RNA structure.

The genetic code, once thought to be rigid, has been found to permit several alternatives in its reading. Interesting alternative relates to the function of the UGA codon. Usually, it acts as a stop codon, but it can also direct the incorporation of the amino acid selenocysteine into a polypeptide. UGA-directed selenocysteine incorporation requires a cis-acting mRNA element called the "selenocysteine insertion sequence" (SECIS) that can form a stem-loop RNA structure. Here we discuss our investigation on the E. coli SECIS. This includes the follows: 1) The nature of the minimal E. coli SECIS. We found that in E. coli only the upper-stem and loop of 17 nucleotides of the SECIS is necessary for selenocysteine incorporation on the condition that it is located in the proper distance from the UGA [34]; 2) The upper stem and loop structure carries a bulged U residue that is required for selenocysteine incorporation [34] because of its interaction with SelB; and 3) We described an extended fdhF SECIS that includes the information for an additional function: The prevention of UGA readthrough under conditions of selenium deficiency [35]. This information is contained in a short mRNA region consisting of a single C residue adjacent to the UGA on its downstream side, and an additional segment consisting of the six nucleotides immediately upstream from it. These two regions act independently and additively and probably through different mechanisms. The single C residue acts as itself; the upstream region acts at the level of the two amino acids, arginine and valine, for which it codes. These two codons at the 5' side of the UGA correspond to the ribosomal E and P sites. Finally, we present a model for the E. coli fdhF SECIS as a multifunctional RNA structure containing three functional elements. Depending on the availability of selenium the SECIS enables one of two alternatives for the translational machinery: Either selenocysteine incorporation into a polypeptide or termination of the polypeptide chain.

Amino Acid Sequence↗
PubMed2001

Localization of Lutheran, a novel laminin receptor, in normal, knockout, and transgenic mice suggests an interaction with laminin alpha5 in vivo.

Laminins are major components of all basement membranes. One laminin that has garnered particular interest, due to its widespread expression pattern and importance during development, is the laminin alpha5 chain. In vitro studies have suggested that the Lutheran blood group glycoprotein/basal cell adhesion molecule (Lu), an Ig superfamily transmembrane protein, is a receptor for laminins containing the alpha5 chain. However, there are no in vivo studies showing that these proteins are capable of interacting in tissues. We have isolated the mouse ortholog of Lu and characterized its expression and localization in mouse tissues. Lu was primarily found on the basal surface of epithelial cells and on muscle cells adjacent to basement membranes containing laminin alpha5. In addition, there was both a dramatic reduction in the basal concentration of Lu in mice lacking laminin alpha5, and a significant increase in Lu protein in transgenic mice overexpressing laminin alpha5. Together, these data provide the first in vivo evidence for an interaction between Lu and laminin alpha5 and support the hypothesis that Lu is a laminin alpha5 receptor. We propose that laminin alpha5 is involved in concentrating Lu on the basal surface of epithelial cells. This may be one mechanism by which basement membrane signals are transmitted to the cell.

Animals↗
PubMed2001

Fibroblast growth factor receptor 2 (Fgfr2) plays an important role in eyelid and skin formation and patterning.

Initiating as protruding ridges above and below the optic vesicle, the eyelids of mice grow across the eye and temporarily fuse in fetal life. Mutations of a number of genes disrupt this developmental process and result in a birth defect, "open-eyelids at birth." Here we show that a critical event for eyelid induction occurs at embryonic day 11.5 (E11.5) when the single cell-layered ectoderm in the presumptive eyelid territory increases proliferation and undergoes morphologic transition to form cube-shaped epithelial cells. Using embryos lacking the Fgfr2 Ig domain III (Fgfr2(DeltaIII/DeltaIII)) generated by tetraploid rescue and chimeric embryo formation approaches, we demonstrate that this event is controlled by Fgfr2 signals as the Fgfr2(DeltaIII/DeltaIII) mutation blocks these changes and results in embryos without eyelids. Fgfr2 and its ligands are differentially expressed in the ectoderm and underlying mesenchyme and function in a reciprocal interacting loop that specifies eyelid development. We also demonstrate that similar defects account for failure of skin formation at early stages. Interestingly, Fgfr2-independent skin formation occurs at E14.5 mutant embryos, resulting in much thinner, yet well-differentiated epidermis. Notably, mutant skin remains thin with decreased hair density after transplantation to wild-type recipients. These data demonstrate an essential role of Fgfr2 in eyelid and skin formation and patterning.

Animals↗
PubMed2001

Direct removal in the mouse of a floxed neo gene from a three-loxP conditional knockout allele by two novel approaches.

The presence in an intron of the ploxP-neo-loxP cassette often results in severe interference with gene expression. Consequently, many investigators selectively remove the ploxP-neo-loxP cassette by transient expression of Cre in ES cells. Although effective, the added manipulation of the ES cells may reduce the likelihood that a clone will be able to transmit via the germline. Therefore, we developed two novel approaches that remove the ploxP-neo-loxP by Cre-mediated recombination in mouse. First, the ploxP-neo-loxP-containing mice were crossed with EIIa-Cre transgenic mice. Second, a Cre-expression plasmid was injected into pronuclei of fertilized eggs bearing the ploxP-neo-loxP allele. Both approaches produced mosaic mice with partial and complete excision. These mosaic mice were then mated, and the neo-less conditional knockout allele was found in the offspring after screening only a few litters. These procedures provide options for removing neo directly in the mouse in addition to the commonly used approach that deletes neo in ES cells.

Alleles↗
PubMed2001

Feature extraction and normalization algorithms for high-density oligonucleotide gene expression array data.

Algorithms for performing feature extraction and normalization on high-density oligonucleotide gene expression arrays, have not been fully explored, and the impact these algorithms have on the downstream analysis is not well understood. Advances in such low-level analysis methods are essential to increase the sensitivity and specificity of detecting whether genes are present and/or differentially expressed. We have developed and implemented a number of algorithms for the analysis of expression array data in a software application, the DNA-Chip Analyzer (dChip). In this report, we describe the algorithms for feature extraction and normalization, and present validation data and comparison results with some of the algorithms currently in use.

Algorithms↗
PubMed2001

Regulation of expression and activity of four PKC isozymes in confluent and mechanically stimulated UMR-108 osteoblastic cells.

The transcript (mRNA), protein levels, enzyme activity, and cellular localization of four protein kinase C (PKC) isozymes identified in rat osteogenic sarcoma cells (UMR-108) were studied at confluent density and during mechanical stress (cyclic stretch). Western blot analysis indicated that growth to confluent density significantly increased the protein levels of cPKC-alpha (11.6-fold), nPKC-delta (5.3-fold), and nPKC-epsilon (22.0-fold) but not aPKC-zeta. Northern blot analysis indicated a significant (2.3-fold) increase in the 10 kb transcript of cPKC-alpha, a slight (1.3-fold) increase in that of nPKC-epsilon but no detectable change in that of the remaining isozymes. Enzyme activity assays of the individually immunoprecipitated isozymes yielded detectable kinase activity only for PKC-alpha, PKC-delta, and PKC-epsilon and only in confluent cells, corroborating the selective increase of these isozymes at confluent density. The UMR-108 cells showed a dramatic orientation response to mechanical stress with cell reshaping and alignment of the cell long axis perpendicular to the axis of force, remodeling of the actin cytoskeleton, and the appearance of multiple peripheral sites which stained for actin, vinculin, and PKC in separate experiments. Longer term mechanical stress beyond 24 h, however, resulted in no significant change in the mRNA level, protein level, or enzyme activity of any of the four PKC isozymes investigated. The results indicate that there are isozyme-selective increases in the protein levels of PKC isozymes of osteoblastic UMR-108 cells upon growth to confluence which may be regulated at the transcriptional or the post-transcriptional level. The results from UMR-108 cells support the earlier proposal (Carvalho RS, Scott JE, Suga DM, Yen EH. 1994. J Bone Miner Res 9(7):999-1011) that PKC could be involved in the early phase of mechanotransduction in osteoblasts through the activation of focal adhesion assembly/disassembly and the remodeling of the actin cytoskeleton.

Actin Cytoskeleton↗
PubMed2001

No direct mitogenic effect of sex hormones on antlerogenic cells detected in vitro.

Deer pedicles, antecedents of antlers, develop from a specialized periosteum (antlerogenic periosteum) which overlies the lateral crest of the deer frontal bone. The initiation of pedicle growth is triggered by androgen hormones. Thus far, it is not known whether pedicle initiation is caused by direct stimulation of androgen hormones on the antlerogenic periosteum or whether some intermediate mechanisms are necessary. The present study took an in vitro approach to investigate whether sex hormones have direct mitogenic effects on primary cultured antlerogenic periosteal cells (antlerogenic cells). Antlerogenic cells were obtained from two 5-month-old red deer calves. The cells were passaged twice and then treated with testosterone, dihydrotestosterone, and estradiol. The proliferation assays showed that no direct mitogenic effects on the second passage antlerogenic cells could be detected with any of the sex hormone treatments (P > 0.05). Testosterone-binding studies showed that at the second passage, specific testosterone-binding sites were present in the antlerogenic cells. Therefore, we conclude that androgens do not have mitogenic effects on antlerogenic cells in vitro. Our results suggest that pedicle formation may not be the result of direct stimulation of androgen hormones on antlerogenic tissue. Instead, androgen hormones may only allow the process to proceed by increasing the sensitivity of antlerogenic cells to mitogens, e.g., some growth factors.

Animals↗
PubMed2001

Seasonal changes of reproductive behavior and fecal steroid concentrations in Père David's deer.

From July 1997 to September 1997 and from March 1998 to July 1998, we studied reproductive behaviors of Père David's deer in Dafeng, China. During the field behavioral observations, we collected fresh voided fecal samples from the Père David's stags and hinds periodically and kept those samples under -20 degrees C until laboratory analysis. We analyzed the fecal testosterone, estradiol, and progesterone concentrations in those samples using radioimmunoassay. During this study, we also recorded 17 types of male reproductive behaviors and nine types of female reproductive behaviors. Reproductive behaviors and the fecal steroid concentrations showed overt seasonal fluctuations. There were statistically significant correlations between some male reproductive behaviors, such as anogenital sniffing, urine sniffing, urine spraying, wallowing, bellowing, antler adorning, Antler swags mud, chasing, herding hinds, chin resting, mounting and copulating, with the fecal testosterone concentrations. These results suggested that seasonal reproductive behaviors in stags are strongly associated with circulating testosterone. We also found that some female reproductive behaviors fluctuate corresponding with changes in fecal estrogen concentrations. Although there was no direct evidence to confirm the correlations between female reproductive behaviors and fecal estrogen in our experiment, we could not rule out that reproductive activities of hinds were largely related to ovarian estrogen secretion, and estrogen is necessary for inducing female reproductive behaviors.

Acclimatization↗
PubMed2001

Effect of pressure on intracardiac backscatter from microbubbles.

The backscatter from sonicated albumin microbubbles (Albunex) was analyzed using acoustic densitometry in an in vitro pulsatile heart model to evaluate the effects of pressure on the backscatter from Albunex, and the cardiac cyclic changes of intracardiac backscatter from sonicated albumin microbubbles in 16 healthy persons were analyzed. It was found that the Albunex microbubbles were compressed in systole and decompressed in diastole, causing corresponding changes of backscatter in cardiac cycle. Although the intensities of backscatter in diastole and systole were related to the concentration of microbubbles, the concentration of microbubbles had no effect on the difference of end-diastolic and end-systolic backscatter. The difference of the backscatter was highly correlated with end-systolic pressure (r = 0.96, P = 0.001). In human studies, we also observed same intracardiac cyclic changes of backscatter from sonicated albumin microbubbles. Our study indicates that it is possible to evaluate the intracardiac pressure non-invasively by analyzing the intracardiac backscatter from the microbubbles with acoustic densitometry.

Acoustics↗
PubMed2001

The study of ovarian artery hemodynamics in patients with infertility by color Doppler energy.

Seventy-four cases of infertility were examined to study the hemodynamics of the bilateral ovarian arteries at 21st day during the corpus luteum phase by color Doppler energy(CDE) and color Doppler flow imaging (CDFI). All the patients were verified by laparoscopy, fallopian tube patency examination and ovarian function test. Twenty-two healthy women served as controls. The results showed that the difference of resistance index(RI) and pulsatility index (PI) of bilateral ovarian arteries between the infertility and the normal controls had statistical significance (P < 0.01), and the PI showed negative correlation with the thickness of endometrium (left side: r = 0.724, P < 0.01; right side: r = 0.756, P < 0.01). The results also showed that CDE was more sensitive than CDFI in displaying the ovarian arteries. It could be concluded that the elevated resistance of ovarian artery during the corpus luteum phase was one of the important factors that resulted in infertility.

Adult↗
PubMed2001

Population data for 12 STR loci in Hong Kong Chinese.

The allele distributions at the 12 short tandem repeat (STR) loci D3S1358, HUMvWA, HUMFIBRA/FGA, HUMTHO1, HUMTPOX, HUMCSF1P0, D5S818, D13S317, D7S820, D8S1179, D21S11 and D18S51 have been determined for 284 unrelated Chinese in Hong Kong. The combined probability of identity for the 12 STR loci was about 4.1 x 10(-14) and the overall probability of excluding paternity 0.999978. None of the 12 loci were found to deviate from Hardy-Weinberg expectations according to the results of the exact test. There was also little evidence for association of alleles between loci. The results demonstrate that the loci are useful for forensic human identification and parentage testing for the Chinese population in Hong Kong.

China↗
PubMed2001

Adhesion, actin cytoskeleton organisation and the spreading of colon adenocarcinoma cells induced by EGF are mediated by alpha2beta1 integrin low clustering through focal adhesion kinase.

Both epidermal growth factor (EGF) and the extracellular matrix components have been implicated in the pathobiology of adenocarcinomas by somewhat poorly understood mechanisms. We have addressed this problem using an in vitro model comprising the colon adenocarcinoma cell line HT29-D4, wherein the role of EGF and type IV collagen on cell adhesion was examined. We demonstrated that the effect of EGF on HT29-D4 cell adhesion was regulated by type IV collagen in a time- and dose-dependent manner. The incorporation of a panel of monoclonal antibodies to integrins alpha1beta1, alpha2beta1 and alpha3beta1 in adhesion medium revealed that EGF-mediated increase in the cell adhesion was mediated essentially by alpha2beta1, and the use of flow cytometry led us to conclude that this EGF effect was mediated by an increase in alpha2beta1 activation and not by an increase in cell surface expression of integrin. An indirect immunofluorescence technique was employed to demonstrate that focal adhesion kinase (FAK) and alpha2beta1 integrin were present in focal complexes in large EGF-induced lamellipodia whereas actin cytoskeleton was organised in small tips that colocalised with FAK. This pattern was observed at early time points (15 min) with a strong FAK tyrosine phosphorylation and with an increase in mitogen-activated protein kinase activity (5-15 min) as measured by immunoprecipitation and immunoblotting. We conclude that at early time points of cell adhesion and spreading, EGF exerted an inside-out regulation of alpha2beta1 integrin in HT29-D4 cells. This regulation seemed to be mediated by EGF-dependent FAK phosphorylation entailing an increase in integrin activation and their recruitment in numerous focal complexes. Furthermore after activation, FAK induced aggregation of actin-associated proteins (paxillin, vinculin and other tyrosine phosphorylated proteins) in focal complexes, leading to organisation of actin cytoskeleton that is involved in lamellipodia formation. Finally, activated alpha2beta1 integrins intervened in all these processes clustered in small focal complexes but not in focal adhesions.

Actins↗
PubMed2001

Concentration-dependent phorbol stimulation of PKCalpha localization at the nucleus or subplasmalemma in A7r5 cells.

The subcellular translocation of PKCalpha was studied in A7r5 cells by confocal microscopy through use of standard immunohistologic staining and PKCalpha-enhanced green fluorescent protein (PKCalpha-EGFP) fusion protein expression. The results from both methods were consistent in indicating that PKCalpha, observed to be diffusely distributed in the unstimulated cell, was translocated primarily to either the perinuclear region of the cell or to subplasmalemmal sites depending on the concentration of phorbol 12, 13 dibutyrate (PDBu) used to activate the response. Translocation of PKCalpha to the perinucleus but not the plasmalemma was blocked by the use of colchicine to disrupt cell microtubules. However, there was little evidence of significant colocalization of PKCalpha with the microtubular cytoskeleton during the interval of translocation. By comparison, cytochalasin B disruption of actin microfilaments had no significant effect on PKCalpha translocation to either the plasmalemma or the perinucleus. The results indicate that the target site of PKCalpha translocation may vary with activating stimulus strength in A7r5 cells and that the translocation of the isoform to perinuclear target loci depends on an intact microtubular cytoskeleton. This suggests that multiple pathways are available for the redistribution of PKCalpha that may employ different mechanisms to regulate the movement and/or docking of the isoform at specific target sites.

Animals↗
PubMed2001

Deer antlerogenic periosteum: a piece of postnatally retained embryonic tissue?

This article reviews the research findings on the piece of periosteum overlying the lateral crest of prepubertal deer frontal bone, known as antlerogenic periosteum (AP). AP was initially discovered by Hartwig and Schrudde in 1974 when searching for the tissue that gives rise to antlers. In their experiment, when AP was transplanted elsewhere on the deer body it formed ectopic antlers. This clearly shows that AP possesses full self-differentiating ability, an attribute that can only be paralleled by embryonic tissue in mammals, like lateral plate mesoderm (LPM). Studies along this line by Goss in the 1980s further demonstrated that AP also holds the patterning information for antler formation. In the 1990s, our group carried out a series of studies on this unique tissue. The results showed that some of the critical features of AP resemble those of embryonic tissues, such as the astonishing growth potential in vivo and in vitro, and rich glycogen content. Histological observations and cell lineage tracing using a genetic marker convincingly demonstrate that pedicles and antlers are the derivatives of AP. Based on these findings, we advanced a hypothesis that AP is a piece of postnatally retained embryonic tissue. Morphological and histological examinations on the presumptive antler growth regions in deer prenatal life showed that the growth of primordial pedicles is initiated in the early pregnant stage (about 55 days) but then ceases (about 100 days) and is subsequently repressed at the late stage of pregnancy. The epidermis overlying the primordial pedicles resembles the apical ectoderm ridge (multicellular layer). These results strongly support our hypothesis. The results from the specific comparison between deer antler formation (from AP in postnatal) and mammalian limb development (from LPM in prenatal) showed that the ontogeny of antlers and limbs are comparable, and that deer antler has the same level of regulative properties as mammalian limbs. We believe that revealing the mechanism underlying the retention of embryonic tissue properties by AP until deer postnatal life will have important implications in biomedical research. Antler formation from AP offers an ideal model to work with in investigating how a self-differentiating system functions.

Animals↗
PubMed2001

Sensitization of adenylate cyclase induced by a dopamine D2 receptor mutant: inverse agonism by D2 receptor antagonists.

Mutations of residues in the third intracellular loops of several G-protein coupled receptors have been shown to confer constitutive activation. The authors investigated the effects of one such mutation in the dopamine D2 receptor. Compared to the wild type D2, the mutant D2 receptor (D2T344K) showed a substantial increase in agonist affinity with affinity for antagonists unchanged. The increased agonist affinity was unaffected by pertussis toxin treatment, indicating it is an intrinsic property of the mutant receptor. The potency of dopamine for acute inhibition of forskolin-stimulated cAMP production in stably expressing Chinese Hamster Ovary (CHO) cells was higher for the mutant than the wild type receptor. CHO cells stably expressing D2T344K displayed enhanced responses to forskolin-stimulated adenylate cyclase activity compared with cells stably expressing the wild type D2 receptor. The increased forskolin responsiveness of adenylate cyclase is similar to the sensitization previously observed with wild type D2 receptor after agonist treatment. Adenylate cyclase responsiveness of CHO cells stably expressing D2T344K receptor was not further increased by agonist treatment. Sensitization was blocked by pertussis toxin and D2 receptor antagonists haloperidol, butaclamol, and clozapine, indicating inverse agonist activity of these compounds at D2T344K. Inverse agonist activity was further demonstrated by the finding that overnight treatment with these compounds drastically increased the density of the mutant receptor but had minimal effect on the density of the wild type receptor. Taken together, these results suggest the authors have generated a constitutively active dopamine D2 receptor capable of sensitizing adenylate cyclase in the absence of agonist activation.

Adenylyl Cyclase Inhibitors↗
PubMed2001

Qian-Kun-Nin, a Chinese herbal medicine formulation, attenuates mitochondrial oxidant stress in cardiomyocytes.

Qian-Kun-Nin is a Chinese herbal medicine formulation used for several indications, including the treatment of cardiovascular diseases. This formulation contains herbs which possess antioxidant properties. In this study, Qian-Kun-Nin's ability to confer protection to cardiomyocytes against reactive oxygen species (ROS) generated during mitochondrial electron transport inhibition was tested. The intracellular fluorescent probe 2',7'-dichlorofluorescin diacetate (DCFH-DA, sensitive to H(2)O(2) and hydroxyl radicals) was used to assess intracellular ROS, and propidium iodide (PI) was used to assess viability in cultured chick embryonic cardiomyocytes. Qian-Kun-Nin significantly attenuated oxidation of DCFH in cells exposed to the mitochondrial site III inhibitor, antimycin A, consistent with a decrease in oxidative stress. These attenuated oxidant levels were associated with improved cell survival. After antimycin A exposure, Qian-Kun-Nin decreased cell death from 51. 6+/-3.3% in untreated cells to 27.3+/-3.8% in treated cells at 2 h. We conclude that Qian-Kun-Nin attenuates oxidant stress and protects cells from lethal oxidant damage during mitochondrial electron transport inhibition, and thus its therapeutic potential in treating cardiovascular diseases may relate to its antioxidant properties.

Analysis of Variance↗