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Biomedical subjects

X Liu

Publications and source records attributed to X Liu.

At least 505 records · Page 28Linked to original sources
PubMed2000

Site-specific NMR monitoring of cis-trans isomerization in the folding of the proline-rich collagen triple helix.

Understanding the folding of the proline-rich collagen triple helix requires consideration of the effects of proline cis-trans isomerization and may shed light on the misfolding of collagen in connective tissue diseases. Folding was monitored in real time by heteronuclear 2D NMR spectroscopy for the (15)N labeled positions in the triple-helical peptide T1-892 [GPAGPAGPVGPAGARGPAGPOGPOGPOGPOGV]. In the equilibrium unfolded monomer form, each labeled residue showed multiple peaks with interconversion rates consistent with cis-trans isomerization of Gly-Pro and Pro-Hyp bonds. Real-time NMR studies on the folding of T1-892 showed slow decay of monomer peaks and a concomitant increase in trimer peaks. Gly25 in the C-terminal rich (Gly-Pro-Hyp)(4) domain folds first, consistent with its being a nucleation domain. Analysis of the kinetics indicates that the folding of Gly25 is biphasic and the slower step represents cis-trans isomerization of imino acids. This illustrates that nucleation is limited by cis-trans isomerization. Monitoring Gly6, Gly10, Ala12, and Gly13 monomer and trimer peaks captures the C- to N-terminal propagation of the triple helix, which is also limited by Gly-Pro cis-trans isomerization events. The zipper-like nature of the propagation process is confirmed by the slower rate of folding of Ala6 compared to Gly13, reflecting the larger number of isomerization events encountered by the more N-terminal Ala6. The cis-trans isomerization events at multiple proline residues is a complex statistical process which can be visualized by these NMR studies.

Amino Acid Sequence↗
PubMed2000

Molecular dissection of TrkA signal transduction pathways mediating differentiation in human neuroblastoma cells.

Activation of the neurotrophin receptor TrkA by its ligand nerve growth factor (NGF) initiates a cascade of signaling events leading to neuronal differentiation in vitro and might play an important role in the differentiation of favorable neuroblastomas (NB) in vivo. To study TrkA signal transduction pathways and their effects on differentiation in NB, we stably expressed wild-type TrkA and five different TrkA mutants in the NGF unresponsive human NB cell line SH-SY5Y. Resulting clones were characterized by TrkA mRNA and protein expression, and by autophosphorylation of the receptor. Introduction of wild-type TrkA restored NGF responsiveness of SH-SY5Y cells, as demonstrated by morphological differentiation, activation of mitogen-activated protein kinases (MAPK) and induction of immediate-early genes. Expression of TrkA in the absence of NGF resulted in growth inhibition of transfectants compared to parental cells, whereas NGF-treatment increased their proliferation rate. Analysis of downstream signal transduction pathways indicated that NGF-induced differentiation was dependent on TrkA kinase activity. Our data suggest that several redundant pathways are present further downstream, but activation of the RAS/MAPK signaling pathway seems to be of major importance for NGF mediated differentiation of NB cells. Our results also show that the signaling effector SH2-B is a substrate of NGF-mediated Trk signaling in NB, whereas it is not activated by NGF in rat pheochromocytoma PC12 cells. This might explain the differences we observed in TrkA signaling between neuroblastoma and PC12 cells. Further insight into TrkA signaling may suggest new options for the treatment of NB.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Generation of mammalian cells stably expressing multiple genes at predetermined levels.

Expression of cloned genes at desired levels in cultured mammalian cells is essential for studying protein function. Controlled levels of expression have been difficult to achieve, especially for cell lines with low transfection efficiency or when expression of multiple genes is required. An internal ribosomal entry site (IRES) has been incorporated into many types of expression vectors to allow simultaneous expression of two genes. However, there has been no systematic quantitative analysis of expression levels in individual cells of genes linked by an IRES, and thus the broad use of these vectors in functional analysis has been limited. We constructed a set of retroviral expression vectors containing an IRES followed by a quantitative selectable marker such as green fluorescent protein (GFP) or truncated cell surface proteins CD2 or CD4. The gene of interest is placed in a multiple cloning site 5' of the IRES sequence under the control of the retroviral long terminal repeat (LTR) promoter. These vectors exploit the approximately 100-fold differences in levels of expression of a retrovirus vector depending on its site of insertion in the host chromosome. We show that the level of expression of the gene downstream of the IRES and the expression level and functional activity of the gene cloned upstream of the IRES are highly correlated in stably infected target cells. This feature makes our vectors extremely useful for the rapid generation of stably transfected cell populations or clonal cell lines expressing specific amounts of a desired protein simply by fluorescent activated cell sorting (FACS) based on the level of expression of the gene downstream of the IRES. We show how these vectors can be used to generate cells expressing high levels of the erythropoietin receptor (EpoR) or a dominant negative Smad3 protein and to generate cells expressing two different cloned proteins, Ski and Smad4. Correlation of a biologic effect with the level of expression of the protein downstream of the IRES provides strong evidence for the function of the protein placed upstream of the IRES.

Animals↗
PubMed2000

Cloning of cytidine triphosphate: phosphocholine cytidylyltransferase mRNA upregulated by a neuropeptide arginine-vasopressin((4-8)) in rat hippocampus.

Neuropeptide arginine-vasopressin((4-8)) (AVP(4-8)) is a metabolite of arginine-vasopressin which has been shown to have potent memory-enhancing activity, facilitate neurite elongation and prolongate cell aging. Identification of differentially expressed genes in hippocampus induced by AVP(4-8) is important for understanding the molecular basis of AVP(4-8) function. Differential display PCR and 5'rapid amplification of cDNA Ends were used. One new full length cDNA encoding rat cytidine triphosphate: phosphocholine cytidylyltransferase (CCT) beta was thus obtained. Northern blot analysis demonstrated that it was upregulated by AVP(4-8) in mature rat hippocampus. The study of tissue distribution with reverse transcription PCR showed that the gene was abundant in brain. Since CCT catalyzes the formation of cytidine diphosphate choline, which was reported to have a beneficial therapeutic effect on Alzheimer's disease, so we speculated that AVP(4-8) may be a potential candidate for treating Alzheimer's disease by upregulating CCT mRNA level.

Amino Acid Sequence↗
PubMed2000

Molecular beacons: a novel DNA probe for nucleic acid and protein studies.

A new concept has been introduced for molecular beacon DNA molecules. Molecular beacons are a new class of oligonucleotides that can report the presence of specific nucleic acids in both homogeneous solutions and at the liquid-solid interface. They emit an intense fluorescent signal only when hybridized to their target DNA or RNA molecules. Biotinylated molecular beacons have been designed and used for the development of ultrasensitive DNA sensors and for DNA molecular interaction studies at a solid-liquid interface. Molecular beacons have also been used to study protein-DNA interactions. They have provided a variety of exciting opportunities in DNA/RNA/protein studies.

Animals↗
PubMed2000

A novel protein MAJN binds to Jak3 and inhibits apoptosis induced by IL-2 deprival.

To find a possible signal interacting with the Jak3 N-terminal, we screened the human peripheral blood cDNA library through both a two-hybrid system and a tyrosine-phosphorylation-modified two-hybrid system using the N-terminal of Jak3 as bait. Results showed that one new homologue of myosin heavy chain, designated MAJN (molecule associated with Jak3 N-terminal), could bind to Jak3 in a tyrosine-phosphorylation-independent manner. The interaction between Jak3 and MAJN was further confirmed by immunoprecipitation in BAF-B03 beta cells. To investigate the function of MAJN, we have constructed the BAF-B03 beta/MAJN cell line that stably expresses MAJN and found that overexpression of MAJN can partially inhibit the apoptosis induced by interleukin-2 deprival. Further studies are needed to elucidate how MAJN executes its function to antagonize BAF-B03beta cell death in the absence of IL-2.

Apoptosis↗
PubMed2000

Axotomized and intact muscle afferents but no skin afferents develop ongoing discharges of dorsal root ganglion origin after peripheral nerve lesion.

After peripheral nerve lesions, some axotomized afferent neurons develop ongoing discharges that originate in the dorsal root ganglion (DRG). We investigated in vivo which functional types of afferent neurons contributed to this ectopic activity. Six to twelve days after the gastrocnemius soleus (GS) nerve supplying skeletal muscle and the sural (SU) nerve supplying skin had been transected (experimental group E1), 20.4% of afferent neurons with myelinated axons projecting into the GS nerve produced ongoing discharges of irregular or bursting pattern. In contrast, all SU neurons were silent. Additional transection of peroneal and tibial nerves (group E2) induced ongoing activity in a similar percentage of GS neurons (22.1%), but their mean discharge frequency was higher (6.0 vs 2.7 Hz), and more of them exhibited bursting discharges (63 vs 17%). When the GS nerve had been left intact while tibial, peroneal, and SU nerve had been transected (group E3), 18.8% of unlesioned GS neurons developed ongoing discharges at a mean frequency of 6.1 Hz; most of them exhibited a bursting pattern. Without a preceding nerve lesion, almost no GS neuron (1.1%) fired spontaneously. Most afferent neurons with ongoing activity had an axonal conduction velocity of 5-30 m/sec indicating that some of these neurons may have had nociceptive function. These findings provide the first evidence that after peripheral nerve injury both axotomized as well as intact afferent neurons supplying skeletal muscle but not skin afferents generate ongoing activity within the DRG, probably because of a yet unknown signal in the DRG triggered by axotomy.

Animals↗
PubMed2000

Regulation of xanthine oxidase by nitric oxide and peroxynitrite.

Xanthine oxidase (XO) is a central mechanism of oxidative injury as occurs following ischemia. During the early period of reperfusion, both nitric oxide (NO(*)) and superoxide (O-*(2)) generation are increased leading to the formation of peroxynitrite (ONOO(-)); however, questions remain regarding the presence and nature of the interactions of NO(*) or ONOO(-) with XO and the role of this process in regulating oxidant generation. Therefore, we determined the dose-dependent effects of NO(*) and ONOO(-) on the O-*(2) generation and enzyme activity of XO, respectively, by EPR spin trapping of O-*(2) using 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide and spectrophotometric assay. ONOO(-) markedly inhibited both O-*(2) generation and XO activity in dose-dependent manner, while NO(*) from NO(*) gas in concentrations up to 200 microM had no effect. Furthermore, we observed that NO(*) donors such as NOR-1 also inhibited O-*(2) generation and XO activity; however, these effects were O-*(2)-dependent and blocked by superoxide dismutase or ONOO(-) scavengers. Finally, we found that ONOO(-) totally abolished the Mo(V) EPR spectrum. These changes were irreversible, suggesting oxidative disruption of the critical molybdenum center of the catalytic site. Thus, ONOO(-) formed in biological systems can feedback and down-regulate XO activity and O-*(2) generation, which in turn may serve to limit further ONOO(-) formation.

Electron Spin Resonance Spectroscopy↗
PubMed2000

Crystal structures of mutant monomeric hexokinase I reveal multiple ADP binding sites and conformational changes relevant to allosteric regulation.

Hexokinase I, the pacemaker of glycolysis in brain tissue, is composed of two structurally similar halves connected by an alpha-helix. The enzyme dimerizes at elevated protein concentrations in solution and in crystal structures; however, almost all published data reflect the properties of a hexokinase I monomer in solution. Crystal structures of mutant forms of recombinant human hexokinase I, presented here, reveal the enzyme monomer for the first time. The mutant hexokinases bind both glucose 6-phosphate and glucose with high affinity to their N and C-terminal halves, and ADP, also with high affinity, to a site near the N terminus of the polypeptide chain. Exposure of the monomer crystals to ADP in the complete absence of glucose 6-phosphate reveals a second binding site for adenine nucleotides at the putative active site (C-half), with conformational changes extending 15 A to the contact interface between the N and C-halves. The structures reveal distinct conformational states for the C-half and a rigid-body rotation of the N-half, as possible elements of a structure-based mechanism for allosteric regulation of catalysis.

Adenosine Diphosphate↗
PubMed2000

Diurnal preference, sleep habits, circadian sleep propensity and melatonin rhythm in healthy human subjects.

After 24-h sleep deprivation, 33 healthy young subjects entered the 10/20 min ultra-short sleep-wake schedule for 26 h. Melatonin rhythm was hourly assessed simultaneously. Results indicated that morning preference was significantly correlated with habitual sleep onset (r=-0.41, P=0.04), habitual sleep offset (r=-0.52, P=0.002), melatonin peak time (r=-0.36, P=0.04), and sleep propensity onset time (r=-0.36, P=0.04). The intervals between habitual sleep mid-point and melatonin peak time and between habitual sleep mid-point and sleep propensity onset time were significantly longer in morning-preference subjects than in evening-preference subjects (P<0.05). These findings suggest that the variance of diurnal preference may be related to differences in phase relations between habitual sleep timing and the circadian pacemaker.

Adult↗
PubMed2000

Morphologic and electrophysiologic maturation in developing dentate gyrus granule cells.

Dentate gyrus granule cells from immature (7-28 days) Sprague-Dawley rats were examined with whole cell patch clamp recordings and biocytin filling in in vitro hippocampal slice preparations. Although recordings were confined to the middle third of the suprapyramidal limb of the dentate, the granule cells exhibited marked variability in their physiologic properties: input resistance (IR) ranged from 250 MOmega to 3 GOmega, and resting membrane potential (RMP) from -82 to -41 mV. Both IR and RMP were inversely correlated with dendritic length, a morphometric indicator of cell maturity. Thus the highest IR cells were the youngest, and maturation was characterized by a progressive decrease in IR, hyperpolarization of RMP, and elongation of the dendritic arbor. When cells were grouped by IR, significant intergroup differences were found in RMP, dendritic length, and number of dendritic terminal branches. Although cells of all IR categories were examined throughout the age spectrum under study, none of the inter-IR group differences was age-dependent. These data suggest that IR provides a reasonable estimate of granule cell maturity and that maturation entails predictable changes in cell properties and morphology. These aspects of maturation correlate with each other, are independent of animal age, and most likely proceed according to a program related to cell birth.

Aging↗
PubMed2000

Sleep loss and daytime sleepiness in the general adult population of Japan.

There are few epidemiological studies on sleep loss and daytime sleepiness in the general adult population of Japan. A total of 4000 adult people, aged 20 and over, were randomly drawn from five areas of Japan, and 3030 individuals were interviewed and completed a questionnaire including information about sleep duration and sleep problems. Overall, 29% slept less than 6 h at night, 23% reported having insufficient sleep, and 6% took sleep enhancing medications. The prevalence rates were 21% for symptoms of insomnia and 15% for excessive daytime sleepiness. Symptoms of insomnia were more prevalent in the elderly, whereas young people were more likely to report short sleep duration, subjective insufficient sleep and excessive daytime sleepiness. A multiple logistic regression model revealed that excessive daytime sleepiness had significant associations with young people, short sleep duration, insomnia symptoms, subjective insufficient sleep and sleep enhancing medication use. Short sleep duration was the strongest predictor of excessive daytime sleepiness. The findings indicate that sleep loss and excessive daytime sleepiness in the Japanese adult population are common, and comparable to those reported in Western countries. Excessive daytime sleepiness in the general adult population seems more likely to be attributed to short sleep duration.

Adult↗
PubMed2000

Morphological assessment of the effect of growth hormone on preantral follicles from 11-day-old mice in an in vitro culture system.

The present study was carried out to verify that the cells attached to the outside of the basement membrane of mechanically isolated follicles are theca cells and to evaluate the effect of growth hormone (GH) on these cells. Preantral follicles, 100-140 micrometer in diameter, were mechanically isolated from 11-day-old BDF1 hybrid immature mice, divided randomly into two groups, and cultured in vitro. One group was treated with 0.1% collagenase immediately after mechanical isolation in an attempt to remove theca cells attached to the outside of the basement membrane. The other group was untreated. Morphological examination revealed that 86.1% of mechanically isolated follicles before collagenase treatment had at least one theca cell around the basement membrane on the single section. However, after collagenase treatment no theca cells remained on the basement membrane of the follicles. Androstenedione secretion as a result of stimulation by 100 ng/ml hCG was significantly higher in the culture medium of the follicles with theca cells than in those of collagenase-pretreated follicles (p < 0.0001), indicating that the cells attached to the outside of the basement membrane were actually functional theca cells, not interstitial cells. To elucidate the effect of GH on theca cells, preantral follicles cultured in the presence of 1.0 mIU/ml GH were morphologically examined. Preantral follicles mechanically isolated from immature mice showed significant proliferation of not only granulosa cells but also theca cells in the presence of GH. In particular, theca cells, which remained dotted on the basement membrane in a small number just after isolation, proliferated and finally formed complete layers after the culture with GH. This is the first report that GH induced the proliferation of theca cells to form morphologically complete layers around the preantral follicle from 11-day-old mice.

Androstenedione↗
PubMed2000

Trp1, a candidate protein for the store-operated Ca(2+) influx mechanism in salivary gland cells.

The trp gene family has been proposed to encode the store-operated Ca(2+) influx (SOC) channel(s). This study examines the role of Trp1 in the SOC mechanism of salivary gland cells. htrp1, htrp3, and Trp1 were detected in the human submandibular gland cell line (HSG). HSG cells stably transfected with htrp1alpha cDNA displayed (i) a higher level of Trp1, (ii) a 3-5-fold increase in SOC (thapsigargin-stimulated Ca(2+) influx), determined by [Ca(2+)](i) and Ca(2+)-activated K(+) channel current measurements, and (iii) similar basal Ca(2+) permeability, and inhibition of SOC by Gd(3+) but not by Zn(2+), as compared with control cells. Importantly, (i) transfection of HSG cells with antisense trp1alpha cDNA decreased endogenous Trp1 level and significantly attenuated SOC, and (ii) transfection of HSG cells with htrp3 cDNA did not increase SOC. These data demonstrate an association between Trp1 and SOC and strongly suggest that Trp1 is involved in this mechanism in HSG cells. Consistent with this suggestion, Trp1 was detected in the plasma membrane region, the proposed site of SOC, of acinar and ductal cells in intact rat submandibular glands. Based on these aggregate data, we propose Trp1 as a candidate protein for the SOC mechanism in salivary gland cells.

Animals↗
PubMed2000

Characterization, chromosomal assignment, and tissue expression of a novel human gene belonging to the ARF GAP family.

We have identified and characterized a novel human ADP-ribosylation factor GTPase-activating protein (ARFGAP1) gene that is related to other members of the ARF GAP family. The full-length cDNA for human ARFGAP1 was cloned following the identification of an EST obtained by large-scale cDNA library sequencing through a Blast search of public databases. Structurally, ARFGAP1 encodes a polypeptide of 516 amino acids, which contained a typical GATA-1-type zinc finger motif (CXXCX(16)CXXC) with the four cysteine residues that are highly conserved among other members of the ARF GAP family. The conserved ARF GAP domain may emphasize the biological importance of this gene. The ARFGAP1 gene, which contained 16 exons ranging from 0.5 to 9.3 kb, was mapped to human chromosome 22q13.2-q13.3 using radiation hybridization and in silico analyses. ARFGAP1 is strongly expressed in endocrine glands and testis. Interestingly, the expression of ARFGAP1 in testis is about sixfold higher than that in ovary, indicating a possible role of ARFGAP1 in the physiological function of sperm. Expression of ARFGAP1 in four human fetal tissues and seven cancer cell lines was also detected.

ADP-Ribosylation Factors↗
PubMed2000

A recombinant antibody-targeted plasminogen activator with high affinity for activated platelets increases thrombolytic potency in vitro and in vivo.

To increase thrombolytic specificity of urokinase (uPA), we engineered a recombinant chimeric plasminogen activator SZ51Hu-scuPA, which consists of a humanized monoclonal antibody (SZ-51Hu) specifically against P-selectin on activated human platelet and a single-chain urokinase (scuPA). The cDNA, encoding scuPA amino acids 1-411, was inserted in 5' end to 3' end orientation immediately after the CH3 of SZ-51Hu heavy-chain sequence in the expression vector alphaLys30. The resulting construct alphaLys30-SZ51VH/Hu-scuPA was used to transfect into SP2/0 murine myeloma cell line, which was pretransfected with SZ51Hu light chain. The fusion protein SZ51Hu-scuPA was expressed at 5 mg/L in the supernatant of cell culture. The fusion protein purified by affinity chromatography had a molecular weight of 160 kDa with fibrinolytic activity of 39,000 IU/mg and its affinity to activated human platelet was 67% of the parent murine mAb SZ-51. The thrombolytic property of the fusion protein was first characterized in an in vitro system, which consists of a 125I-fibrin-labeled human plasma clot containing different concentrations of human platelets suspended in citrated human plasma. Fifty percent lysis was reached with SZ51Hu-scuPA in 1 hour at a concentration of 20 IU/mL or in 2 hours at a concentration of 10 IU/ mL, which was much faster than uPA at the same concentration. The maximal lysis of the clots by SZ51Hu-scuPA was 4.1 to 8.4 times more potent than that by uPA. The fusion protein was further characterized in the hamster pulmonary embolism model with clots prepared from fresh platelet-rich human plasma containing 125I-labeled fibrinogen. The thrombolytic activity of SZ51-scuPA was 3.9 times more potent than that of uPA at 2,000 IU/kg in this model. Almost no significant fibrinogen breakdown was observed either in vitro and in vivo.

Animals↗
PubMed2000

Overexpression of M68/DcR3 in human gastrointestinal tract tumors independent of gene amplification and its location in a four-gene cluster.

Fas-mediated apoptosis is an important regulator of cell survival, and abnormalities in this system have been shown to result in a number of human pathological conditions. A secreted member of the tumor necrosis factor receptor superfamily, DcR3, was recently reported to be amplified in human lung and colon cancers as a negative regulator of Fas-mediated apoptosis. We identified this gene, which we call M68. M68 genomic DNA, mRNA, and protein levels were examined in a series of human gastrointestinal tract tumors. Using M68 immunohistochemistry and a scoring system similar to that used for HER-2/neu, we found that M68 protein was overexpressed in 30 of 68 (44%) human adenocarcinomas of the esophagus, stomach, colon, and rectum. Tumors examined by Northern blot revealed M68 mRNA highly elevated in a similar fraction of primary tumors from the same gastrointestinal tract regions, as well as in the colon adenocarcinoma cell lines SW480 and SW1116. Further, we found M68 protein to be overexpressed in a substantial number of tumors in which gene amplification could not be detected by fluorescence in situ hybridization or quantitative genomic PCR, suggesting that overexpression of M68 may precede amplification in tumors. Finally, we find that M68 lies within a four-gene cluster that includes a novel helicase-like gene (NHL) related to RAD3/ERCC2, a plasma membrane Ras-related GTPase and a member of the stathmin family, amplification or overexpression of which may also contribute to cell growth and tumor progression.

ADP-Ribosylation Factors↗