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Y Luo

Publications and source records attributed to Y Luo.

At least 289 records · Page 16Linked to original sources
PubMed1997

Compromised mitochondrial function leads to increased cytosolic calcium and to activation of MAP kinases.

We have investigated in rat pheochromacytoma PC12 cells the activation of the mitogen-activated protein kinases ERK1 and ERK2 by the mitochondrial uncoupler carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP). This treatment slowly decreases ATP levels to 30% of control, whereas the internal calcium level rises very rapidly to 250% of control, derived from internal stores. Tyrosine phosphorylation of ERK1 and ERK2 increases gradually, starting after 5 min of treatment, to reach a maximum at 30 min; the kinase activity reaches 250% when measured after 1 hr of treatment. The drop in ATP levels is slower still. Comparison of the time courses of the rapid rise in cytosolic calcium with the slower increase in ERK1 and ERK2 activation suggests one or more intermediate stages in this pathway. Chelation of cytosolic calcium with dimethyl bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid abolished the FCCP-stimulated rise in internal calcium, as well as the tyrosine phosphorylation and the activation of the ERKs. Surprisingly, caffeine, which releases calcium from different internal stores, did not increase the tyrosine phosphorylation and did not activate the ERKs. The FCCP effect on calcium storage may be related to mitochondrial dysfunction in Alzheimer disease, which might result in ineffective buffering of cytosolic calcium that leads to mitogen-activated protein kinase activation and subsequent protein phosphorylations.

Animals↗
PubMed1997

Murine astrocytes express a functional chemokine receptor.

Elevated levels of chemokines have been observed in various diseases of the CNS. Little is known, however, about how these chemokines affect parenchymal cells of the CNS. The current studies examine astrocyte chemotaxis to the mouse chemokine macrophage inflammatory protein-1alpha (MIP-1alpha). Murine astrocytes demonstrate directed migration along a chemical gradient in response to 10(-10)-10(-8) M MIP-1alpha. Peak chemotactic responses are noted at 10(-9) M. MIP-1alpha-induced astrocyte migration is specifically inhibitable with pertussis toxin, suggesting a role for Galphai proteins in the signaling process. RT-PCR and in situ hybridization were used to identify expression of the murine CCR1 MIP-1alpha receptor on astrocytes. Astrocytes contain mRNA for CCR1, but messages for CCR4 and the orphan chemokine receptor MIP-1alphaR-like#1 were not detected. The combined results suggest that a functional chemokine receptor is expressed on resident cells of the CNS. We speculate that the interactions of chemokines with astrocytes are involved in inflammatory reactions of the CNS.

Animals↗
PubMed1997

A myoglobin variant with a polar substitution in a conserved hydrophobic cluster in the heme binding pocket.

Well-ordered internal amino acids can contribute significantly to the stability of proteins. To investigate the importance of the hydrophobic packing interface between helices G and H in the proximal heme pocket of horse heart myoglobin, the highly conserved amino acid, Leu104, was substituted with asparagine, a polar amino acid of similar size. The Leu104Asn mutant protein and its recombinant wild-type horse heart myoglobin counterpart were expressed from synthetic genes in Escherichia coli. Thermal denaturation of these two recombinant myoglobins, as studied by measurement of circular dichroism ellipticity at 222 nm, revealed that the Leu104Asn mutant had a significantly lower t(m) (71.8 +/- 1 degree C, pH 7.0) than recombinant wild-type myoglobin (81.3 +/- 1 degree C, pH 7.0). To examine the extent to which this 10 degrees C decrease in thermal stability was associated with structural perturbations, X-ray diffraction techniques were used to determine the three-dimensional structures of both the recombinant wild-type and Leu104Asn myoglobins to 0.17 nm resolution. Refinement of these structures gave final crystallographic R-factors of 16.0% and 17.9%, respectively. Structural comparison of the natural and recombinant wild-type myoglobins, together with absorption spectroscopic and electron paramagnetic resonance (EPR) analyses, confirmed the proper expression and folding of the recombinant protein in E. coli. Surprisingly, despite the decreased thermal stability of the Leu104Asn mutant, there are no significant structural differences between the mutant and wild-type myoglobins. EPR and absorption spectroscopic analyses further confirmed the similar nature of the heme iron centres in both proteins. Thus, the introduction of an energetically unfavourable change in side chain polarity at position 104 into a hydrophobic environment that does not support the hydrogen bonding potential of the mutant asparagine appears to perturb important stabilizing helix-helix and heme-protein interactions. The induced structural destabilization is thereby reflected by a significant decrease in the t(m) of horse heart myoglobin.

Animals↗
PubMed1997

Functional expression of the CXC-chemokine receptor-4/fusin on mouse microglial cells and astrocytes.

The mRNA for the seven-transmembrane-spanning G protein-coupled receptor fusin/CXCR-4 is expressed in primary mouse astrocyte cultures and the transformed mouse microglial cell line, N9. Cell surface expression of fusin in these cells was confirmed by staining with a polyclonal anti-fusin Ab. The functional capacity of this chemokine receptor was examined by evaluating the calcium responses following stimulation of glial cells with the CXC-chemokine, stromal-derived cell factor-1alpha (SDF-1alpha). Both astrocytes and microglial cells mobilized calcium following stimulation with chemically synthesized SDF-1alpha. SDF-1alpha- and carbachol-mediated calcium responses of astrocytes were partially inhibited by treatment with pertussis toxin (PTx), suggesting receptor coupling to a combination of G alpha(i) and other G proteins. In contrast, the calcium responses of microglial cells to SDF-1alpha were completely PTx sensitive, while responses to carbachol stimulation were PTx resistant. The ability of SDF-1alpha to induce glial cell migration was also examined. Synthetic SDF-1alpha was a potent chemoattractant for mouse microglial cells at ligand concentrations of 10 to 500 ng/ml; peak responses were noted at 100 ng/ml. In contrast, astrocytes did not migrate toward a gradient of SDF-1alpha. The failure of SDF-1alpha to induce astrocyte migration was specific, as another chemokine, macrophage inflammatory protein-1alpha, triggered astrocyte chemotaxis.

Animals↗
PubMed1997

Backfoot is a novel homeobox gene expressed in the mesenchyme of developing hind limb.

Homeobox genes play important roles in pattern formation during development. Here, we report the cloning and temporal and spatial expression patterns of a novel homeobox gene Backfoot (BFT for the human gene, and Bft for the mouse gene), whose expression reveals an early molecular distinction between forelimb and hind limb. BFT was identified as a sequence-specific DNA-binding protein. In addition to the homeodomain, it shares a carboxyl-terminal peptide motif with other paired-like homeodomain proteins. Northern hybridization analysis of RNAs from human tissues revealed that human BFT is highly expressed in adult skeletal muscle and bladder. During midgestation embryogenesis, mouse Bft is expressed in the developing hind limb buds, mandibular arches, and Rathke's pouch. The expression of Bft begins prior to the appearance of hind limb buds in mesenchyme but is never observed in forelimbs. At later stages of limb development, the expression is progressively restricted to perichondrial regions, most likely in tendons and ligaments. The timing and pattern of expression suggest that Bft plays multiple roles in hind limb patterning, branchial arch development, and pituitary development. Bft is likely identical to a mouse gene, Ptx1, that was recently isolated by Lamonerie et al. ([1996] Genes Dev. 10:1284-1295) and that has been suggested to play a role in pituitary development.

Adult↗
PubMed1997

C-myc deregulation during transformation induction: involvement of 7SK RNA.

The process of oncogenic transformation has been widely studied but is still poorly understood. We have focused on the mechanism of deregulation of the c-myc gene during transformation of a temperature-sensitive SV40-transformed mouse cell line. Run-on transcription assays showed that the two c-myc minor promoters, P1 and P3, are transiently activated following induction of transformation and that peak activation of both promoters is preceded by a large increase in transcription of a small RNA (7SK). To test the possibility that this RNA might participate in promoter activation, we transfected cells with sense and antisense oligodeoxynucleotides corresponding to different regions of the 7SK RNA predicted to be accessible within the RNP particle. Out of 14 oligos tested, inhibition of activation of P1 and/or P3 was observed with four antisense oligonucleotides corresponding to looped regions in the putative 7SK secondary structure. To identify c-myc promoter sequences which might serve as targets for 7SK activity, we carried out mobility-shift assays with either whole or 7SK-depleted cell extracts. The CT element of the c-myc promoter formed a 7SK-dependent complex which could be competed only with the same antisense 7SK oligo that suppressed P1 and P3 activation in vivo. Taken together these results suggest that 7SK RNP participates in transformation-dependent c-myc deregulation.

3T3 Cells↗
PubMed1997

Minimum length of sequence homology required for in vivo cloning by homologous recombination in yeast.

With efficient homologous recombination in Saccharomyces cerevisiae, a rapid in vivo cloning technique has been available. Here we demonstrated that 30 bp of a homologous sequence at each end of a DNA fragment is sufficient to integrate the fragment into a linearized plasmid in yeast. To obtain a high yield of recombination transformants, however, more than 60 bp are desirable. Interestingly, we observed that 20 bp of homology at one end of a DNA fragment is sufficient for efficient recombination provided that the other end contains 80 bp of homologous sequence. Some applications, including high-throughput transferring of EST inserts to the yeast expression systems for the Human Genome Project, are discussed.

Animals↗
PubMed1997

Regulation of peroxisomal fatty acyl-CoA oxidase in the yeast. Saccharomyces cerevisiae.

Peroxisomes are specialized organelles found in most eukaryote cells, where their major functions are in cellular respiration and fatty acid oxidation. Proliferation of this organelle, and induction of peroxisomal enzymes, is a phenomenon that occurs in diverse species, and is stimulated by a number of physiological and pharmacological stimuli. A large number of chemically diverse compounds, including hypolipidemic drugs and industrial plasticizers, have been shown to cause peroxisome proliferation and the induction of peroxisomal enzymes in rodents. Chronic exposure to these compounds produces hepatocellular carcinomas, however, the mechanism by which this tumorigenic event occurs is unknown. In the yeast Saccharomyces cerevisiae peroxisomes are induced when a fatty acid such as oleate is supplied as a carbon source in the growth medium. In addition, many peroxisomal enzymes are induced by growth on oleate; these include enzymes of the peroxisomal beta-oxidation cycle. This regulation occurs at the transcription level, and is controlled by specific trans-acting factors. The research in our laboratory has focused on the mechanisms involved in this regulation, and on the identification and characterization of the proteins involved. Our recent results, and current research directions are summarized.

Acyl-CoA Oxidase↗
PubMed1997

Clinical application of retrograde island flap pedicled with lateral plantar artery.

The purpose of the study is to identify a repair procedure for the ulcers or defect of the anterior lateral plantar region. The defects of anterior lateral plantar region after resection of the obstinate ulcer, painful corns and callosity were repaired by the retrograde island flap pedicled with lateral plantar artery. One to 10 years following up revealed that all the 10 flaps in 9 patients survived after operation. Patients walked freely without pain and claudication. The flap did not show any sign of damages of the flap even if the patients walked for a long time. It is concluded that the retrograde island flap pedicled with lateral plantar artery is the best choice in repairing the defects of anterior lateral plantar region.

Adolescent↗
PubMed1997

Somatic embryogenesis in cultured immature zygotic embryos and leaf protoplasts of Arabidopsis thaliana ecotypes.

Immature zygotic embryos of six ecotypes (Nd-0, Ler, C24, Col-0, Nossen, Ws-2) of Arabidopsis thaliana (L.) Heynh. were cultured in vitro. The same ecotypes, except Nossen, were used for studies on leaf protoplast culture. Experimental conditions for the induction of somatic embryos were established in both culture systems. In the case of immature zygotic embryos, the parameters investigated were the influence of developmental stage of the explant, the ecotypes used, and various concentrations and combinations of growth regulatory substances (phytohormones). In the ecotype Ler, structures were discovered which were very similar to those found in the early stages of zygotic embryo-genesis: globular structures at the end of a suspensor-like single file of cells were frequently observed. In the case of leaf protoplasts, high efficiencies of colony formation and plant regeneration occurred in Ws-2 and C24. A novel type of cell division pattern was found in Col-0 and C24, again highly reminiscent of the early division patterns in zygotic embryos. Similarities and differences between zygotic and somatic embryogenesis are discussed.

2,4-Dichlorophenoxyacetic Acid↗
PubMed1997

Effects of fractionated 5-aminolevulinic acid administration on tissue levels of protoporphyrin in vivo.

The photodynamic therapy (PDT) of malignant tissues can be achieved via the administration of 5-aminolevulinic acid (ALA), which is naturally converted to the photoreactive substance protoporphyrin IX (PP). This study compares bolus with fractionated ALA dosing in order to determine whether one of these methods results in a higher tissue concentration of PP. Mice bearing a subcutaneously implanted colon-26 tumor were treated with ALA (200 mg kg-1), given intravenously either as a single bolus or as three equally divided doses at 50 min intervals. Tissue samples of tumor, kidney, skin, liver, skeletal muscle, colon and plasma were obtained 2, 3, 4 and 6 h later for the analysis of PP concentrations. Fractionated dosing results in significantly higher concentrations of PP at 4 and 6 h for kidney, 3 and 6 h for skin, 3 h for colon and 6 h for liver. In contrast, fractionated dosing has no significant effect on the PP concentrations of muscle and plasma. Fractionated dosing results in a significantly greater PP concentration in the tumor at 3 h relative to that observed for the bolus dose. However, from a consideration of the time of PP measurement, it is concluded that fractionated dosing may not cause a significant increase in the PP concentration in colon-26 tumors relative to that observed for the bolus dose.

Aminolevulinic Acid↗
PubMed1997

Cooperativity of folding of the apomyoglobin pH 4 intermediate studied by glycine and proline mutations.

The apomyoglobin pH 4 folding intermediate contains the A, G, and H helices of myoglobin. Helix destabilizing mutations in the A and G helices are used to test whether the pH 4 folding intermediate of apomyoglobin folds cooperatively. Single glycine or proline mutations destabilize the intermediate substantially, showing that intrinsic helix propensities are important for stability of the intermediate. The A and G helices interact to stabilize each other, as shown by the effect of mutations in the G helix on the unfolding of the A helix, which can be monitored by tryptophan fluorescence. Wild type and the most stable mutant unfold in a two-state reaction, as shown by superposition of the unfolding curves measured by two probes (far-UV circular dichroism and Trp fluorescence), while unfolding of the less stable mutants is more complex. Cooperativity and stability of folding are linked also when stabilizing anions (sulphate, perchlorate) are used to adjust stability.

Animals↗
PubMed1997

Identification and characterization of human genes encoding Hprp3p and Hprp4p, interacting components of the spliceosome.

Nuclear RNA splicing occurs in an RNA-protein complex, termed the spliceosome. U4/U6 snRNP is one of four essential small nuclear ribonucleoprotein (snRNP) particles (U1, U2, U5 and U4/U6) present in the spliceosome. U4/U6 snRNP contains two snRNAs (U4 and U6) and a number of proteins. We report here the identification and characterization of two human genes encoding U4/U6-associated splicing factors, Hprp3p and Hprp4p, respectively. Hprp3p is a 77 kDa protein, which is homologous to the Saccharomyces cerevisiae splicing factor Prp3p. Amino acid sequence analysis revealed two putative homologues in Caenorhabditis elegans and Schizosaccharomyces pombe. Polyclonal antibodies against Hprp3p were generated with His-tagged Hprp3p over-produced in Escherichia coli . This splicing factor can co-immunoprecipitate with U4, U6 and U5 snRNAs, suggesting that it is present in the U4/U6.U5 tri-snRNP. Hprp4p is a 58 kDa protein homologous to yeast splicing factor Prp4p. Like yeast Prp4p, the human homologue contains repeats homologous to the beta-subunit of G-proteins. These repeats are called WD repeats because there is a highly conserved dipeptide of tryptophan and aspartic acid present at the end of each repeat. The primary amino acid sequence homology between human Hprp4p and yeast Prp4p led to the discovery of two additional WD repeats in yeast Prp4p. Structural homology between these human and yeast splicing factors and the beta-subunit of G-proteins has been identified by sequence-similarity comparison and analysis of the protein folding by threading. Structural models of Hprp4p and Prp4p with a seven-blade beta-propeller topology have been generated based on the structure of beta-transducin. Hprp3p and Hprp4p have been shown to interact with each other and the first 100 amino acids of Hprp3p are not essential for this interaction. These experiments suggest that both Hprp3p and Hprp4p are components of human spliceosomes.

Amino Acid Sequence↗
PubMed1997

Initiation of apoptosis versus necrosis by photodynamic therapy with chloroaluminum phthalocyanine.

While chloroaluminum phthalocyanine is a highly effective photosensitizer of murine leukemia P388 or L1210 cells, the mode of cell death varies as a function of the PDT dose. When cells were incubated with 0.3 microM of the sensitizer, a light dose of 45 mJ cm-2 (670 +/- 5 nm) yielded a 90% apoptotic cell population within 60 min. The sensitizer localized throughout the cytoplasm and catalyzed both lysosomal and mitochondrial photodamage at this light dose. Higher light doses yielded progressively more membrane photodamage and inhibited the apoptotic response as determined by the examination of Höchst dye HO 33342-labeled nuclei, DNA fragmentation on gels and a poly(adenosylribose) polymerase (PARP)-cleavage assay. Pulse-field gel electrophoresis revealed nonspecific DNA degradation to particles > or = 50 kbp at the higher PDT doses but neither PARP cleavage nor apoptotic nuclei.

Animals↗
PubMed1997

Localization and efficacy analysis of the phototherapeutic lutetium texaphyrin (PCI-0123) in the murine EMT6 sarcoma model.

Lutetium texaphyrin (PCI-0123) is a pure, water-soluble photodynamic therapy (PDT) agent that is activated by tissue-penetrating far red light. The sensitizer is highly fluorescent and exhibits a strong, broad emission signal at 750 nm. In vitro cellular uptake studies revealed an increase in sensitizer retention with incubation time. Confocal laser scanning microscopy demonstrated that the intracellular localization site of PCI-0123 is the lysosomes. Ensuing illumination of the EMT6 cells led to lysosomal breakup, extensive cytoplasmic blebbing and subsequent cell death. Noninvasive spectral imaging analysis of PCI-0123 fluorescence depicted selective drug uptake, compared to surrounding normal tissue, in EMT6 mammary sarcomas syngeneic to BALB/c mice. The PCI-0123 PDT was shown to effectively treat the EMT6 murine sarcoma. Irradiation (732 nm light) 3 h postintravenous injection of 10 mumol PCI-0123 per kg gave 100% cures (no evidence of cancer), whereas light exposure at 5 h resulted in 75% cures. Hematoxylin and eosin histologic examination of photoirradiated tumors indicated apoptosis of the EMT6 neoplasms at early times post-PDT progressing, with time, to extensive necrotic areas. Gel electrophoresis of extracted photoirradiated tumors showed the typical apoptotic DNA ladder pattern that increased in intensity following PDT treatment.

Animals↗
PubMed1997

The role of subcellular localization in initiation of apoptosis by photodynamic therapy.

Rapid initiation of apoptosis can be induced by photodynamic therapy, depending on the cell line and sensitizer employed. In this study, we evaluated the photodynamic responses to two structurally related photosensitizing agents, using the P388 murine leukemia cell line in culture. Photodamage mediated by tin etiopurpurin involved lysosomes and mitochondria and yielded a rapid apoptotic response; apoptotic nuclei were observed within 60 min after PDT. A drug analog, tin octaethylpurpurin amidine, targeted lysosomes, mitochondria and cell membranes; apoptotic nuclei were not observed until 24 h after PDT. These results, together with other recent reports, are consistent with the hypothesis that membrane photodamage can delay or prevent an apoptotic response to PDT.

Animals↗
PubMed1997

Cloning and characterization of the major outer membrane protein gene (ompH) of Pasteurella multocida X-73.

The major outer membrane protein (OmpH) of Pasteurella multocida X-73 was purified by selective extraction with detergents, followed by size exclusion chromatography. The planar lipid bilayer assay showed that OmpH has pore-forming function. The average single channel conductance in 1.0 M KCl was 0.62 nS. The gene (ompH) encoding OmpH has been isolated and sequenced by construction of a genomic library and PCR techniques. The coding region of this gene is 1,059 bp long. The predicted primary protein is composed of 353 amino acids, with a 20-amino-acid signal peptide. The mature protein is composed of 333 amino acids with a molecular mass of 36.665 kDa. The ompH gene encoding mature protein has been expressed in Escherichia coli by using a regulatable expression system. The ompH gene was distributed among 15 P. multocida serotypes and strain CU. Protection studies showed that OmpH was able to induce homologous protection in chickens. These findings demonstrate that OmpH is a protective outer membrane porin of strain X-73 and is conserved among P. multocida somatic serotypes.

Amino Acid Sequence↗