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X J Chen

Publications and source records attributed to X J Chen.

At least 55 records · Page 3Linked to original sources
PubMed1998

Cloning, sequencing and disruption of the ARG8 gene encoding acetylornithine aminotransferase in the petite-negative yeast Kluyveromyces lactis.

A recombinant plasmid was isolated from a Kluyveromyces lactis genomic DNA library which complements a Saccharomyces cerevisiae arg8 mutant defective in the gene encoding acetylornithine aminotransferase. The complementation activity was found to reside within a 2.0 kb DNA fragment. Nucleotide sequence analysis revealed an open reading frame able to encode a 423-residue protein sharing 68.1% and 35.0% sequence identities with the products of the ARG8 and argD genes of S. cerevisiae and Escherichia coli. That the cloned gene, KlARG8, is the functional equivalent of S. cerevisiae ARG8 was supported by a gene disruption experiment which showed that K. lactis strains carrying a deleted chromosomal copy of KlARG8 are auxotrophic for arginine.

Base Sequence↗
PubMed1998

MR microscopy of lung airways with hyperpolarized 3He.

A technique using hyperpolarized (HP) 3He to image the small airways of the lung by using moderate flip angles and a short scanning period during early inspiration is demonstrated. Flip angles (alpha) ranging from 10-90 degrees were used in guinea pig experiments with scanning during the entire inspiration period. A second series acquired data throughout a short window of the ventilatory cycle with alpha = 45 degrees. The success of the animal studies has motivated implementation of similar imaging techniques in the clinical arena. Human studies involved imaging over the total inspiration period with alpha approximately 10 degrees. The first series of guinea pig experiments demonstrated that larger flip angles (50-90 degrees) destroy the magnetization before it reaches the smaller airways. At moderate flip angles (20-40 degrees), airway branching down to the fourth generation was apparent. Fifth-order branchings were seen in the images of the second series. The trachea down to fourth generation pulmonary airway branching, along with some distal air spaces, was seen in the human lung images.

Animals↗
PubMed1998

Hyperpolarized 3He NMR lineshape measurements in the live guinea pig lung.

Spatially localized lineshapes of hyperpolarized (HP) 3He in guinea pig lungs have been measured in vivo. Three different axial slice locations, each containing different compositions of airway sizes and orientations, were studied. Gas peaks from major bronchi (2 ppm) and alveoli (-2 ppm) were distinguished. The gas phase spectra show structural features that are a result of frequency shifts caused by bulk magnetic susceptibility. For a given slice, the spectral lineshapes reflect the airway composition within the slice location, according to theory. The peak assignments given here also agree with previous studies done by Wagshul et al. with HP 129Xe. At each of the slice locations, data were acquired during two phases of the breathing cycle, resulting in a relative frequency shift of approximately 0.3 ppm in the superior slices. Spectra obtained over a number of breaths show the dynamics of the gas buildup in the lung and provide further evidence supporting the peak assignments.

Animals↗
PubMed1998

Signal dynamics in magnetic resonance imaging of the lung with hyperpolarized noble gases.

The nonequilibrium bulk magnetic moment of hyperpolarized (HP) noble gases generated by optical pumping has unique characteristics. Based on the Bloch equations, a model was developed describing the signal dynamics of HP gases used in magnetic resonance imaging (MRI) of the lung with special consideration to the breathing cycle. Experimental verification included extensive investigations with HP 3He and 129Xe during both inspiration and held breath in live guinea pigs. Radial acquisition was used to investigate the view variations with a temporal resolution of 5 ms. Agreement between theoretical predictions and in vivo results was excellent. Additionally, information about effects from noble gas diffusion and spin-lattice relaxation was obtained. In vivo results for T1 were 28.8 +/- 1.8 s for 3He and 31.3 +/- 1.8 s for 129Xe. Comparison with in vitro data indicated that relaxation in the pulmonary gas space is dominated by dipolar coupling with molecular oxygen. The results provide a quantitative basis for optimizing pulse sequence design in HP gas MRI of the lung.

Animals↗
PubMed1998

Allele-specific expression of the Mgi- phenotype on disruption of the F1-ATPase delta-subunit gene in Kluyveromyces lactis.

Kluyveromyces lactis is a petite-negative yeast that does not form viable mitochondrial genome-deletion mutants (petites) when treated with DNA-targeting drugs. Loss of mtDNA is lethal for this yeast but mutations at three loci termed MGI, for mitochondrial genome integrity, can suppress this lethality. The three loci encode the alpha-, beta- and gamma-subunits of mitochondrial F1-ATPase. In this study we report the isolation and characterization of the KlATPdelta gene encoding the delta-subunit of F1-ATPase. The deduced protein contains 158 amino acids showing 72% identity to the protein from Saccharomyces cerevisiae and a putative mitochondrial targeting sequence of 23 amino acids. Disruption of the gene causes cells to become respiratory deficient while the introduction of ATPdelta from S. cerevisiae restores growth on glycerol. Cells with a disrupted ATPdelta gene, like strains with disruptions of alpha-, beta- and gamma-F1-subunits, do not produce petite mutants when treated with ethidium bromide. However, unlike strains with disruptions in the three largest F1-subunits, disruption of ATPdelta in the presence of some mgi alleles does not abolish the Mgi- phenotype. By contrast, elimination of ATPdelta in other mgi strains removes resistance to ethidium bromide and rho0 mutants are not formed. Hence the ATPdelta subunit of F1-ATPase, while not mandatory for a Mgi- phenotype, aids some mgi alleles in suppressing rho0 lethality.

Alleles↗
PubMed1998

Suppression of rho0 lethality by mitochondrial ATP synthase F1 mutations in Kluyveromyces lactis occurs in the absence of F0.

Specific mgi mutations in the alpha, beta or gamma subunits of the mitochondrial F1-ATPase have previously been found to suppress rho0 lethality in the petite-negative yeast Kluyveromyces lactis. To determine whether the suppressive activity of the altered F1 is dependent on the F0 sector of ATP synthase, we isolated and disrupted the genes KlATP4, 5 and 7, the three nuclear genes encoding subunits b, OSCP and d. Strains disrupted for any one, or all three of these genes are respiration deficient and have reduced viability. However a strain devoid of the three nuclear genes is still unable to lose mitochondrial DNA, whereas a mgi mutant with the three genes inactivated remains petite-positive. In the latter case, rho0 mutants can be isolated, upon treatment with ethidium bromide, that lack six major F0 subunits, namely the nucleus-encoded subunits b, OSCP and d, and the mitochondrially encoded Atp6, 8 and 9p. Production of rho0 mutants indicates that an F1-complex carrying a mgi mutation can assemble in the absence of F0 subunits and that suppression of rho0 lethality is an intrinsic property of the altered F1 particle.

Alleles↗
PubMed1998

Nitric oxide inhibits lung sodium transport through a cGMP-mediated inhibition of epithelial cation channels.

We used the patch-clamp technique to study the effect of nitric oxide (NO) on a cation channel in rat type II pneumocytes [alveolar type II (AT II) cells]. Single-channel recordings from the apical surface of AT II cells in primary culture showed a predominant cation channel with a conductance of 20.6 +/- 1.1 (SE) pS (n = 9 cell-attached patches) and Na(+)-to-K+ selectivity of 0.97 +/- 0.07 (n = 7 cell-attached patches). An NO donor, S-nitrosoglutathione (GSNO; 100 microM), inhibited the basal cation-channel activity by 43% [open probability (Po), control 0.28 +/- 0.05 vs. GSNO 0.16 +/- 0.03; P < 0.001; n = 16 cell-attached patches], with no significant change in the conductance. GSNO reduced the Po by reducing channel mean open and increasing mean closed times. GSNO inhibition was reversed by washout. The inhibitory effect of NO was confirmed by using a second donor of NO, S-nitroso-N-acetylpenicillamine (100 microM; Po, control 0.53 +/- 0.05 vs. S-nitroso-N-acetylpenicillamine 0.31 +/- 0.04; -42%; P < 0.05; n = 5 cell-attached patches). The GSNO effect was blocked by methylene blue (a blocker of guanylyl cyclase; 100 microM), suggesting a role for cGMP. The permeable analog of cGMP, 8-bromo-cGMP (8-BrcGMP; 1 mM), inhibited the cation channel in a manner similar to GSNO (Po, control 0.38 +/- 0.06 vs. 8-BrcGMP 0.09 +/- 0.02; P < 0.05; n = 7 cell-attached patches). Pretreatment of cells with 1 microM KT-5823 (a blocker of protein kinase G) abolished the inhibitory effect of GSNO. The NO inhibition of channels was not due to changes in cell viability. Intracellular cGMP was found to be elevated in AT II cells treated with NO (control 13.4 +/- 3.6 vs. GSNO 25.4 +/- 4.1 fmol/ml; P < 0.05; n = 6 cell-attached patches). We conclude that NO suppresses the activity of an Na(+)-permeant cation channel on the apical surface of AT II cells. This action appears to be mediated by a cGMP-dependent protein kinase.

Animals↗
PubMed1997

Dynamics of magnetization in hyperpolarized gas MRI of the lung.

The magnetization in hyperpolarized gas (HP) MRI is generated by laser polarization that is independent of the magnet and imaging process. As a consequence, there is no equilibrium magnetization during the image acquisition. The competing processes of gas inflow and depolarization of the spins lead to large changes in signal as one samples k-space. A model is developed of dynamic changes in polarization of hyperpolarized 3He during infusion and in vivo imaging of the lung and verified experimentally in a live guinea pig. Projection encoding is used to measure the view-to-view variation with temporal resolution < 4 ms. Large excitation angles effectively sample the magnetization in the early stages of inflow, highlighting larger airways, while smaller excitation angles produce images of the more distal spaces. The work provides a basis for pulse sequences designed to effectively exploit HP MRI in the lung.

Animals↗
PubMed1997

Mitochondrial ATP synthase subunit 9 is not required for viability of the petite-negative yeast Kluyveromyces lactis.

Specific mutations in nuclear MGI genes encoding the alpha, beta and gamma subunits of the mitochondrial inner membrane F1-ATPase complex allow mitochondrial DNA (mtDNA) to be lost from K. lactis. In the absence of a mutation in any of these three nuclear genes, loss of mtDNA is lethal. These results imply that mtDNA encodes a gene that is essential. Likely candidates for such an essential role are the ATP6, 8 and 9 genes coding for proteins of the ATP synthase-F0 component. The present study removes ATP9 from contention as a vital mitochondrial gene because in a respiratory deficient mutant, Gly- 3. 9, lacking a nuclear mgi mutation, we have found that a rearrangement in mtDNA has deleted 22 amino acids from the carboxy terminus of the 75 amino-acid subunit-9 protein. Rearrangement in mtDNA has occurred by recombination at a 23-bp repeated sequence in the introns of the ATP9 and large ribosomal RNA (LSU) subunit genes. These two introns, of 394 (ATP9) and 410 (LSU) nucleotides, both belong to group 1.

Amino Acid Sequence↗
PubMed1997

Cloning and characterization of the lipoyl-protein ligase gene LIPB from the yeast Kluyveromyces lactis: synergistic respiratory deficiency due to mutations in LIPB and mitochondrial F1-ATPase subunits.

The mgi1-4 and mgi2-1 mutants of the petite-negative yeast Kluyveromyces lactis have mutations in the beta- and alpha-subunits of the mitochondrial F1-ATPase, respectively. The mutants are respiratory competent but can form petites with deletions in mitochondrial DNA. In this study a cryptic nuclear mutation (lipB-1) was identified which, in combination with the mgi alleles, displays a synergistic respiratory-deficient phenotype on glycerol medium. The gene defined by the mutation was cloned and shown to encode a polypeptide of 332 amino acids with an N-terminal sequence characteristic of a mitochondrial targeting signal. The deduced protein shares 27% sequence identity with the product of the Escherichia coli lipB gene, which encodes a lipoyl-protein ligase involved in the attachment of lipoyl groups to lipoate-dependent apoproteins. A K. lactis strain carrying a disrupted lipB allele is severely compromised for growth on glycerol medium. The growth defect cannot be rescued by addition of lipoic acid, but cell growth can be restored on medium containing ethanol plus succinate. In addition, it was observed that lipB mutants of K. lactis, unlike the wild-type, are unable to utilize glycine as sole nitrogen source, indicating that activity of the glycine decarboxylase complex (GDC) is also affected. Taken together, these findings suggest that LIPB is the main determinant of the lipoyl-protein ligase activity required for lipoylation of enzymes such as alpha-ketoacid dehydrogenases and GDC.

Amino Acid Sequence↗
PubMed1997

Single-site mutations in the conserved alternating-arginine region affect ionic channels formed by CryIAa, a Bacillus thuringiensis toxin.

The role of the third domain of CryIAa, a Bacillus thuringiensis insecticidal toxin, in toxin-induced membrane permeabilization in a receptor-free environment was investigated. Planar lipid bilayer experiments were conducted with the parental toxin and five proteins obtained by site-directed mutagenesis in block 4, an arginine-rich, highly conserved region of the protein. Four mutants were constructed by replacing the first arginine in position 21 by a lysine (R521K), a glutamine (R521Q), a histidine (R521H), or a glutamic acid (R521E). A fifth mutant was obtained by replacing the fourth arginine by a lysine (R527K). Like CryIAa, the mutants formed cation-selective channels. A limited but significant reduction in channel conductance was observed for all mutants except R521H. The effect was more dramatic for the voltage dependence of the channels formed by R521K and R521Q, which was reversed compared to that of the parental toxin. This study provides the first direct evidence of a functional role for domain III in membrane permeabilization. Our results suggest that residues of the positive arginine face of block 4 interact with domain I, the putative pore-forming region of CryIAa.

Amino Acid Sequence↗
PubMed1997

[High performance liquid chromatographic assay for aceclofenac in plasma and its pharmacokinetics in dogs].

A sensitive and simplified high performance liquid chromatographic procedure has been developed for the quantification of aceclofenac and its major metabolite in dog plasma. The recovery of aceclofenac was 91.3%-96.9%. The relative standard deviation was 3.69%-8.13%. The calibration curve was linear in the range from 0.050 microgram.ml-1 to 51.2 micrograms.ml-1 with gamma = 0.9998. The detection limit of the method is 10 ng.ml-1 for aceclofenac. Plasma concentrations of both aceclofenac and its major metabolite were simultaneously determined. After oral medication of aceclofenac to dogs at the dose of 2.5 mg.kg-1, the drug was absorbed rapidly and reached its peak level in plasma at about 12 min. The plasma drug concentration-time course conformed to a 2-compartment open model with a first order absorption. The mean value of T1/2 alpha and T1/2 beta for aceclofenac were 2.5 min and 137 min respectively. Plasma diclofenac concentration, a major metabolite of aceclofenac, reached its maximum at about 110 min, the mean Cmax for diclofenac was 3.2 micrograms.ml-1. The mean terminal elimination half-life for diclofenac was 140 min.

Animals↗
PubMed1996

Probing the mechanism of action of Bacillus thuringiensis insecticidal proteins by site-directed mutagenesis--a minireview.

The current model of the mechanism of action of several Bacillus thuringiensis insecticidal crystal proteins (Cry) is reviewed and tested by site-directed mutagenesis experiments. Amino acid (aa) residues were substituted in each of the three domains of Cry toxins and the effects on toxin stability, binding to receptors, irreversible insertion into the membrane, and ion channel activity were examined. Mutant proteins with aa altered on the putative membrane-proximal surface of domain I are affected in insertion into the membrane and toxicity, but not in binding to the receptor. Alterations in the putative receptor-binding loops of domain II show an effect on the initial (reversible) binding to the receptor when certain aa are altered, while affecting irreversible binding when other aa are altered. Mutant proteins with aa altered in a conserved track of aa of domain III have altered ion channel properties, as measured by the voltage clamping of insect midguts and the K+ permeability of brush border membrane vesicles. In summary, domain I is involved in insertion into the membrane and affects ion channel function, domain II is involved in receptor binding and insertion into the membrane, and domain III is involved ion channel function, receptor binding, and insertion into the membrane.

Bacillus thuringiensis↗
PubMed1996

A vital function for mitochondrial DNA in the petite-negative yeast Kluyveromyces lactis.

Petite-negative yeasts do not form viable respiratory-deficient mutants on treatment with DNA-targeting drugs that readily eliminate the mitochondrial DNA (mtDNA) from petite-positive yeasts. However, in the petite-negative yeast Kluyveromyces lactis, specific mutations in the nuclear genes MG12 and MG15 encoding the alpha- and gamma-subunits of the mitochondrial F1-ATPase, allow mtDNA to be lost. In this study we show that wild-type K. lactis does not survive in the absence of its mitochondrial genome and that the function of mgi mutations is to suppress lethality caused by loss of mtDNA. Firstly, we find that loss of a multicopy plasmid bearing a mgi allele readily occurs from a wild-type strain with functional mtDNA but is not tolerated in the absence of mtDNA. Secondly, we cloned the K. lactis homologue of the Saccharomyces cerevisiae mitochondrial genome maintenance gene MGM101, and disrupted one of the two copies in a diploid. Following sporulation, we find that segregants containing the disrupted gene form minicolonies containing 6-8000 inviable cells. By contrast, disruption of MGM101 is not lethal in a haploid mgi strain with a specific mutation in a subunit of the mitochondrial F1-ATPase. These observations suggest that mtDNA in K. lactis encodes a vital function which may reside in one of the three mitochondrially encoded subunits of Fo.

Amino Acid Sequence↗
PubMed1996

Low- and high-copy-number shuttle vectors for replication in the budding yeast Kluyveromyces lactis.

Four sets of plasmid vectors for the budding yeast Kluyveromyces lactis (Kl) have been constructed. All plasmids are pUC19-based shuttle vectors having multiple unique sites in their multiple cloning site (MCS) within the bacterial lacZ gene. The first set of vectors contains Klori, the origin of replication for Kl isolated from Kluyveromyces plasmid pKD1, and one of the selectable nutritional markers, URA3, TRP1 or LEU2. These markers from the yeast, Saccharomyces cerevisiae (Sc), can complement the uraA1, trp1 and leu2 mutations of Kl. The second set of vectors, in addition to Klori, contains the ARS (autonomously replicating sequence) and centromeric sequences of Sc, and are able to replicate in both Sc and Kl. The third group of plasmids is centromeric vectors that are maintained in Kl at low copy number. The last family of vectors was designed for gene overexpression. As they contain the bacterial kanamycin-resistance-encoding gene (kan), plasmid copy number can be amplified to over 100 copies per cell in Kl by growing cells in the presence of the antibiotic G418 (Geneticin). This type of vector has been used to study the high-copy-lethality phenotype of a truncated version of the Kl MGI2 gene encoding the alpha-subunit of the mitochondrial F1F0-ATP synthase.

Cloning, Molecular↗
PubMed1996

A new point mutation in the nuclear gene of yeast mitochondrial RNA polymerase, RPO41, identifies a functionally important amino-acid residue in a protein region conserved among mitochondrial core enzymes.

The core enzyme of mitochondrial RNA polymerase in yeast is homologous to those of bacteriophages T3, T7 and SP6. In previous studies the identification of the first conditional yeast mutant for this enzyme helped to identify the corresponding specificity factor and to elucidate their interaction inside mitochondria. In the present study we report the identification of a second nuclear mutation located in the gene for mitochondrial RNA polymerase. A comparison of the two temperature-sensitive mutants demonstrates that the new mutant has a phenotype distinct from the first one and characterizes a new important domain of the enzyme. Two different suppressor genes which both rescue the first mutant do not abolish the defect of the second one and, in addition, an extremely high instability of mitochondrial genomes is observed in the new mutant. The enzymatic defect is caused by a single nucleotide exchange which results in the replacement of the serine938 residue by phenylalanine. This amino acid is located in the middle part of the protein in an as yet poorly characterized region that is not highly conserved between mitochondrial core enzymes and bacteriophage-type RNA polymerases. However, the affected amino acid and the respective protein domain are specific for mitochondrial RNA polymerase core enzymes and may help to define enzymatic functions specific for the mitochondrial transcription apparatus.

Amino Acid Sequence↗