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Y Lu

Publications and source records attributed to Y Lu.

At least 847 records · Page 47Linked to original sources
PubMed1993

Structurally engineered cytochromes with unusual ligand-binding properties: expression of Saccharomyces cerevisiae Met-80-->Ala iso-1-cytochrome c.

A strategy has been developed to express and purify a recombinant, nonfunctional axial-ligand mutant of iso-1-cytochrome c (Met-80-->Ala) in Saccharomyces cerevisiae in quantities necessary for extensive biophysical characterization. It involves coexpressing in the same plasmid (YEp213) the nonfunctional gene with a functional gene copy for complementation in a selective medium. The functional gene encodes a product with an engineered metal-chelating dihistidine site (His-39 and Leu-58-->His) that enables efficient separation of the two isoforms by immobilized metal-affinity chromatography. The purified Met-80-->Ala protein possesses a binding site for dioxygen and other exogenous ligands. Absorption spectra of several derivatives of this mutant show striking similarities to those of corresponding derivatives of horseradish peroxidase, myoglobin, and cytochrome P450. The use of a dual-gene vector for cytochrome c expression together with metal-affinity separation opens the way for the engineering of variants with dramatically altered structural and catalytic properties.

Alanine↗
PubMed1993

Lateral gap junction connections between retinal amacrine cells summating sustained responses.

Vertebrate retinal amacrine cells produce transient or sustained responses. Sustained depolarizing amacrine cells in the dace retina were identified by their intracellularly recorded responses to light flashes. The response amplitude produced a notable spatial summation which exceeded that of individual dendritic arbors. When sustained type amacrine cells were intracellularly injected with Lucifer Yellow and biocytin, there was extensive transfer of biocytin, but not Lucifer Yellow, to surrounding cells with similar cellular morphology. Ultrastructural analysis of the interconnections by electron microscopy revealed the presence of gap junctions at the contact area, which did not include conventional synapses. Present results demonstrate that sustained response amacrine cells make direct electrical connections between the cells of the same type and electrical coupling may contribute to extension of their receptive fields.

Animals↗
PubMed1993

Acute promyelocytic leukemia: clinical relevance of two major PML-RAR alpha isoforms and detection of minimal residual disease by retrotranscriptase/polymerase chain reaction to predict relapse.

Recent data have shown that the PML-RAR alpha fusion gene resulting from translocation t(15;17) is a highly reliable molecular marker of acute promyelocytic leukemia (APL). In this study performed on 97 Chinese patients with APL, the retrotranscriptase/polymerase chain reaction (RT/PCR) was used to evaluate the clinical relevance of the long (L) or short (S) PML-RAR alpha fusion mRNA isoforms and to study minimal residual disease during clinical remission (CR). There were more early deaths during the all-trans retinoic acid (ATRA) induction treatment and more relapses within 2 years of CR in the S-type (6 of 19 cases) than in the L-type group (2 of 33 cases) (P < .025). Among 12 cases analyzed before and after the ATRA-induced CR, 9 cases (75%) showed positive RT/PCR, whereas only 3 cases showed a negative result, justifying the need for chemotherapy after ATRA-induced CR. Eleven of 62 APL patients in CR, after ATRA-induced CR and chemotherapy consolidation (follow-up, from 3 to 72 months), showed positive RT/PCR. Five of them relapsed within 1 to 6 months after the positive test; one converted to negative after further chemotherapy; and 5 remained in CR status without further PCR data. However, the latter 5 cases all received further intensive consolidation therapy after the PCR positivity. These results show that a positive RT/PCR of PML-RAR alpha is a sensitive predictor of relapse in APL.

Adolescent↗
PubMed1993

Increased feeding after treatment with fructose, but not glucose, antimetabolites in rats with dopamine-depleting brain lesions.

Rats were given dopamine-depleting lesions either as adults or as neonates. Compared with intact controls, lesioned rats failed to increase their food intake after acute administration of the glucoprivic agent, 2-deoxy-D-glucose. In contrast, both lesioned and control rats increased food intake after acute treatment with a peripherally-acting fructose antimetabolite, 2,5-anhydro-D-mannitol. These data suggest that the failure of lesioned rats to respond behaviorally to glucoprivation is related to inhibition of glycolysis in the brain, rather than the acute nature of the metabolic challenge.

Animals↗
PubMed1993

Identification and partial characterization of an ectoATPase expressed by human natural killer cells.

An extracellular membrane-associated ectoATPase has been identified on the human natural killer cell line NK3.3. The enzyme is distinct from other classes of ATPases, kinases, and phosphatases. NK3.3 ectoATPase demonstrated a Km for ATP of 41 microM and a Vmax of 0.2 mumol/min and required both Ca2+ and Mg2+ for maximal activity. Purine and pyrimidine nucleotides were competitive inhibitors of the catalytic reaction. Inhibition increased with the addition of increasing negative charge of the phosphate side chain and was also dependent on contributions from the nucleoside. NK3.3 ectoATPase activity was inhibited by reaction with the affinity label [p-(fluorosulfonyl)benzoyl]-5'-adenosine (5'-FSBA), which is shown to modify the enzyme at or near the ATP-binding domain. Photoaffinity labeling of intact NK3.3 cells with [alpha-32P]-8-azidoATP demonstrated an ATP-binding protein of 68-80 kDa unique to NK3.3 cells. A positive correlation was observed between the ability of the various nucleotides to block photoincorporation into the 68-80-kDa protein and their ability to inhibit ectoATPase activity. NK3.3 cells which were made ectoATPase-deficient by reaction with 5'-FSBA demonstrated that this enzyme does not have a major role in the protection of this cytolytic effector cell from the possible lytic effects of extracellular ATP.

Adenosine Triphosphatases↗
PubMed1993

Cloning and expression of a novel human DNA binding protein, PO-GA.

We have cloned a novel human DNA-binding protein from a HeLa cDNA expression library using the cognate DNA binding site of a transcription factor, PO-B. Further hybridization screening with the initial clone produced contiguous cDNA sequence of 4508 bp and a complete open reading frame encoding a 128 kDa protein, PO-GA. Northern analysis revealed a wide distribution of PO-GA mRNA in most human tissue. However, PO-GA mRNA levels were lower in lung and kidney and undetectable in placental tissue. A DNA-binding fragment of PO-GA expressed in E. Coli bound selectively to the PO-B element and other GA-rich double-stranded DNA sequences and to certain single-stranded DNA sequences. PO-GA has regions of homology to E. coli and yeast DNA ligases, and to proteins involved in DNA repair. Thus, in addition to a potential role in transcription, PO-GA may also be involved in DNA repair or replication.

Amino Acid Sequence↗
PubMed1993

Induction of N-acetylglucosaminyltransferase V by elevated expression of activated or proto-Ha-ras oncogenes.

Viral infection of cultured cells with transforming viruses causes an increase in cell-surface N-linked beta 1-6 (GlcNAc beta 1-6Man) branching of complex-type oligosaccharides. Similar observations have been made after transfection of cells with activated oncogenes, which is associated with an induction of tumorigenic and metastatic properties. In this study, the effects of transfection of both activated and proto-Ha-ras oncogenes into NIH3T3 cells were analyzed. The results showed that, in comparison with NIH3T3 cells, both ras transfectants have increased sensitivity to the cytotoxic action of L-PHA. An increase in beta 1-6 branching and an increased level of N-acetylglucosaminyltransferase V (GlcNAc-T V), the enzyme which initiates the beta 1-6 branching were also observed. The levels of GlcNAc-T I and beta 1-4 Gal-T remained unchanged in activated Ha-ras transfected NIH3T3 cells. These data suggest that a specific induction of GlcNAc-T V occurs after transfection with either the proto- or activated Ha-ras oncogenes, which is responsible for the increased beta 1-6 branching previously observed.

3T3 Cells↗
PubMed1993

Isolation of oligomannose-type glycans from bean glycoproteins.

We have isolated individual oligosaccharyl-asparagine derivatives from the total soluble glycoproteins from kidney beens (Phaseolus vulgaris) and from lima beans (Phaseolus limensis). The protein/glycoprotein mixture was digested exhaustively by pronase, and the glycan-containing fractions were separated from free amino acids and peptides by gel filtration. The oligosaccharyl-asparagine derivatives were finally fractionated on Dowex 50 (C. C. Huang, H.E. Meyer, and R. Montgomery, Carbohydr. Res. 13, 127-137, 1970), and the individual fractions were characterized by mass spectrometry, NMR, and ion exchange chromatography. With the procedures described, only oligomannose derivatives were obtained from the beans. In the case of kidney beans, six different derivatives were observed and characterized, Man9GlcNAc2Asn, two positional isomers of Man8GlcNAc2Asn, two positional isomers of Man7GlcNAc2Asn, and Man6GlcNAc2Asn. Under identical conditions the lima beans yielded primarily the Man9GlcNAc2Asn derivative along with a small amount of the two Man8GlcNAc2Asn derivatives. The oligomannose structures can be isolated in reasonable quantities (2-20 mg) from about 200 g of dry beans.

Asparagine↗
PubMed1993

Point mutations are associated with a gene duplication leading to the bloodstream reexpression of a trypanosome metacyclic VSG.

African trypanosomes evade the immune response of their hosts by sequentially expressing different variant surface glycoproteins (VSGs). We isolated a bloodstream trypanosome clone of Trypanosoma brucei rhodesiense that expresses a VSG normally present during the metacyclic stage of the parasite in the insect vector. Associated with the bloodstream reexpression of this metacyclic VSG is a gene conversion in which the duplicated, expressed gene of 1650 nt contains 11 scattered point mutations when compared with its donor gene. Analysis of an uncloned population of bloodstream trypanosomes revealed another VSG reexpressor of the same donor gene in which the coding region had undergone 24 point mutations. The mutations are unique to the duplicated gene and appear to be nontemplated. The generation of these mutations provides a way for the trypanosome to increase further its antigenic diversity.

Amino Acid Sequence↗
PubMed1993

On the regulation of K+ uniport in intact mitochondria by adenine nucleotides and nucleotide analogs.

Respiring mitochondria drive the electrophoretic uptake of K+ and other cations. In the presence of permeant acids this transport leads to mitochondrial swelling if it is not compensated by electroneutral K+/H+ exchange mediated by the K+/H+ antiporter. The mechanism of influx has yet to be established; however, evidence is accumulating that in addition to leak pathways a specific K+ channel or uniporter may be involved. We examine some of the properties of K+ uniport which are consistent with the existence of a specific ATP-regulated K+ channel. In contrast to the K+/H+ antiporter, K+ uniport shows little dependence on pH. K+ uniport is, however, very sensitive to inhibition by adenine nucleotides. The maximum percent inhibition is increased from 40 to 60% by treatment of mitochondria with N-ethylmaleimide (30 nmol/mg) which stimulates K+ uniport 3.6-fold. N-Ethylmaleimide, however, has no effect on the IC50 values which are 0.5, 2.3, and 8 microM for ADP, ATP, and AMP, respectively. GDP has no effect, while carboxyatractyloside is found to inhibit. The nucleotide analogs Cibacron blue 3GA and erythrosin B exhibit three effects on K+ uniport. Low doses partially inhibit K uniport (I50 = 0.13 microM Cibacron Blue), while higher doses stimulate (EC50 = 13 microM Cibacron Blue). Stimulation is especially apparent in N-ethylmaleimide-treated mitochondria. These analogs also antagonize inhibition by ATP. Since the EC50 values for this antagonism for these two drugs are similar, while the IC50 values for inhibition of ATP transport differ by a factor of five, we suggest that inhibition of K+ uniport by ATP is not mediated via the adenine nucleotide translocase. These data are consistent with the existence of an ATP-regulated K+ channel in the inner mitochondrial membrane.

Adenine Nucleotides↗
PubMed1993

Histological organization of the right and left atrioventricular valves of the chicken heart and their relationship to the atrioventricular Purkinje ring and the middle bundle branch.

In the avian heart the right and left atrioventricular (AV) valves not only exhibit their own special anatomical characteristics, but they also are in close proximity to the conduction system. The right AV valve is a single, spiral plane of myocardium, in remarkable contrast to the fibrous structure characteristic of the mammalian tricuspid valve. A ring of Purkinje tissue encircles the avian right AV orifice and connects to the muscular valve. The chicken has no crista supraventricularis, its right AV valve serving that function as well as opening and closing the right AV orifice. The left AV valve consists of three leaflets instead of the two typical of mammalian hearts. Its anterior and posterior leaflets are small; its large aortic (medial) leaflet merges with the bases of both the left and noncoronary cusps of the aortic valve by fibrous tissue, resembling that of the mammalian heart. However, unlike in mammals, there is a slim cylinder of continuous myocardium coursing parallel to this fibrous junction. This unusual arc of myocardium in the chicken serves to complete an entire subaortic ring of myocardium and is thus potentially capable of constricting the outflow tract of the chicken's left ventricle. The middle bundle branch connects with both the muscle arch and the AV Purkinje ring. Thus the myocardium in or near both AV valves (and the left ventricular outflow tract) in the chicken heart is so arranged that it may receive direct early activation from the conduction system.

Animals↗
PubMed1993

Cardiac conduction system in the chicken: gross anatomy plus light and electron microscopy.

Twenty-three chicken hearts were used to study the cardiac conduction system by light and electron microscopy. In addition to a sinus node, atrioventricular node (AVN), His bundle, left and right bundle branches (LBB, RBB), the chicken also has an AV Purkinje ring and a special middle bundle branch (MBB). The sinus node lies near the base of the lower portion of the right sinoatrial valve. The AV node is just above the tricuspid valve and anterior to the coronary sinus. The His bundle descends from the anterior and inferior margin of the AV node into the interventricular septum, then dividing into right, left and middle branches some distance below the septal crest. The middle bundle branch turns posteriorly toward the root of the aorta. The AV Purkinje ring originates from the proximal AV node and then encircles the right AV orifice, joining the MBB to form a figure-of-eight loop. The chicken conduction system contains four types of myocytes: 1) The P cell is small and rounded, with a relatively large nucleus and sparse myofibrils. 2) The transitional cell is slender and full of myofibrils. 3) The Purkinje-like cell resembles the typical Purkinje cell, but is smaller and darker. 4) The Purkinje cell is found in the His bundle, its branches, and the periarterial and subendocardial Purkinje network.

Animals↗
PubMed1993

Association of the p56lck protein tyrosine kinase with the Fc gamma RIIIA/CD16 complex in human natural killer cells.

The multimeric Fc gamma RIIIA (CD16) complex is expressed on the surface of natural killer (NK) cells and is composed of a 50-70-kDa transmembrane glycoprotein Fc gamma receptor (CD16), the T cell receptor (TCR)-zeta chain, and the Fc epsilon RI gamma chain. Cross-linking Fc gamma RIIIA initiates the rapid tyrosine phosphorylation of multiple substrates including the zeta subunit and causes subsequent cell activation and antibody-dependent cellular cytotoxicity (ADCC). The subunits of the Fc gamma RIIIA complex lack intrinsic protein tyrosine kinase (PTK) activity, suggesting that receptor-induced tyrosine phosphorylation events are mediated by a nonreceptor PTK. We report here that the human Fc gamma RIIIA is complexed with p56lck, a src-family PTK previously found associated with the CD4 and CD8 receptors on T cells. Upon engagement of the CD16 receptor, p56lck is rapidly (within 30 s) and transiently phosphorylated on tyrosine residues. Several Fc gamma RIIIA-associated proteins are identified in immune complex kinase assays including the TCR-zeta subunit, a p70-90 zeta-associated protein (ZAP), p50a (acidic) and p50b (basic), and p56lck. We demonstrate that the src-family protein tyrosine kinase inhibitor, herbimycin A, blocks increased intracellular calcium levels and ADCC caused by CD16 cross-linking on NK3.3 cells. Likewise cross-linking CD16 with the protein tyrosine phosphatase CD45, abrogates CD16-induced calcium mobilization. These data suggest that p56lck is physically associated with Fc gamma RIIIA (CD16) and functions to mediate signaling events related to the control of NK cellular cytotoxicity.

Antibody-Dependent Cell Cytotoxicity↗
PubMed1993

Molecular aspects of early stages of breast cancer progression.

It is clear that breast cancer progression is associated with inactivation of a number of different recessive oncogenes. The most widely evaluated tumor suppressor gene, p53, is mutated in approximately 30-50% of sporadic breast cancers. Mutations usually occur early in malignant progression. Loss of heterozygosity (LOH) studies have identified numerous chromosomal regions where other recessive oncogenes relevant to breast cancer may be located. Each LOH is seen in a varying proportion of breast cancers and may appear either early or late in progression. High-grade ductal carcinoma in situ (DCIS) and invasive carcinoma have similar genetic lesions, showing that aberrations can occur before invasive disease. Direct evidence that the same aberrations can be acquired later in progression comes from a study of multiple metastases from the same patient; other studies found that primary invasive cancers are characterized by marked intratumor heterogeneity for each lesion examined. The model we propose to account for these results hypothesizes that multiple genetic lesions can accomplish each phenotype required for malignancy (i.e., dysregulated proliferation, invasion, angiogenesis, etc.) and that, for a given tumor, at least one aberrant gene for each phenotypic change is stochastically selected. Biological heterogeneity of breast cancer results from the stochastic acquisition of various genetic aberrations. We further propose that the lymphocytic reaction in high-grade DCIS may select for aggressive tumor subpopulations capable of escaping immune surveillance. Another aspect of tumor heterogeneity may be the multiple mechanisms employed by various tumors to escape immune surveillance.

Breast Neoplasms↗
PubMed1993

6-substituted 3,4-benzocoumarins: a new structural class of inducers and inhibitors of CYP1A1-dependent activity.

A new synthetic route was utilized to prepare 6-substituted 3,4-benzocoumarins where the substituents were iodo, fluoro, trifluoromethyl, bromo, chloro, isopropyl, ethyl, t-butyl, methyl, hydrogen, amino, phenyl, or nitro; 3,4-naphthocoumarin was also synthesized. The relative affinities of these congeners for the aryl hydrocarbon (Ah) receptor were determined using rat hepatic cytosol and 2,3,7,8-[3H]tetrachlorodibenzo-p-dioxin ([3H]TCDD) as the radioligand. In addition, the Ah receptor agonist activity of the 6-substituted 3,4-benzocoumarins was determined from their concentration-dependent induction of ethoxyresorufin O-deethylase (EROD) activity. In contrast with many other structural classes of halogenated aromatics, there was not a correlation between the structure-binding versus structure induction relationships for the 6-substituted 3,4-benzocoumarins. These results suggested that some of these congeners may exhibit partial Ah receptor antagonist activities and this was investigated by determining the inhibitory effects of 6-substituted 3,4-benzocoumarins on TCDD-induced EROD activity in rat hepatoma H4II E cells in culture. Only four compounds (6-isopropyl, 6-phenyl, 6-fluoro, and 6-t-butyl) inhibited the TCDD-induced response (21.7 to 64.4% inhibition) and the mechanism of action of the most active inhibitor, 6-t-butyl-3,4-benzocoumarin, was further investigated. In contrast, with other partial Ah receptor antagonists such as alpha-naphthoflavone, cotreatment of rat hepatoma H4II E cells with 1 nM TCDD plus 1 and 10 microM 6-t-butyl-3,4-benzocoumarin did not result in decreased levels of the Ah receptor complex (liganded with TCDD). In addition, there was not significant inhibition of TCDD-induced CYP1A1 mRNA levels or protein as determined by Northern and Western blot analyses. The results suggest that 6-t-butyl-3,4-benzocoumarin or one of its metabolites is a post-translational inhibitor of CYP1A1-dependent enzyme (EROD) activity in this cell line and thus represents a novel Ah receptor-independent inhibition of CYP1A1.

Animals↗
PubMed1993

Expression of chimeric human transferrin-chloramphenicol acetyltransferase genes in liver and brain of transgenic mice during development.

Transferrin (TF) gene expression is tissue specific and is regulated during development. Transgenic mice have been developed which carry 1.2 or 0.67 kb of the TF 5' flanking region of the human TF gene fused to the bacterial chloramphenicol acetyltransferase (CAT) gene. The onset of expression of the chimeric human TF-CAT transgenes in liver and brain during development has been studied in these transgenic mice. In brain, the TF(0.67)CAT transgene began to express between 5 and 10 days after birth; in liver, the TF(0.67)CAT transgene was turned on between 10 and 20 days after birth. Endogenous mouse TF mRNA levels in liver and brain have also been measured during development by Northern analysis. In brain, the developmental expression pattern of the TF(0.67)CAT transgene is the same as the mouse endogenous TF gene; in liver, the transgene is turned on later than the endogenous mouse TF gene. DNA-protein mobility shift assays and DNase I footprinting analyses were conducted in the region of -621 to -409 bp of the human TF gene by using TF-CAT expressing liver nuclear extract from 27-day-old mice and nonexpressing liver nuclear extract from 7-day-old mice. The level of protein-DNA complex formation is several times higher in the expressing extracts, and the region from -481 to -463 bp of human TF gene is protected by the expressing extract but not the nonexpressing extracts. As demonstrated by this and other studies, the transgenic mouse model furnishes a unique opportunity to analyze developmental regulation of human transgenes.

Animals↗