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Y Bai

Publications and source records attributed to Y Bai.

At least 91 records · Page 5Linked to original sources
PubMed1999

Two proteins share immunological epitopes on the tumor-associated antigen 17-1A.

The mouse monoclonal antibody (mAb) 17-1A which recognizes the tumor-associated antigen 17-1A (also called EGP-40 or EpCAM) was successfully used in adjuvant therapy for colorectal carcinoma. In the 17-1A antigen analysis, we isolated not only a protein of 33 kDa (P33) which was reported as the tumor associated antigen 17-1A, but also a protein of 65 kDa (P65) using affinity chromatography from cell lysates of HCT, and another protein of 50 kDa (P50) from lysates of human colorectal tumor tissues. The mAbs 17-1A and M79 (mAb M79 recognizes a different epitope on the 17-1A antigen) both could bind P33 and P50, but only M79 bound to P65 in an enzyme-linked immunosorbant assay (ELISA). These results indicate that P33 and P50 share at least two epitopes, and a common immunological epitope exists among P33, P50 and P65, suggesting that the two new proteins (P50 and P65) are related to the tumor-associated antigen 17-1A.

Animals↗
PubMed1999

Control of 3' splice site choice in vivo by ASF/SF2 and hnRNP A1.

The constitutive splicing factor ASF/SF2 has been shown to affect the choice between alternative splice sites by favoring the proximal as opposed to the distal choice. HnRNP A1 antagonizes ASF/SF2 by promoting the distal choice for competing 5' splice sites. We have tested the in vivo effects of these proteins on alternative 3' splice site choices. Cotransfection of a dihydrofolate reductase-calcitonin chimeric construct togetherwith a plasmid specifying the SR protein ASF/SF2 into cells of several mammalian lines increased use of a proximal 3' splice site, resulting in the inclusion of a terminal calcitonin exon. This stimulation of 3' proximal splicing was antagonized by cotransfection with an hnRNP A1 plasmid. This effect of hnRNP A1 in promoting distal splicing was also seen in an hnRNP A1-deficient MEL cell line. A similar effect of hnRNP A1 was demonstrated with mutant hamster adenine phosphoribosyltransferase (aprt) transcripts that are normally constitutively spliced, suggesting that hnRNP A1 may be a general inhibitor of proximal splicing. Intron size also influenced splice site choice in mutant aprt transcripts, with larger introns favoring proximal splicing. These results support the idea that the ratios of particular but general splicing factors and hnRNPs play a role in alternative splicing.

3' Untranslated Regions↗
PubMed1999

Kinetic evidence for an on-pathway intermediate in the folding of cytochrome c.

An early folding event of cytochrome c populates a helix-containing intermediate (INC) because of a pH-dependent misligation between the heme iron and nonnative ligands in the unfolded state (U). For folding to proceed, the nonnative ligation error must first be corrected. It is not known whether I is on-pathway, with folding to the native state (N) as in U <-->INC <--> N, or whether the I must first move back through the U and then fold to the N through some alternative path (INC <--> U <--> N). By means of a kinetic test, it is shown here that the cytochrome c I does not first unfold to U. The method used provides an experimental criterion for rejecting the off-pathway I <--> U <--> N option.

Animals↗
PubMed1999

Mechanism of SNAP potentiating antiproliferative effect of calcitonin gene-related peptide in cultured vascular smooth muscle cells.

We previously showed that CGRP inhibits cell proliferation which correlates with an elevation of cAMP levels in rabbit aortic vascular smooth muscle cells (VSMCs). The present study determined the effects of S-nitroso-N-acetylpenicillamine (SNAP, a nitric oxide donor) on CGRP-induced antiproliferative effects and cellular mechanism in cultured rabbit aortic VSMCs. The cells (in fifth-sixth passage) were exposed to 2.5% fetal bovine serum for 24 h in the presence or absence of SNAP, hCGRP or both.(3)H-thymidine incorporation was used to measure DNA synthesis. The results showed that SNAP (60-100 microm) significantly inhibited the proliferation and elevated cGMP levels in cultured rabbit aortic VSMCs. In combination, however, SNAP (30 microm) potentiated hCGRP (10-100 n m)-induced antiproliferation. SNAP (30 microm) and hCGRP (10-100 n m) or forskolin (10 microm), an activator of adenylate cyclase, caused more than additive cAMP elevations, but not cGMP elevations, in these cells. Quazinone, an inhibitor of cGMP-inhibited-phosphodiesterase (cGI-PDE, PDE3), or SNAP plus quazinone caused a similar potentiation as SNAP of the hCGRP-induced elevations of cAMP levels. The data indicate that SNAP-induced potentiation of CGRP's effects likely involves inhibition of cGI-PDE, thus allowing enhanced accumulation of cAMP that mediates the antiproliferative effects of hCGRP in cultured rabbit aortic VSMCs.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1999

Equilibrium amide hydrogen exchange and protein folding kinetics.

The classical Linderstrøm-Lang hydrogen exchange (HX) model is extended to describe the relationship between the HX behaviors (EX1 and EX2) and protein folding kinetics for the amide protons that can only exchange by global unfolding in a three-state system including native (N), intermediate (I), and unfolded (U) states. For these slowly exchanging amide protons, it is shown that the existence of an intermediate (I) has no effect on the HX behavior in an off-pathway three-state system (I<-->U<-->N). On the other hand, in an on-pathway three-state system (U<-->I<-->N), the existence of a stable folding intermediate has profound effect on the HX behavior. It is shown that fast refolding from the unfolded state to the stable intermediate state alone does not guarantee EX2 behavior. The rate of refolding from the intermediate state to the native state also plays a crucial role in determining whether EX1 or EX2 behavior should occur. This is mainly due to the fact that only amide protons in the native state are observed in the hydrogen exchange experiment. These new concepts suggest that caution needs to be taken if one tries to derive the kinetic events of protein folding from equilibrium hydrogen exchange experiments.

Amides↗
PubMed1999

A novel allele of RAD52 that causes severe DNA repair and recombination deficiencies only in the absence of RAD51 or RAD59.

With the use of an intrachromosomal inverted repeat as a recombination reporter, we have shown that mitotic recombination is dependent on the RAD52 gene, but reduced only fivefold by mutation of RAD51. RAD59, a component of the RAD51-independent pathway, was identified previously by screening for mutations that reduced inverted-repeat recombination in a rad51 strain. Here we describe a rad52 mutation, rad52R70K, that also reduced recombination synergistically in a rad51 background. The phenotype of the rad52R70K strain, which includes weak gamma-ray sensitivity, a fourfold reduction in the rate of inverted-repeat recombination, elevated allelic recombination, sporulation proficiency, and a reduction in the efficiency of mating-type switching and single-strand annealing, was similar to that observed for deletion of the RAD59 gene. However, rad52R70K rad59 double mutants showed synergistic defects in ionizing radiation resistance, sporulation, and mating-type switching. These results suggest that Rad52 and Rad59 have partially overlapping functions and that Rad59 can substitute for this function of Rad52 in a RAD51 rad52R70K strain.

Alleles↗
PubMed1999

The CCR4 and CAF1 proteins of the CCR4-NOT complex are physically and functionally separated from NOT2, NOT4, and NOT5.

The CCR4-NOT complex (1 mDa in size), consisting of the proteins CCR4, CAF1, and NOT1 to NOT5, regulates gene expression both positively and negatively and is distinct from other large transcriptional complexes in Saccharomyces cerevisiae such as SNF/SWI, TFIID, SAGA, and RNA polymerase II holoenzyme. The physical and genetic interactions between the components of the CCR4-NOT complex were investigated in order to gain insight into how this complex affects the expression of diverse genes and processes. The CAF1 protein was found to be absolutely required for CCR4 association with the NOT proteins, and CCR4 and CAF1, in turn, physically interacted with NOT1 through its central amino acid region from positions 667 to 1152. The NOT3, NOT4, and NOT5 proteins had no significant effect on the association of CCR4, CAF1, and NOT1 with each other. In contrast, the NOT2, NOT4, and NOT5 interacted with the C-terminal region (residues 1490 to 2108) of NOT1 in which NOT2 and NOT5 physically associated in the absence of CAF1, NOT3, and NOT4. These and other data indicate that the physical ordering of these proteins in the complex is CCR4-CAF1-NOT1-(NOT2, NOT5), with NOT4 and NOT3 more peripheral to NOT2 and NOT5. The physical separation of CCR4 and CAF1 from other components of the CCR4-NOT complex correlated with genetic analysis indicating partially separate functions for these two groups of proteins. ccr4 or caf1 deletion suppressed the increased 3-aminotriazole resistance phenotype conferred by not mutations, resulted in opposite effects on gene expression as compared to several not mutations, and resulted in a number of synthetic phenotypes in combination with not mutations. These results define the CCR4-NOT complex as consisting of at least two physically and functionally separated groups of proteins.

Binding Sites↗
PubMed1999

UVB and gamma-radiation induce the expression of mRNAs encoding the ribosomal subunit L13A in rat keratinocytes.

Ultraviolet B radiation produces an array of cellular perturbations in the skin. We isolated a keratinocyte cDNA encoding the rat 60S ribosomal subunit protein L13a following differential cDNA library screening with UVB-enriched probes. In contrast to the reported structure of liver L13a, the keratinocyte L13a cDNA contains a longer 3'-untranslated region. Northern blot analysis detected two L13a mRNA transcripts, approximately 800 bp and approximately 1.2 kb, in keratinocytes and a variety of rat tissues. Both L13a mRNA transcripts were induced by UVB irradiation, forskolin and gamma-irradiation. In contrast, no induction of L13a mRNA transcript levels was observed following exposure of keratinocytes to 12-O-tetradecanoylphorbol-13-acetate, serum and the DNA damage-inducing agents methyl methanesulfonate or 4-nitroquinoline-N-oxide. These observations suggest that increased expression of ribosomal subunit genes may be a molecular component of the keratinocyte response to UVB in particular and not part of a nonspecific response to DNA damage.

Animals↗
PubMed1999

[Studies on DNA-DNA hybridization and 16S rDNA sequence of rhizobia isolated from Shapotou desert soil in Ningxia autonomous region of China].

Based on the previous studies on numerical taxonomy and multilocus enzymes electrophoresis patterns of the 12 rhizobial strains isolated from Shapotou region, the contents of G + C mol%, DNA-DNA relatedness and 16SrDNA sequence of the representative strain were tested. The DNA G + C content of the members of this group ranged from 56.4 to 62.2. The values of DNA-DNA hybridization within the group were above 70%, and relatendness between representative strains of this group and known rhizobial species was below 66.6%. The full-length of 16S rDNA sequence of representative strain N220 was compared with the type strains of all known rhizobia species and related bacteria by the PHLIP version 3.572c composed a unrooted phylogenetic tree, the strain N220, R. galegea, two unnamed rhizobial strains(SH19312, SH22623) and three A-grobacterium strains constituted a branch in this tree. The similarity values of 16S rDNA sequence between strain N220 and other strains in this branch were above 95%.

Base Sequence↗
PubMed1999

[Study on the inclusion effect of menthol with beta-cyclodextrin].

Indusion effect of beta-CD with menthol was studied by three methods of preparation. Determination of physical constants and quantitative analysis proved that the inclusion compounds of beta-CD with menthol were formed, which was found to be 1:1 and 2:3. The results of thermal analysis demmonstrated that the gasifiation temperature of the inclusion compounds was by far higher than that of the free menthol. It was considered that the inclusion compound must be disconnected to be free, and then it can be determined by gas chromatography in the same condition of the free menthol.

Chromatography, Gas↗
PubMed1998

Cultured sympathetic neurons express functional interleukin-1 receptors.

Since recent work has identified an apoptotic pathway in sympathetic neurons that is mediated by autocrine interleukin-1 (IL-1), we investigated whether cultured sympathetic neurons possess functional IL-1 receptors. Cultured sympathetic neurons express levels of IL-1RI and IL-1RAcP mRNAs consistent with signal transduction. Neurons stimulated with IL-1 demonstrate enhanced p65 NF-kappaB nuclear translocation and enhanced NF-kappaB DNA binding activity, with at least p65 and p50 subunits participating in the DNA binding activity. RNA differential display identified several neuronal mRNAs regulated by IL-1, including a member of the reticulon family. We conclude that IL-1 stimulates a potential component of a neuronal secretory pathway.

Animals↗
PubMed1998

The mtDNA-encoded ND6 subunit of mitochondrial NADH dehydrogenase is essential for the assembly of the membrane arm and the respiratory function of the enzyme.

Seven of the approximately 40 subunits of the mammalian respiratory NADH dehydrogenase (Complex I) are encoded in mitochondrial DNA (mtDNA). Their function is almost completely unknown. In this work, a novel selection scheme has led to the isolation of a mouse A9 cell derivative defective in NADH dehydrogenase activity. This cell line carries a near-homoplasmic frameshift mutation in the mtDNA gene for the ND6 subunit resulting in an almost complete absence of this polypeptide, while lacking any mutation in the other mtDNA-encoded subunits of the enzyme complex. Both the functional defect and the mutation were transferred with the mutant mitochondria into mtDNA-less (rho0) mouse LL/2-m21 cells, pointing to the pure mitochondrial genetic origin of the defect. A detailed biosynthetic and functional analysis of the original mutant and of the rho0 cell transformants revealed that the mutation causes a loss of assembly of the mtDNA-encoded subunits of the enzyme and, correspondingly, a reduction in malate/glutamate-dependent respiration in digitonin-permeabilized cells by approximately 90% and a decrease in NADH:Q1 oxidoreductase activity in mitochondrial extracts by approximately 99%. Furthermore, the ND6(-) cells, in contrast to the parental cells, completely fail to grow in a medium containing galactose instead of glucose, indicating a serious impairment in oxidative phosphorylation function. These observations provide the first evidence of the essential role of the ND6 subunit in the respiratory function of Complex I and give some insights into the pathogenic mechanism of the known disease-causing ND6 gene mutations.

Animals↗
PubMed1998

Quantitative imaging of TATA-binding protein in living yeast cells.

We describe the quantitative monitoring of TATA-binding protein (TBP) localization and expression in living Saccharomyces cerevisiae cells. We replaced the endogenous TBP with a green fluorescent protein (GFP) x TBP fusion, which was imaged quantitatively by laser scanning confocal microscopy (LSCM). When GFP x TBP expression was altered by using various promoters, the levels measured by LSCM correlated well with the levels determined by immunoblot of whole cell extract protein. These results show that GFP x TBP imaging not only offers a method of measurement equivalent to a more conventional technique but also provides real-time quantitation in living cells and subcellular localization information. Time-lapse confocal imaging of GFP x TBP in mitotic yeast cells revealed that it remains localized to the nucleus and displays an asymmetric distribution (1:0.7) between mother and daughter cells. Based on this and data from a mutant which underexpresses GFP x TBP, we suggest that intracellular levels of TBP are near rate-limiting for growth and viability.

Cell Nucleus↗
PubMed1998

Dhh1p, a putative RNA helicase, associates with the general transcription factors Pop2p and Ccr4p from Saccharomyces cerevisiae.

The POP2 (Caf1) protein in Saccharomyces cerevisiae affects a variety of transcriptional processes and is a component of the Ccr4p complex. We have isolated five multicopy suppressor genes of a pop2 deletion mutation: CCR4, DHH1 (a putative RNA helicase), PKC1, STM1, and MPT5 (multicopy suppressor of pop two). Overexpression of either the CCR4 or DHH1 genes effectively suppressed phenotypes associated with pop2 mutant cells; overexpression of PKC1, STM1, or MPT5 genes produced only partial suppression. Disruption of the CCR4 or DHH1 genes resulted in phenotypes similar to those observed for pop2 cells. In addition, overexpression of the DHH1 gene also suppressed the ccr4 mutation, suggesting a close relationship between the POP2, CCR4, and DHH1 genes. Two-hybrid analysis and coimmunoprecipitation experiments revealed that Pop2p and Dhh1p interact physically, and these and other data suggest that Dhh1p is also a component of the Ccr4p complex. Finally, we investigated the genetic interaction between factors associated with POP2 and the PKC1 pathway. The temperature-sensitive growth defect of dhh1 or mpt5 cells was suppressed by overexpression of PKC1, and the defect of mpk1 cells was suppressed by overexpression of MPT5. These results and phenotypic analysis of double mutants from the POP2 and PKC1 pathways suggested that the POP2 and the PKC1 pathways are independent but have some overlapping functions.

Bacterial Proteins↗
PubMed1998

Molecular cloning, characterization, and potential roles of cytosolic and mitochondrial aldehyde dehydrogenases in ethanol metabolism in Saccharomyces cerevisiae.

The full-length DNAs for two Saccharomyces cerevisiae aldehyde dehydrogenase (ALDH) genes were cloned and expressed in Escherichia coli. A 2,744-bp DNA fragment contained an open reading frame encoding cytosolic ALDH1, with 500 amino acids, which was located on chromosome XVI. A 2,661-bp DNA fragment contained an open reading frame encoding mitochondrial ALDH5, with 519 amino acids, of which the N-terminal 23 amino acids were identified as the putative leader sequence. The ALDH5 gene was located on chromosome V. The commercial ALDH (designated ALDH2) was partially sequenced and appears to be a mitochondrial enzyme encoded by a gene located on chromosome XV. The recombinant ALDH1 enzyme was found to be essentially NADP dependent, while the ALDH5 enzyme could utilize either NADP or NAD as a cofactor. The activity of ALDH1 was stimulated two- to fourfold by divalent cations but was unaffected by K+ ions. In contrast, the activity of ALDH5 increased in the presence of K+ ions: 15-fold with NADP and 40-fold with NAD, respectively. Activity staining of isoelectric focusing gels showed that cytosolic ALDH1 contributed 30 to 70% of the overall activity, depending on the cofactor used, while mitochondrial ALDH2 contributed the rest. Neither ALDH5 nor the other ALDH-like proteins identified from the genomic sequence contributed to the in vitro oxidation of acetaldehyde. To evaluate the physiological roles of these three ALDH isoenzymes, the genes encoding cytosolic ALDH1 and mitochondrial ALDH2 and ALDH5 were disrupted in the genome of strain TWY397 separately or simultaneously. The growth of single-disruption delta ald1 and delta ald2 strains on ethanol was marginally slower than that of the parent strain. The delta ald1 delta ald2 double-disruption strain failed to grow on glucose alone, but growth was restored by the addition of acetate, indicating that both ALDHs might catalyze the oxidation of acetaldehyde produced during fermentation. The double-disruption strain grew very slowly on ethanol. The role of mitochondrial ALDH5 in acetaldehyde metabolism has not been defined but appears to be unimportant.

Acetaldehyde↗
PubMed1998

Isolation of Rhodospirillum centenum mutants defective in phototactic colony motility by transposon mutagenesis.

The purple photosynthetic bacterium Rhodospirillum centenum is capable of forming swarm colonies that rapidly migrate toward or away from light, depending on the wavelength of excitation. To identify components specific for photoperception, we conducted mini-Tn5-mediated mutagenesis and screened approximately 23,000 transposition events for mutants that failed to respond to either continuous illumination or to a step down in light intensity. A majority of the ca. 250 mutants identified lost the ability to form motile swarm cells on an agar surface. These cells appeared to contain defects in the synthesis or assembly of surface-induced lateral flagella. Another large fraction of mutants that were unresponsive to light were shown to be defective in the formation of a functional photosynthetic apparatus. Several photosensory mutants also were obtained with defects in the perception and transmission of light signals. Twelve mutants in this class were shown to contain disruptions in a chemotaxis operon, and five mutants contained disruptions of components unique to photoperception. It was shown that screening for photosensory defective R. centenum swarm colonies is an effective method for genetic dissection of the mechanism of light sensing in eubacteria.

Chemotaxis↗