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Biomedical subjects

F Y Wu

Publications and source records attributed to F Y Wu.

At least 55 records · Page 3Linked to original sources
PubMed1993

Comparison of antitumor activity of vitamins K1, K2 and K3 on human tumor cells by two (MTT and SRB) cell viability assays.

Vitamin K (VK) congeners (VK1, VK2, and VK3) have been used as antihemorrhagic agents, while VK3 has also been found to inhibit growth in various rodent and human tumor cells. We have compared the antitumor activities of vitamin K1, K2, and K3 against a panel of human cancer cell lines. For each test agent, a dose-response profile was generated by using an MTT (3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) and an SRB (sulforhodamine B) assay. Both assays yielded similar results. The respective ID50 values of VK3 in five hepatoma cell lines, HA59T, HA22T, PLC, HepG2, and Hep3B, of increasing differentiation state, were 42, 36, 28, 27, and 20 microM. For nasopharyngeal carcinoma (CG1), leukemia (U937), oral epidermoid carcinoma (KB), and breast carcinoma (BC-M1) cells, the ID50 values of VK3 were 26, 15, 25, and 33 microM, respectively. For all the above cells, the ID50 values of VK1 ranged from 6 to 9 mM, and the ID50 values of VK2 ranged from 1 to 2 mM. Thus, the relative potencies of antitumor activity of VK3 compared to VK2 and to VK1 are about 60- and 300-fold, respectively. These results support the preference for use of VK3 over VK1 and VK2 in cancer therapy.

Antineoplastic Agents↗
PubMed1993

Vitamin K3-induced cell cycle arrest and apoptotic cell death are accompanied by altered expression of c-fos and c-myc in nasopharyngeal carcinoma cells.

Vitamin K3 is known to inhibit the growth of various rodent and human tumor cells. However, the molecular mechanism of its action is still elusive. We have found that vitamin K3 induces cell cycle arrest and apoptotic cell death in nasopharyngeal carcinoma (NPC) cells, as evaluated by flow cytometry and DNA gel electrophoresis. Involvement of c-fos and c-myc proto-oncogenes and expression of their proto-oncoproteins in VK3-induced cell cycle arrest and apoptosis were also investigated. Northern blot analysis of NPC cells treated with 50 microM VK3 showed that c-fos was transiently induced for 60 min after treatment, while c-myc was persistently induced for 1-9 h after drug treatment. Western blot analysis also showed that c-Fos was induced at 4-6 h and at 1-4 h after treatment with 50 microM and 200 microM VK3 respectively, while c-Myc was induced at 1-6 h and at 4-6 h, respectively, after such treatments. These results suggest that the expression of c-fos and c-myc may play an important role in the signaling mechanism of VK3-induced growth arrest and apoptotic cell death.

Actins↗
PubMed1992

The structure of the zinc sites of Escherichia coli DNA-dependent RNA polymerase.

X-ray absorption spectroscopy is ideally suited for the investigation of the electronic structure and the local environment (approximately 5 A) of specific atoms in biomolecules. While the edge region provides information about the valence state of the absorbing atom, the chemical identity of neighboring atoms, and the coordination geometry, the extended x-ray absorption fine structure region contains information about the number and average distance of neighboring atoms and their relative disorder. The development of sensitive detection methods has allowed studies using near physiological concentrations (as low as approximately 100 microM). RNA polymerase from Escherichia coli contains two zinc atoms: one tightly bound in the beta' subunit, the subunit that participates in template binding, and the other loosely bound in the beta subunit, the subunit that participates in substrate binding. X-ray absorption studies of these zinc sites in the native protein and of the zinc site in the beta' subunit after removal of the zinc in the beta subunit site by p-(hydroxymercuri)benzenesulfonate (Giedroc, D. P., and Coleman, J. E. (1986) Biochemistry 25, 4969-4978) indicate that both zinc sites have octahedral coordination. The zinc in the beta' subunit site has four sulfur ligands at an average distance of 2.36 +/- 0.02 A and two oxygen (or nitrogen) ligands at an average distance of 2.23 +/- 0.02 A. The beta subunit zinc site has five sulfur ligands at an average distance of 2.38 +/- 0.01 A and one histidine nitrogen ligand at 2.14 +/- 0.02 A. These results are in general agreement with earlier biochemical and spectroscopic studies.

Absorptiometry, Photon↗
PubMed1992

Proximity between nucleotide/dinucleotide and metal ion binding sites in DNA-dependent RNA polymerase from Escherichia coli.

In order to understand translocation in transcription, it is important to develop a continuous functional assay for RNA polymerase (RNAP) activity in vitro. Fluorescent derivatives of ATP, UTP, UpA, and CpA with aminonaphthalene-5-sulfonic acid (AmNS) attached to the nucleotide triphosphates via a gamma-phosphoramidate bond or to the dinucleotide monophosphates via a 5'-secondary amine linkage were synthesized [Tyagi, S.C., & Wu, F.Y.-H. (1987) J. Biol. Chem. 262, 10684-10688]. The fluorescent emission spectra of (5'-AmNS)UpA, (5'-AmNS)CpA, (gamma-AmNS)ATP, and (gamma-AmNS)UTP overlap the absorption spectrum of co-substituted RNA polymerase (Co-RNAP) and ensure fluorescence resonance energy transfer (FRET) between the fluorescent analog and Co(II) in Co-RNAP. The binding constants at a single site for (gamma-AmNS)ATP, (gamma-AmNS)UTP, (5'-AmNS)UpA, and (5'-AmNS)CpA were observed to be 7.11, 5.26, 0.52, and 0.61 microM, respectively, in Co-RNAP and 5.70, 3.42, 0.12, and 0.21 microM, respectively, in Zn-RNAP. (8-AmTEMPO)ATP, with the spin probe AmTEMPO attached to the C-8 position at ATP [Tyagi, S.C. (1991) J. Biol. Chem. 266, 17936-17940], and Mn(3'-OCH3)UTP were synthesized. Mn-(II)-substituted RNA polymerase (Mn-RNAP) is prepared. The single site binding constants for (8-Am-TEMPO)ATP and Mn(3'-OCH3)UTP were 3.58 and 2.35 microM in Zn-RNAP and 5.77 and 3.43 microM in Mn-RNAP, respectively. These results indicate that dinucleotides bind much more tightly than mononucleotides to RNAP and that the binding constants are roughly the same for both Co- and Zn-substituted RNAP.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗
PubMed1992

Stable expression of functional human cytomegalovirus immediate-early proteins IE1 and IE2 in HeLa cells.

The major immediate-early (IE) genes 1 and 2 of human cytomegalovirus (HCMV) encode proteins that regulate the expression of HCMV genes as well as some other viral and cellular genes. In order to study the expression and function of these IE gene products, we established several HeLa cell lines that stably expressed the 68-kD IE1 protein, the 82-kD IE2 protein, or both proteins. The IE proteins expressed in these cell lines were biologically active, as shown by transient chloramphenicol acetyltransferase assays. Transcription from the major IE promoter was augmented in the IE1-expressing cells, while transcription from the HCMV early gene UL84 promoter was activated in the IE2-expressing cells. In addition, we found that the IE2-expressing cells established colonies in soft agarose more efficiently than the parental HeLa and the IE1-expressing cells. Furthermore, expression of both the IE1 and IE2 proteins was increased by treatment of these cell lines with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate. Thus, our cell lines provide a useful system to study the regulation of IE gene expression in human cells as well as to study transaction by HCMV IE proteins on various viral and cellular genes.

Cytomegalovirus↗
PubMed1992

Differential induction of c-fos and c-jun proto-oncogenes and AP-1 activity by tumor promoter 12-O-tetradecanoyl phorbol 13-acetate in cells at different stages of tumor promotion in vitro.

A two-stage (initiation and promotion) model of chemical transformation of mouse embryonic fibroblasts was used to elucidate the molecular mechanisms of tumor promotion in vitro. C3H10T1/2 cells which had been initiated with a subcarcinogenic dose (0.5 micrograms ml-1) of benzo[a]pyrene (B[a]P) were isolated after 12-O-tetradecanoyl phorbol 13-acetate (TPA) treatment lasting 12, 24 or 36 days. These series of partially promoted cells were designated T-12, T-24 and T-36 cells. T-12 and T-24 cells exhibited higher anchorage-independent growth on soft agar in the presence of TPA than did the initiated or T-36 cells. Cytosolic protein kinase C (PKC) in the resting state was slightly depleted in T-24 and T-36 cells. Proteolytic down-regulation of membrane-bound PKC was enhanced in T-12 cells compared with the initiated cells after 3 h of TPA treatment. Induction of c-fos and c-jun proto-oncogene expression increased two- to threefold in T-12 cells. Moreover, the basal level of c-fos mRNA progressively increased in T-12, T-24 and T-36 cells. As compared with other cell types, T-12 cells had the highest AP-1 DNA-binding activity at both the basal level and at 30 min after TPA treatment. These results indicate that deregulation of the TPA-induced cellular responses occurs in the cells at various stages of tumor promotion, and might be associated with transforming processes.

Animals↗
PubMed1991

Conversion of the E1A Cys4 zinc finger to a nonfunctional His2,Cys2 zinc finger by a single point mutation.

Trans-activation by the adenovirus E1A 289R protein requires a zinc finger defined by Cys-154, Cys-157, Cys-171, and Cys-174. Whereas individually replacing the four cysteine residues with serines resulted in a loss of transactivation, only three of the Cys----Ser mutants (C157S, C171S, and C174S) lost the ability to bind Zn(II). X-ray absorption fine structure analysis revealed that, in the wild-type protein, Zn(II) is coordinated by four cysteine residues whereas in the C154S mutant, Zn(II) is coordinated by two histidines and two cysteines. The mutant protein probably retains, as ligands, two cysteines on the right side of the zinc finger (Cys-171 and Cys-174) and recruits two of the four histidines on the left side (His-149, His-152, His-158, and His-160), despite the presence of Cys-157. This finding may shed light on the general structural requirements of zinc fingers.

Adenoviridae↗
PubMed1991

Mung bean nuclease cleavage pattern at a polypurine.polypyrimidine sequence upstream from the mouse metallothionein-I gene.

Mung bean nuclease, an enzyme specific for single-stranded DNA, was used to probe a non-B DNA structure present in the mouse metallothionein-I gene. The region sensitive to the enzyme was constituted by a 128 base-pair long polypurine.polypyrimidine sequence located at 1.2-kb from the start of transcription. A detailed analysis of the mung bean nuclease cleavage pattern revealed that: (i) under conditions of supercoiling and low pH a triplex structure was formed, (ii) the triplex was flanked by a sequence with the potential of forming a Z-DNA structure, (iii) most of the enzymatic activity was localized at some of the junctions between double-stranded and triple-stranded DNA and at mismatches in the triplex, (iv) no unpaired bases were observed in the loop or outside the triplex, and (v) the triplex was present in more than one configuration.

Animals↗
PubMed1991

1H NMR study of the interaction of ATP with Escherichia coli RNA polymerase containing in vivo-incorporated Co(II).

The DNA-dependent RNA polymerase containing two intrinsic cobalt ions (Co2-RPase) instead of the naturally occurring zinc was purified from Escherichia coli cells grown in zinc-depleted, cobalt-enriched media. Longitudinal NMR relaxation rates of the H2 and H8 protons of ATP were measured in the absence and presence of up to 92 microM Co2-RPase. No enhancement of the proton relaxation rates was observed in the presence of cobalt-containing enzyme, suggesting that the ATP substrate does not undergo rapid exchange at a site close to either of the intrinsic cobalt ions. This result is in contrast to that previously observed when Co2+ was incorporated into RPase by an in vitro procedure involving partial urea denaturation of the protein.

Adenosine Triphosphate↗
PubMed1991

[Quinolones in the treatment of acute amebic dysentery].

75 patients with acute ame bic dysentery treated with pipemidic acid (PPA) alone and 11 patients with norfloxacin (NFX) alone constituted two study groups and 30 patients treated with metronidazole (MNZ) formed a control group. It was found that the cure rates of PPA, NFX and MNZ were similar (P greater than 0.05), being 98.67%, 100% and 93.33%, respectively; the number of days to become afebrile and to stop abdominal pain in these three groups was also similar (P greater than 0.05); the number of days to stop diarrhea in PPA group was similar to that in NFX (P greater than 0.05), but it was significantly shorter in these two groups than that in the controls (P less than 0.01 and 0.05). As to adverse drug reactions, PPA had the least, NFX was similar to PPA (P greater than 0.05), but MNZ had significantly more than PPA (P less than 0.01).

Adolescent↗
PubMed1991

The in vitro and in vivo cytotoxicity of menadione (vitamin K3) against rat transplantable hepatoma induced by 3'-methyl-4-dimethyl-aminoazobenzene.

Menadione (vitamin K3, 2-methyl-1,4-naphthoquinone) is a synthetic derivative of napthoquinone. Its ability to inhibit cell growth in a wide variety of and human tumor cell types, and in rat hepatocytes has been recognized. Using a rat transplantable hepatoma model, we have evaluated the cytotoxic activity of menadione in hepatoma cells. Tumor cells in culture were sensitive to menadione treatment. The ID50 of drug is 3.4 microM as shown by a colorimetric MTT (3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Tumor-bearing rats were randomized into the treatment (n = 16) and control (n = 15) groups. Rats in the treatment group received intraperitoneal injection of menadione (10 mg/2 ml) once a week for four times; the control group received 2 ml water instead. None of the control rats survived after the 17th day following the start of treatment, while 5 out of the 16 treated rats responded well and survived long-term (greater than 60 days). Medadione was shown to inhibit actively the growth of hepatoma cells in vitro as well as in vivo.

Animals↗
PubMed1989

Zinc release from Xenopus transcription factor IIIA induced by chemical modifications.

Xenopus transcription factor IIIA (TFIIIA) contains two tightly bound intrinsic Zn2+ ions that are released through treatment with either p-(hydroxymercuri)benzenesulfonate (PMPS) or diethyl pyrocarbonate (DEP) as monitored by the metallochromic indicator 4-(2-pyridylazo)resorcinol (PAR). The inactivation of TFIIIA by DEP as detected by an in vitro 5S RNA gene transcription assay was correlated with the extent of modification of histidine residues and Zn2+ release. Following reaction with PMPS, the 7S particle was dissociated into free TFIIIA and 5S RNA. This dissociation could be correlated with the extent of modification of cysteine residues as well as the Zn2+ release. The dissociation of the 7S particle was reversed by the addition of excess thiol reagent. However, the reversibility could be inhibited by EDTA, suggesting that Zn2+ was required for the binding of TFIIIA to 5S RNA. In the presence of PMPS- or DEP-modified TFIIIA or Zn2+-depleted TFIIIA, the fluorescence emission maximum of the hydrophobic probe, 8-anilinonaphthalenesulfonate, was blue-shifted by 30 nm, while only less than a 10-nm blue shift was observed in the presence of either the 7S particle or TFIIIA. These results indicate that the two Zn2+ ions in TFIIIA are coordinated with the cysteine and histidine residues and are required for maintenance of the proper conformation of TFIIIA.

Animals↗
PubMed1988

The 289-amino acid E1A protein of adenovirus binds zinc in a region that is important for trans-activation.

The E1A gene of adenovirus type 5 encodes two major proteins of 289 and 243 amino acid residues, which are identical except that the larger protein has an internal stretch of 46 amino acids required for efficient trans-activation of early viral promoters. This domain contains a consensus zinc finger motif (Cys-Xaa2-Cys-Xaa13-Cys-Xaa2-Cys) in which the cysteine residues serve as postulated ligands. Atomic absorption spectrophotometry applied to bacterially expressed E1A proteins revealed that the 289-amino acid protein binds one zinc ion, whereas the 243-amino acid protein binds no zinc. Replacing individual cysteine residues of the finger with other amino acids destroyed the trans-activating ability of the 289-amino acid protein, even when structurally or functionally conserved amino acids were substituted. These results strongly suggest that the zinc finger of the 46-amino acid domain is intimately linked to the ability of the large E1A protein to stimulate transcription of E1A-inducible promoters. Furthermore, zinc binding to one of the mutant finger proteins suggests either that only a precise finger structure formed by the tetrahedral coordination of zinc to the four consensus ligands is required for trans-activation or, possibly, that one of several neighboring histidine residues in various combinations with three of the consensus cysteine residues normally coordinates zinc. How the zinc finger in E1A might interact with DNA or protein to bring about trans-activation is discussed.

Adenovirus Early Proteins↗
PubMed1988

[On the analysis of actual state of contamination with asbestos in the autopsy cases in Hokkaido].

The exposure of asbestos was surveyed in the 92 autopsy cases, including 60 cases of lung cancer with the digestion method of lung tissue. More or less asbestos body was found in 94.6% of cases examined. There was no correlation between numbers of asbestos bodies and histological types of lung cancer. In the group of pulmonary fibrosis, the pleural adhesion was more intense in the cases with increased number of asbestos bodies. More than 80 asbestos bodies per 1g of wet lung tissue were found in 4 cases, but there was no significant relation between occupation and the grade of asbestos contamination.

Aged↗
PubMed1987

Fluorescence resonance energy transfer studies on the proximity relationship between the intrinsic metal ion and substrate binding sites of Escherichia coli RNA polymerase.

DNA-dependent RNA polymerase from Escherichia coli contains 2 mol of zinc/mol of holoenzyme (alpha 2 beta beta' sigma) with one zinc each in the beta and beta' subunits. A new method to substitute selectively the zinc in the beta subunit was developed by the inactivation of RNA polymerase with 0.25 M NaNO3, 1 M NaCl, 1 mM diaminocyclohexane tetraacetic acid, and 0.1 mM dithiothreitol followed by reconstitution with Co(II), Cd(II), or Cu(II). The hybrid Co-Zn, Cd-Zn, or Cu-Zn RNA polymerase thus obtained retains, respectively, 91, 88, and 50% enzyme activity of the reconstituted Zn-Zn RNA polymerase. Co-Zn RNA polymerase exhibits absorption maxima at 395 and 465 nm, and Cu-Zn RNA polymerase at 637 nm (epsilon = 815 M-1 cm-1). 1-Aminonaphthalene-5-sulfonic acid (AmNS) derivatives of ATP, UTP, and dinucleoside monophosphates (diNMPs), UpA or ApU, were synthesized with AmNS attached to NTP via a gamma-phosphoamidate bond or to diNMPs via a 5'-secondary amine linkage. Since the fluorescence emission maxima of (5'-AmNS)UpA, (gamma-AmNS)ATP, and (gamma-AmNS)UTP at 445, 464, and 464 nm, respectively, when excited at 340 nm, overlap the 465-nm absorption band of Co-Zn RNA polymerase, the spatial relationship between fluorescence substrate analogs and the intrinsic Co(II) in Co-Zn RNA polymerase was studied by fluorescence resonance energy transfer technique. The fluorescence of the initiator, (5'-AmNS)UpA, and elongator, (gamma-AmNS)UTP, of the RNA chain, was quenched 20.3 and 7.1%, by the addition of saturation concentration of Zn-Zn RNA polymerase, and 21.3 and 14.7%, respectively, by the addition of template, poly(dA-dT). The fluorescence of (5'-AmNS)UpA and (gamma-AmNS)UTP was quenched 81.8 and 80.6%, respectively, by the addition of the saturation concentration of Co-Zn RNA polymerase in the absence of template, and 82.7 and 82.9% in the presence of template. On the basis of respective Ro values of 21.3 and 21.9 A for the (5'-AmNS)UpA-Co and (gamma-AmNS)UTP-Co pairs, the distances from Co(II) to the initiation site and to the elongation site were calculated to be 17.4 and 17.5 A, respectively, in the absence and 17.2 and 17.4 A in the presence of template.

Binding Sites↗
PubMed1987

Synthesis and characterization of fluorescent dinucleotide substrate for the DNA-dependent RNA polymerase from Escherichia coli.

New fluorescent derivatives of dinucleoside monophosphates, (5'-AmNS)UpA/ApU/GpU/CpA, with a fluorophore, 1-aminonaphthalene-5-sulfonic acid (AmNS), attached to the first nucleotide of the dinucleoside monophosphates via a 5'-secondary amine linkage were synthesized in good yield. The chemical structure of (5'-AmNS)ApU was proved by the phosphodiesterase digestion followed by Whatman No. 3MM paper chromatographic and spectroscopic analysis of the digested products. The ability of these analogs to be incorporated into the 5' terminus of RNA chain forming fluorescent oligonucleotides by Escherichia coli RNA polymerase was studied in the presence of a synthetic DNA template. The enzymatic reaction of (5'-AmNS)UpA and [3H]UTP in the presence of poly(dA-dT) yielded (5'-AmNS)UpAp[3H]U in greater than 30% yield with the Km values of 5 and 2.5 microM and Vmax values of 17 and 25 nmol/min/mg of enzyme for (5'-AmNS)UpA and UpA, respectively. The structure of this fluorescent trinucleotide was identified by RNase A digestion and paper chromatographic analysis of the digested products. (5'-AmNS)UpA or (5'-AmNS)ApU exhibits two absorption maxima around 270 and 340-350 nm and a fluorescent emission maximum at 445 nm when excited at 340 nm. These spectral characteristics permit their use as energy donors for the transfer of energy to the intrinsic cobalt of the cobalt-substituted RNA polymerases. Upon hydrolysis of the phosphodiester bond of these analogs by venom phosphodiesterase, the absorption at 340 and 270 nm increased by 5 and 20%, respectively, while their fluorescence at 445 nm was enhanced by 25%. Thus, these analogs can be used for studying the dynamics of initiation and elongation reactions catalyzed by DNA-dependent RNA polymerases by absorption and fluorescence spectroscopies.

DNA-Directed RNA Polymerases↗