Genetic polymorphism of conjugating enzymes and cancer risk: GSTM1, GSTT1, NAT1 and NAT2.
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Alleles↗
Biomedical subjects
Y Huang
Publications and source records attributed to Y Huang.
Enhanced growth inhibition and differentiation of fluorodeoxyuridine-treated human colon carcinoma cells by phenylbutyrate.
The effect of phenylbutyrate (PB), a nontoxic differentiation inducer, in human colon carcinoma cell lines treated with 5-fluorodeoxyuridine (FUdR) was evaluated. Two HT-29 human colon carcinoma subclones (U4 well differentiated and U9 poorly differentiated) were equally growth inhibited by 16 h of FUdR (0.2 microM) treatment but recovered cell growth in 3-6 days after the removal of FUdR. PB as a single agent had minimal effect on cell growth, but after FUdR treatment, PB inhibited cell growth for 12 days. The inhibition of cell growth in FUdR-treated cells by PB was more sustained in U4 than U9 cells and was associated with an increased and sustained expression of p21waf1 protein, secretion of transforming growth factor beta1, mediators of p53-dependent or -independent G1 cell cycle arrest, and an increase in the alkaline phosphatase activity as well, considered a marker of differentiation in colon carcinoma cells. These effects of PB were seen only in FUdR-pretreated cells because PB alone had minimal effect on the expression of these genes. The sequential use of FUdR followed by PB in patients with colon carcinoma should be explored because two subclones of HT29, irrespective of their state of differentiation, respond to this clinically achievable regimen.
Alkaline Phosphatase↗
Relation of optical coherence tomography to microanatomy in normal and rd chickens.
PURPOSE: To elucidate the relation between optical coherence tomography (OCT) scans and retinal histology in normal and retinal degeneration (rd) chickens. METHODS: Retinas from adult normal and rd chickens were examined in vivo with OCT at 850 nm and compared quantitatively with stained cryosections of unfixed retinas from the same locations. RESULTS: The nerve fiber layer (NFL) and inner plexiform layer (IPL) show homogeneous backscatter throughout their thicknesses. NFL reflectivity is approximately 0.6 log units higher than that of the IPL. The inner nuclear layer shows a low reflectivity; the properties of reflections from ganglion cell and outer nuclear layers are indeterminate. The outer retina and choroid form a large reflective complex. Photoreceptor inner segments produce the highest of these reflections in normal chicken retinas, approximately 1.5 log units higher than that of the IPL. The retinal pigment epithelium also has a relatively large backscatter coefficient and is the dominant reflector in rd retinas that lack photoreceptors. Choroidal pigment produces an intermediate level of backscatter and is the largest attenuator of signal at 850 nm. CONCLUSIONS: Quantified OCT signals have a predictable relationship to histology and pathology in chicken retinas. The results from rd retinas represent a first step toward in vivo quantitation of retinal structure in retinal degenerative disease.
Animals↗
Retinal degenerations with truncation mutations in the cone-rod homeobox (CRX) gene.
PURPOSE: To define the phenotypes of retinal degenerations associated with mutations in the gene encoding CRX (cone-rod homeobox), a photoreceptor-specific transcription factor. METHODS: Heterozygotes with the E168 [delta1 bp], E168 [delta2 bp], or G217 [delta1 bp] CRXgene mutation were studied clinically, with visual function tests, including rod and cone perimetry and electroretinography (ERG), and with optical coherence tomography (OCT). RESULTS: Clinical diagnoses included autosomal dominant cone-rod dystrophy in one family (E168 [delta1 bp] mutation) and simplex Leber congenital amaurosis in two families (E168 [delta2 bp], G217 [delta1 bp] mutations). In the family with the E168 [delta1 bp] mutation, two siblings had relatively mild disease expression in the third decade of life. The central retinas of these two patients had profound loss of rod and short wavelength cone function; long/middle wavelength cone thresholds were elevated at fixation, but there were greater paracentral than central abnormalities. Peripheral retinal dysfunction was evident by psychophysics and by maximum amplitude loss for rod- and cone-isolated ERG photoreceptor responses. OCT cross-sectional reflectance images showed decreased central retinal thickness consistent with photoreceptor loss. An additional member of this family (E168 [delta1 bp] mutation) and two other patients (representing E168 [delta2 bp] and G217 [delta1 bp] mutations) had a severe phenotype with retina-wide loss of function and islands of function remaining only in the temporal periphery. CONCLUSIONS: Truncation mutations in CRX are associated with retinopathies that share phenotypic features but vary in disease severity. The disease mechanism could involve abnormal photoreceptor development compounded by a disturbance in the maintenance of photoreceptors in the mature retina.
Adult↗
p27Kip1 overexpression causes apoptotic death of mammalian cells.
p27KiP1, a member of the Cip/Kip family of cyclin-dependent kinase (cdk) inhibitors, has been implicated in mediating G1 arrest in response to a variety of growth inhibitory signals. Its importance in regulating cell growth is emphasized by the fact that mice lacking p27Kip1 are abnormally large and display hyperplasia of multiple tissues. However, these mice retain the ability to undergo G1 arrest in response to growth inhibitory signals, suggesting that p27KiP1 may serve other functions important for controlling tissue growth. In the present study, we utilized an adenoviral vector-based expression system to examine the consequences of p27Kip1 overexpression in the human carcinoma cell lines A549, HeLa and RKO, in human melanoma SK-MEL-110 cells, in human lung fibroblasts IMR90 and in the rat fibroblast line Rat1. We demonstrate that overexpression of p27Kip1 leads to apoptotic cell death in all cell types, and further show that ectopic expression of Bcl-2 can protect HeLa cells from apoptosis mediated by p27Kip1 overexpression. To our knowledge, this is the first study demonstrating that p27Kip1 can induce apoptosis. Our findings provide new insight into the possible functions of this growth regulatory protein, and support the potential utility of gene therapeutic approaches aimed at elevating p27Kip1 expression for treatment of human cancers.
Apoptosis↗
The use of exfoliative cell samples to map clonal genetic alterations in the oral epithelium of high-risk patients.
Although it is widely accepted that clonal genetic alterations are an essential component of tumor progression, little is known of the distribution of such changes in high-risk lesions or how such clones are altered over time. We explored the feasibility of using exfoliative cells collected by scraping the mucosal surface to detect allelic loss in oral lesions of 22 patients (14 squamous cell carcinomas, 2 carcinomas in situ, and 6 dysplasias). The data show that the patterns of allelic loss observed in these samples closely represent those observed in biopsies of the same region. Furthermore, early indications are that this approach can be used to detect recurrent outgrowth of clones of altered cells in patients after therapy.
Adult↗
Impact of lower atmospheric carbon dioxide on tropical mountain ecosystems
Carbon-isotope values of bulk organic matter from high-altitude lakes on Mount Kenya and Mount Elgon, East Africa, were 10 to 14 per mil higher during glacial times than they are today. Compound-specific isotope analyses of leaf waxes and algal biomarkers show that organisms possessing CO2-concentrating mechanisms, including C4 grasses and freshwater algae, were primarily responsible for this large increase. Carbon limitation due to lower ambient CO2 partial pressures had a significant impact on the distribution of forest on the tropical mountains, in addition to climate. Hence, tree line elevation should not be used to infer palaeotemperatures.
Journal Article↗
Genetic interactions with Rap1 and Ras1 reveal a second function for the fat facets deubiquitinating enzyme in Drosophila eye development.
The Drosophila fat facets gene encodes a deubiquitinating enzyme that regulates a cell communication pathway essential very early in eye development, prior to facet assembly, to limit the number of photoreceptor cells in each facet of the compound eye to eight. The Fat facets protein facilitates the production of a signal in cells outside the developing facets that inhibits neural development of particular facet precursor cells. Novel gain-of-function mutations in the Drosophila Rap1 and Ras1 genes are described herein that interact genetically with fat facets mutations. Analysis of these genetic interactions reveals that Fat facets has an additional function later in eye development involving Rap1 and Ras1 proteins. Moreover, the results suggest that undifferentiated cells outside the facet continue to influence facet assembly later in eye development.
Amino Acid Sequence↗
Determinants of ribose specificity in RNA polymerization: effects of Mn2+ and deoxynucleoside monophosphate incorporation into transcripts.
The catalytic specificity of T7 RNA polymerase (RNAP) for ribonucleoside triphosphates vs deoxynucleoside triphosphates {(kcat/Km)rNTP/(kcat/Km)dNTP} during transcript elongation is approximately 80. Mutation of tyrosine 639 to phenylalanine reduces specificity by a factor of approximately 20 and largely eliminates the Km difference between rNTPs and dNTPs. The remaining specificity factor of approximately 4 is kcat-mediated and is nearly eliminated if Mn2+ is substituted for Mg2+ in the reaction. Mn2+ substitution does not significantly affect the Km difference between rNTPs and dNTPs. Mn2+ substitution also enhances the activity of poorly active mutant enzymes carrying nonconservative substitutions in the active site, and its effects are generally consistent with the Mn2+-catalyzed reaction being less restrictive in its requirements for alignment of the reactive groups. In addition to discrimination occurring at the level of nucleoside monophosphate (NMP) incorporation, it is also found that transcripts containing deoxynucleoside monophosphates (dNMPs) are more poorly extended than transcripts of canonical structure, though a severe barrier to transcript extension is seen only when the 3' region of the transcript is heavily substituted with dNMPs. The barrier to extension of transcripts heavily substituted with dNMPs is reduced for sequences known to be amenable to forming A-like helices and is larger for sequences that resist transformation from B-form DNA.DNA structures. The barrier to extension of dNMP-substituted transcripts is also reduced by solution conditions known to destabilize B-form DNA and to stabilize A-form structures. These observations imply a requirement for a non-B-form, possibly A-like, conformation in the transcript.template hybrid that is disrupted when the transcript is of predominantly deoxyribose structure.
Bacteriophage T7↗
Pulsed-dose-rate brachytherapy: design of convenient (daytime-only) schedules.
PURPOSE: To design pulsed-brachytherapy (PDR) protocols that are expected to be at least as clinically efficacious (in terms of both tumor control and late sequelae) as continuous low-dose-rate (CLDR) regimens, but that involve irradiation only during extended office hours. Both interstitial and intracavitary brachytherapy protocols are considered. METHODS AND MATERIALS: The linear quadratic formalism was used in which the late normal tissue damage and tumor control for one protocol relative to another are assumed to be determined primarily by the level of cellular survival. PDR schedules were designed in which pulses are delivered during "extended office hours" (8 A.M. to 8 P.M.) with no irradiation overnight. Generally, the proposed PDR regimes last the same number of treatment days as the corresponding CLDR regimen, but the PDR treatment lasts longer on the final day (i.e., until 8 P.M.). PDR doses were calculated such as to produce a tumor control which is equivalent to standard CLDR protocols, and the corresponding predicted late complication rate was compared with that for CLDR. Ranges of plausible values for the half-times of sublethal damage repair for tumors and for late-responding normal tissues were considered. RESULTS: As has been previously shown, the efficacy of PDR relative to CLDR depends considerably on the repair rates for sublethal damage repair. Clinical and experimental evidence suggests that average repair half-times for early effects (e.g., tumor control) are less than about a half hour, and for late sequelae are more than about an hour. If these estimates are correct, daytime PDR regimes can usually be designed which take the same number of days as the corresponding CLDR regimen, but have comparable or better therapeutic ratios than CLDR. CONCLUSION: Protocols for PDR can be designed to involve irradiation only during extended office hours, that are likely to result in clinical results comparable or better than CLDR, for any expected combination of the repair half-times of early- and late-responding tissues. The suggested protocols allow all of the advantages of a computerized remote-controlled afterloader while preserving the benefits of low dose rate. In addition, the protocols could allow the patient to go home overnight, or to stay overnight in an adjacent medical inn or hospital-associated hotel, rather than in a hospital bed-which could have major economic benefits. In such an economic situation, an extra treatment day for the daytime PDR could well be considered, which would virtually guarantee an improved clinical advantage relative to CLDR.
Brachytherapy↗
HIV type 1 subtypes, coreceptor usage, and CCR5 polymorphism.
Identification of the chemokine receptors CCR5 and CXCR4 as the major coreceptors for HIV-1 entry has greatly assisted our understanding of HIV-1 pathogenesis, transmission, and tropism. However, most of our current knowledge on coreceptor usage comes from studies using HIV-1 strains or env genes derived from the genetic subtype B predominant in North America and western Europe. In this report, the coreceptor usage of 20 primary viral isolates representative of genetic subtypes A, B, C, D, E, and group O was examined. Thirty-nine full-length CCR5 sequences from individuals of diverse geographic origins were also obtained to examine the possible effect of CCR5 polymorphism on HIV-1 subtype distribution. Our results indicate that (1) CCR5 and CXCR4 serve as the two major coreceptors for viruses belonging to HIV-1 subtypes A, B, C, D, E, and group O, whereas other chemokine receptors such as CCR2b and CCR3 play only a minor role in facilitating viral entry into stimulated PBMCs; (2) the coreceptor usage is determined by the viral phenotype rather than its genotype because all NSI strains, irrespective of their subtype classification, utilize CCR5, whereas all SI strains are able to use CXCR4; and (3) there is no geographic clustering of CCR5 polymorphism in different ethnic populations, suggesting that CCR5 diversity is not the underlying explanation for differences in the spread of different HIV-1 subtypes. Therefore, the uneven worldwide distribution of HIV-1 subtypes is more likely the result of stochastic dissemination.
Amino Acid Sequence↗
Neurofibrillary lesions in experimental aluminum-induced encephalopathy and Alzheimer's disease share immunoreactivity for amyloid precursor protein, A beta, alpha 1-antichymotrypsin and ubiquitin-protein conjugates.
Neurofibrillary tangles of Alzheimer's disease contain predominantly tau protein and to a lesser degree amyloid precursor protein (APP), A beta protein, alpha 1-antichymotrypsin (ACT) and ubiquitin. Previously we have demonstrated the presence of phosphorylated tau and neurofilament proteins in neurofibrillary degeneration (NFD) induced by aluminum (Al) maltolate in rabbits [Savory et al., Brain Res. 669 (1995) 325-329; Savory et al., Brain Res. 707 (1996) 272-281]. Using the same animal system we have now detected APP, A beta, ACT and ubiquitin-like immunoreactivities in NFD-bearing neurons, often colocalizing in the NFD. Diffuse cytoplasmic staining for APP, A beta and ubiquitin was also present in neurons without NFD from Al maltolate-treated rabbits. This study provides additional support for immunochemical similarities between Al-induced NFD in rabbits and the neurofibrillary tangles in human subjects with Alzheimer's disease.
Aluminum↗
Pro-apoptotic effect of the c-Abl tyrosine kinase in the cellular response to 1-beta-D-arabinofuranosylcytosine.
Treatment of cells with the antimetabolite 1-beta-D-arabinofuranosylcytosine (ara-C) and other genotoxic agents is associated with activation of the c-Abl protein tyrosine kinase. The functional role of c-Abl in the response to DNA damage, however, remains unclear. The present studies demonstrate that cells expressing a dominant negative, kinase-inactive c-Abl (K-R) are resistant to killing by ara-C. The expression of c-Abl (K-R) blocked ara-C-induced apoptosis by a mechanism that is at least in part independent of the p53 tumor suppressor. Cells null for c-Abl also exhibited resistance to induction of apoptosis. These findings provide support for a pro-apoptotic function of c-Abl in the response to certain genotoxic drugs.
Apoptosis↗
An artificial cytochrome P450 that hydroxylates unactivated carbons with regio- and stereoselectivity and useful catalytic turnovers.
A catalyst has been synthesized comprising a manganese porphyrin carrying four beta-cyclodextrin groups. It catalyzes the hydroxylation of substrates of appropriate size carrying tert-butylphenyl groups that can hydrophobically bind into the cyclodextrin cavities. In one example as many as 650 catalytic turnovers are seen before the catalyst is oxidatively destroyed, and with a rate comparable to that of typical cytochrome P450 enzymes. In another example, a steroid derivative is regio- and stereoselectively hydroxylated at a single unactivated carbon atom, but more slowly and with fewer turnovers. The carbon attacked is not the most chemically reactive, and the selectivity is determined by the geometry of the catalyst-substrate complex. Nonbinding substrates are not reactive under the conditions used, and substrates with more flexible binding geometries give more than a single product.
Catalysis↗
Inhibition of phosphatidylinositol 3-kinase by c-Abl in the genotoxic stress response.
Activation of phosphatidylinositol (PI) 3-kinase by growth factors results in phosphorylation of phosphatidylinositol lipids at the D3 position. Although PI 3-kinase is essential to cell survival, little is known about mechanisms that negatively regulate this activity. Here we show that the c-Abl tyrosine kinase interacts directly with the p85 subunit of PI 3-kinase. Activation of c-Abl by ionizing radiation exposure is associated with c-Abl-dependent phosphorylation of PI 3-kinase. We also show that phosphorylation of p85 by c-Abl inhibits PI 3-kinase activity in vitro and in irradiated cells. These findings indicate that c-Abl negatively regulates PI 3-kinase in the stress response to DNA damage.
Humans↗
Expression of CYP2E1 during embryogenesis and fetogenesis in human cephalic tissues: implications for the fetal alcohol syndrome.
Reverse transcription and the polymerase chain reaction (RT-PCR) with oligonucleotide primers designed to target cDNA nucleotides 1241-1357 corresponding to exons 8 (3' end) and 9 (5' end) in human genomic CYP2E1 detected consistently strong signals in 9 of 10 prenatal human brains. Cephalic tissues analyzed were between 54 and 78 days of gestation. RT-PCR signals for expression of CYP2E1 in corresponding human hepatic or adrenal tissues were weaker or, with only 2 exceptions, undetectable. Attempts to approximate levels of P4502E1 mRNA with Northern blots and RNase protection assays indicated that levels in human prenatal whole brain tissues tended to increase as a function of gestational age but, at the early stages investigated, were far lower than the constitutive levels in hepatic tissues of adult humans or male rats. Localized, P4502E1-dependent cephalic bioactivation of ethanol, with associated generation of several reactive chemical species, could contribute significantly to the etiology of neuroembryotoxic effects of prenatal ethanol exposure.
Adult↗
The mouse histone H2a gene contains a small element that facilitates cytoplasmic accumulation of intronless gene transcripts and of unspliced HIV-1-related mRNAs.
Histone mRNAs are naturally intronless and accumulate efficiently in the cytoplasm. To learn whether there are cis-acting sequences within histone genes that allow efficient cytoplasmic accumulation of RNAs, we made recombinant constructs in which sequences from the mouse H2a gene were cloned into a human beta-globin cDNA. By using transient transfection and RNase protection analysis, we demonstrate here that a 100-bp sequence within the H2a coding region permits efficient cytoplasmic accumulation of the globin cDNA transcripts. We also show that this sequence appears to suppress splicing and can functionally replace Rev and the Rev-responsive element in the cytoplasmic accumulation of unspliced HIV-1-related mRNAs. Like the Rev-responsive element, this sequence acts in an orientation-dependent manner. We thus propose that the sequence identified here may be a member of the cis-acting elements that facilitate the cytoplasmic accumulation of naturally intronless gene transcripts.
Animals↗
Thermodynamic stability of the asymmetric doubly-ligated hemoglobin tetramer (alpha+CNbeta+CN)(alphabeta): methodological and mechanistic issues.
Free energy contributions to cooperativity by the eight ligation intermediates of human hemoglobin (Hb) have been characterized extensively using six oxygenation analogs [cf. Huang et al. (1996) Biophys. J. 71, 2094-2105, Table 2]. These unprecedented data bses have strongly supported the molecular code mechanism of Hb cooperativity [Ackers et al. (1992) science 255, 54-83]. The present study addresses a recent argument against this work [Shibayama et al. (1997) Biochemistry 36, 4375-4381] based on "free energy" determinations for a doubly-ligated species of the CN-met analog. Shibayama et al. (1997) have claimed that, in the hybridization experiments that have been used to determine free energy of the asymmetric "species[21]" tetramer, a portion of the bound cyanide is allegedly released from CN-met Hb during the incubation with deoxy Hb that is used to achieve hybrid equilibrium. These authors have claimed that cyanide release has resulted in extensive electron exchange between heme sites of the hybridizing sample, leading to incorrect evaluation of the equilibrium species population by the cryogenic techniques that have been employed. In this report, we demonstrate that neither appreciable cyanide loss nor electron exchange occurs with the methods that have been used extensively by our two laboratories for these equilibrium determinations [Perrella et al. (1990) Biophys. Chem. 35, 97-103; Daugherty et al. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 1110-1114]. An alternative experiment, which Shibayama et al. (1997) have carried out to illustrate their claim, does not evaluate a thermodynamic equilibrium property of the species [21] hybrid. The relevance of their newly-estimated "free energy" is therefore unclear. Nevertheless, Shibayama et al. (1997) have claimed that their proposed "free energy" (which is approximately 1.3 kcal more positive than the free energy of -11.4 kcal found independently by our two laboratories) renders invalid the molecular code mechanism of hemoglobin cooperativity. This representation is utterly without foundation since a free energy even more positive than suggested by Shibayama et al. (1997) would be fully consistent with the molecular code mechanism.
Animals↗