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Y N Chang

Publications and source records attributed to Y N Chang.

31 records · Page 2Linked to original sources
PubMed1992

The basic RNA-binding domain of HIV-2 Tat contributes to preferential trans-activation of a TAR2-containing LTR.

Human immunodeficiency viruses HIV-1 and HIV-2 encode a Tat protein that trans-activates the respective viral genome through RNA targets (TAR1 and TAR2). Tat-1 and Tat-2 have considerable homology. However, an interesting biological observation has been that Tat-1 activates the HIV-1 and HIV-2 LTRs equally while Tat-2 activates the former, in comparison to the latter, poorly. Here, we present evidence that it is the TAR2 RNA target together with the basic RNA-binding protein domain of Tat-2 that dictate this non-reciprocity in trans-activation.

Amino Acid Sequence↗
PubMed1992

Prostaglandin E1 and dibutyryl cyclic AMP enhance platelet resistance to deformation.

The effect of prostaglandin E1 (PGE1) on platelets is mediated through the PGE1 receptor and the consequent maintenance of the platelet's discoid shape. The effects of PGE1 and dibutyryl cAMP (dbcAMP) on the deformability of human platelets were studied. Deformability tests based upon the micropipette aspiration on the platelets were performed by using pipettes with radii (Rp) of 0.26-0.36 microns. The time course of the extension length (Dp, in microns) of the platelets in response to aspiration with a negative pressure (delta P) of 5 cm H2 O (delta P x Rp = 0.15 dynes/cm) was analyzed. PGE1 treatment (0.1 microM) resulted in a decrease of platelet deformability as compared with results obtained for apparently non-activated, control platelets. The deformation index, i.e., Dp/Rp (PGE1-treated)/Dp/Rp (control), was significantly reduced to 0.90 +/- 0.04. DbcAMP treatment also significantly decreased the deformability of platelets and this decrease was dbcAMP dose dependent. In contrast, colchicine- or cytochalasin D-treated platelets increased deformability. PGE1-treated platelets had a higher [cAMP]i than controls. Platelets treated with PGE1 or dbcAMP showed a reduced [Ca2+]i increment induced by thrombin as compared to non-treated controls. These results indicate that PGE1 and dbcAMP treatment of platelets is accompanied by an enhancement of platelet resistance to deformation. The increased [cAMP]i and low [Ca2+]i after PGE1 treatment may limit the rearrangement of cytoskeleton and thus enhance platelet resistance to deformation.

Alprostadil↗
PubMed1991

Functional domains of Epstein-Barr virus nuclear antigen EBNA-1.

The Epstein-Barr virus (EBV)-encoded latency product EBNA-1 is functionally pleiotropic, being required for replication of the episomal form of the EBV genome and having a role in the regulation of latency transcription. EBNA-1 is a direct DNA-binding protein, and both replication and transactivation are dependent on the interaction of EBNA-1 with its cognate DNA recognition sequences. To better understand EBNA-1 function, we have further characterized the DNA-binding domain of EBNA-1 and have examined the contributions of other domains of the protein to EBNA-1 transactivation activity. A Bal31 deletional analysis of the carboxy-terminal region of EBNA-1 identified a core DNA-binding domain located between amino acids 493 and 584. Column chromatographic, sedimentation, and cross-linking studies indicated that EBNA-1 exists in solution as a dimer. Mobility retardation assays using in vitro-translated variants of EBNA-1 showed that the active DNA-binding form of EBNA-1 is also a dimer. In short-term cotransfections, a pFRTK-CAT target containing EBNA-1-binding sites from the EBV origin of plasmid replication, ori-P, was transactivated by a carboxy-terminal EBNA-1 construction (amino acids 450 to 641) that also carried a c-myc nuclear localization signal. These reconstruction experiments demonstrated that a transactivation domain exists within the carboxy-terminal region of EBNA-1, that transactivation is more efficient when a nuclear localization signal is present, and that the natural karyophilic signal lies outside of the carboxy-terminal 191 amino acids. To identify the EBNA-1 nuclear localization signal, small oligonucleotides representing EBNA-1 sequences that encode clusters of basic peptides were transferred into two different vectors expressing cytoplasmic proteins (pyruvate kinase and herpes simplex virus delta IE175 protein) and the cellular locations of the fusion constructions were determined by immunofluorescence staining of transfected cells. In this way we identified a functional nuclear localization signal, Leu-Lys-Arg-Pro-Arg-Ser-Pro-Ser-Ser, encompassing amino acids 379 to 386 of the EBNA-1 protein.

Amino Acid Sequence↗
PubMed1991

The functionally active IE2 immediate-early regulatory protein of human cytomegalovirus is an 80-kilodalton polypeptide that contains two distinct activator domains and a duplicated nuclear localization signal.

The IE2 region of the human cytomegalovirus (CMV) strain Towne major immediate-early (MIE) gene encodes a transcriptional transactivator that stimulates expression from a variety of heterologous target promoters but specifically down-regulates its own promoter. By immunofluorescence and Western immunoblot analysis with monospecific peptide antisera, we found that human CMV MIE exon 5 encodes four overlapping polypeptides, two present at immediate-early times (80 and 55 kDa) and two others detected only at late times after infection (55 and 40 kDa). However, only the 80-kDa version (579 amino acids), which is derived from the small upstream exons 2 and 3 fused to the intact exon 5 region, was functionally active in both transactivation and autoregulation as assessed by cotransfection experiments. These results confirm the corrected assignment of the coding capacity of the exon 5 region based on amino acid homology with the equivalent IE2 protein from simian CMV (Colburn). In transient DNA transfection assays, IE2 expression plasmids also produced a predominant full-length 80-kDa protein, which was localized in a distinctive reticular pattern in the nucleus. Two short basic nuclear localization signals in IE2 were identified by deletion analysis and by conversion of a test cytoplasmic herpes simplex virus protein into a form that localized in the nucleus after insertion of either of these two human CMV motifs. Functional assays with MIE region plasmids containing deletions or truncations in exon 5 revealed that both transactivation and autoregulation required several distinct domains within the COOH half of the IE2 protein, whereas a region between codons 99 and 194 could be discarded. Three segments at the NH2 end of the protein between codons 1 to 85, 86 to 98, and 195 to 290 were also essential for transactivation but played no role in autoregulation. Finally, in domain swap experiments, GAL4-fusion proteins containing either an NH2-terminal 51-amino-acid domain from exon 3 (codons 25 to 85) or the COOH-terminal 33-amino-acid domain from exon 5 (codons 544 to 579) identified two distinct activator domains from IE2, both of which have acidic characteristics.

Amino Acid Sequence↗
PubMed1990

The palindromic series I repeats in the simian cytomegalovirus major immediate-early promoter behave as both strong basal enhancers and cyclic AMP response elements.

A 600-base-pair (bp) enhancer region upstream from the major IE94 gene of simian cytomegalovirus (SCMV) produces very strong basal expression of associated gene products. This domain consists of multiple sets of interspersed repetitive elements, including 11 copies of a conserved 16-bp palindromic sequence with the consensus CCATTGACGTCAATGG. These series I repeats contain an 8-bp core TGACGTCA that resembles the cyclic AMP (cAMP) response element (CRE) of cellular genes. In transient chloramphenicol acetyltransferase assays in K562 human erythroleukemia cells, a set of deleted variants of the IE94 promoter all responded up to 15-fold to induction by cAMP. However, successive removal of most of the SCMV 16-bp motifs reduced basal expression over 20-fold. The cAMP stimulation was also manifested at the steady-state RNA level after SCMV infection of K562 cells and was detectable within 1.5 h after treatment of DNA-transfected cells. Addition of a single 30-bp oligonucleotide encompassing the 16-bp palindrome conveyed up to 10-fold cAMP responsiveness onto a heterologous weak promoter but had no effect on basal expression. In contrast, two or more adjacent copies produced 20- to 40-fold increases in basal expression and provided greater than 200-fold activation in the presence of cAMP. Similar effects were obtained when the oligonucleotides were placed in a downstream location relative to the reporter gene. Studies with mutant oligonucleotides revealed that both the core CRE and the flanking sequence portions of the 16-bp elements were essential for enhancer function. Both components were also important for maximum cAMP responsiveness. Band shift assays with fractionated nuclear extracts from Raji lymphocytes revealed multiple competable complexes with cellular DNA-binding factors that recognized the series I elements. Three distinct CREB-like factors were detected that required only the core 8-bp elements for binding. We conclude that the 16-bp series I repeats provide a major contribution to the constitutive enhancer properties of the IE94 promoter and also act as functional CREs. The cAMP response properties appear likely to play a key role in reactivation of the virus from a latent state in appropriately differentiating cell types.

Animals↗
PubMed1990

The Epstein-Barr virus Zta transactivator: a member of the bZIP family with unique DNA-binding specificity and a dimerization domain that lacks the characteristic heptad leucine zipper motif.

Introduction of the zta gene of Epstein-Barr virus into latently infected B cells leads to induction of the entire lytic cycle program of the virus. The Zta gene product is a sequence-specific DNA-binding protein of 35 kilodaltons that behaves as a specific transcriptional transactivator in transient cotransfection assays. All known Zta-responsive target promoters contain one or more members of a family of consensus-binding sites known as ZREs. On the basis of the presence of limited amino acid similarity within a basic carboxy-terminal domain, Zta has been proposed to be a highly divergent member of the c-Jun/c-Fos/GCN4 family of AP-1-binding proteins. We show here that in vitro-translated Zta and the Jun:Fos proteins have overlapping but distinct target DNA-binding specificies; both recognize canonical AP-1 sites, but only Zta recognizes ZRE sites and only Jun:Fos recognizes CRE sites. The relative binding affinity of Zta for oligonucleotides containing the 7-base-pair c-Fos AP-1 site TGAGTCA was twofold greater than that for the ZRE core motifs TGAGCAA, TG TGCAA, and TGAGTAA, but 10-fold greater than that for TGTGTCA, as measured by gel mobility retardation and competition DNA-binding assays. Cross-linking and cotranslational heterodimerization assays showed that like GCN4, Zta forms a stable homodimer in both its DNA-bound and unbound forms. Furthermore, we show that a potential coiled-coil helical domain adjacent to the basic domain of Zta can substitute for the leucine zipper of c-Fos to produce a DNA-binding protein that has a very stringent target DNA specificity and can only recognize symmetric 9-base-pair AP-1 sites (ATGAGTCAT). Therefore, despite the absence of the repeated heptad leucine zipper motifs, the Zta protein retains the characteristic features of a juxtaposed basic region and an exactly aligned coiled-coil alpha-helical dimerization domain of the bZIP class of transcriptional regulatory factors.

Amino Acid Sequence↗
PubMed1990

Effects of PGE1 on platelet deformability.

Prostaglandin E1 (PGE1) is frequently used as a stabilizing agent while isolating platelets. The platelet-stabilizing effect of PGE1 is mediate through the PGE1 receptor and the consequent increase of intracellular cAMP. The discoid shape of platelet is thus maintained. Recently, we have studied the effect of PGE1 on platelet deformability with micropipette aspiration technics, using pipettes with radii (Rp) of 0.26-0.36 micron. Platelets, obtained from platelet-rich plasma, and suspended in Hank's buffer containing BSA, glucose, adenosine and theophylline, were subjected for micropipette aspiration test within 2 hr after sampling. In general, PGE1 treated platelets (greater than 75%) remained as non-activated form as revealed under video microscopy and maintained their discoid shape for as long as 6 hr. The time course of the extension length (Dp, in micron) of the platelets in response to aspiration with a negative pressure of 5 cm H2O (tension = 0.15 dynes/cm) was analyzed by using a digital image analyzer system (IRIS 3120 with IP-512, with a resolution of 0.076 microns). A computer program based on the algorithm of real-time reading of image intensity was developed to facilitate real-time data acquisition. The data of Dp/Rp vs time (sec) were fitted by using a second-order equation. The deformability of PGE1 (0.1 microM) treated platelet was decreased significantly as compared with control. These results indicate that the platelet stabilization by PGE1 is accompanied by a decrease in platelet deformability.

Alprostadil↗
PubMed1989

Deviations from expected frequencies of CpG dinucleotides in herpesvirus DNAs may be diagnostic of differences in the states of their latent genomes.

The DNA sequences of genomes from G + C-rich and A + T-rich lymphotropic herpesviruses [i.e. gammaherpesviruses; Epstein-Barr virus and herpesvirus saimiri (HVS)] are deficient in CpG dinucleotides and contain an excess of TpG and CpA dinucleotides relative to frequencies predicted from their mononucleotide compositions. In contrast, for sequences from genomes of G + C-rich and A + T-rich neurotropic herpesviruses (i.e. alphaherpesviruses; herpes simplex virus and varicellazoster virus) and human cytomegalovirus (HCMV; a betaherpesvirus) the mean observed frequencies of these dinucleotides are close to those expected from their mononucleotide compositions. Comparisons between DNA sequences that encode proteins conserved in all these viruses also show that sequences of these lymphotropic viruses are CpG-deficient whereas the homologous genes from the neurotropic viruses and the HCMV are not. Analyses of local variations in dinucleotide frequencies reveal some occurrences of clustered CpG dinucleotides in generally deficient genomes (e.g. upstream of the thymidylate synthase gene of HVS) and locally CpG-deficient regions within some generally non-deficient genomes (e.g. the major immediate early genes of human, simian and murine CMVs). A relative deficiency in CpG and an excess of TpG and CpA dinucleotides is a diagnostic feature of higher eukaryotic DNA sequences that have been subjected to methylation of cytosine residues in CpG doublets with the resulting increase in mutations to give TpG (and thereby its complement, CpA). The available evidence implicates the latent genome as the site of methylation of these herpesviruses. We conclude that in the neurotropic herpesviruses the normal latent precursors to infectious progeny are not methylated whereas there is local methylation of the immediate early locus in the latent genomes of CMVs, and the latent genomes of these lymphotropic herpesviruses are extensively methylated.

Base Sequence↗
PubMed1989

Identification of immediate-early-type cis-response elements in the promoter for the ribonucleotide reductase large subunit from herpes simplex virus type 2.

Regulation of the expression of the herpes simplex virus (HSV) type 2 large subunit of ribonucleotide reductase (ICP10) gene was studied directly by immunofluorescence or by chloramphenicol acetyltransferase analysis with hybrid ICP10 promoter constructions. In Vero cells, cotransfection with DNA encoding HSV IE110 or Vmw65 proteins or HCMV IE2 enhanced expression at least 10-fold. In contrast, expression was minimally enhanced by DNA encoding IE175 at low doses and slightly reduced at high doses. IE110-mediated trans-activation was minimal in primary astrocytes and cells from line 293. However, Vmw65 enhanced expression 20-fold in all cell types. cis-Response elements in the ICP10 promoter include a TAATGARAT-like element and other sequences associated with regulation of IE gene expression and potential SP-1, consensus AP-1, and octamer transcription factor 1 binding elements. Factors that bind to the ICP10 promoter were identified in mock and HSV-infected cell extracts. DNA-protein complex formation, presumably involving Vmw65, was demonstrated by gel retardation analysis with mixtures of uninfected cell nuclear extracts and virion lysates. The octamer transcription factor 1 motif (ATGCAAAT) was necessary for optimal Vmw65 binding to the ICP10 promoter as evidenced by competition experiments with oligonucleotides overlapping the consensus IE110 promoter virion response element. The data suggest that ICP10 can be regulated as an immediate-early gene.

Animals↗
PubMed1986

Nucleotide sequence and transcription of a human tRNA gene cluster with four genes.

A bacteriophage lambda clone containing a 20-kb human DNA segment was isolated and found to harbor a cluster of four tRNA genes. An 8.2-kb HindIII subfragment encompassing the genes was cloned into pBR322 for restriction mapping and DNA sequence analysis. The genes were found to be arranged as two tandem pairs, separated by 3 kb. A proline tRNAAGG gene is separated from a leucine tRNAAAG gene by a 724-bp intergenic region in the first pair, and a second proline tRNAAGG gene is 316 bp from a threonine tRNAUGU gene in the second pair, with the leucine tRNA gene being of opposite polarity to the other three genes. A putative Alu-like element was found to occur within a 2.0-kb DNA fragment, at least 0.7 kb from the tRNA gene cluster. The coding sequences of the two proline tRNAAGG genes are identical. The coding regions of all four tRNA genes contain consensus internal split promoter sequences and do not have intervening sequences nor the CCA trinucleotide found in mature tRNAs. The 3'-flanking regions of these four tRNA genes have normal RNA polymerase III termination sites of at least four consecutive T nucleotides. No apparent homologies occur between the 5'-flanking regions of these genes. All four tRNA genes are accurately transcribed in an in vitro HeLa cell-free system, and the RNase T1 fingerprints of the mature-sized tRNA transcripts were found to be consistent with the DNA sequences of the genes.

Base Sequence↗
PubMed1986

The first powerful steps in strategic development.

In an ever-changing business climate, the need for strategically oriented management increases, for the organization that is insensitive and unresponsive to its environment will not survive. The authors of this "Action Plan" examine the challenges facing the health industry and outline a series of actions for initiating strategic-driven management.

Economic Competition↗
PubMed1984

Application of the compartmentalization/airlock concept to aircraft and tolerance of lung to rapid decompression.

The incidence of pulmonary hemorrhage of different laboratory animals undergoing rapid decompression was found to be markedly decreased by applying the compartmentalization/airlock concept to simulated pressurized aircraft. It was observed that, in protected rabbit, mouse, and rat lungs, 6 of 24 (25%), 7 of 24 (29%), and 6 of 24 (25%), respectively, exhibited a few petechial hemorrhages following rapid decompression. In unprotected animals, however, all 72 lungs showed slight-to-very-severe degrees of decompression-induced hemorrhages. The percent of mortality of the unprotected animals undergoing rapid decompression was 47%, while there were no deaths in protected animals. The incidence of such pulmonary hemorrhages and the mortality of experimental animals indicate that compartmentalization, combined with an adequate airlock, would be of great value in protection against accidental decompression of pressurized aircraft.

Aircraft↗