Biomedical subjects

Y Kang

Publications and source records attributed to Y Kang.

At least 109 records · Page 6Linked to original sources

PubMed1999

Arrhythmic firing in dopamine neurons of rat substantia nigra evoked by activation of subthalamic neurons.

Intracellular recordings were made from dopaminergic neurons of the rat substantia nigra compacta (SNc) in in vitro slice preparations to study the synaptic influence from the subthalamic nucleus (STh). After microstimulation of STh, monosynaptic excitatory postsynaptic potentials (EPSPs) were produced in dopaminergic neurons. STh-induced EPSPs were composed of 6-cyano-7-nitroquinoxalene-2,3-dione- and 2-amino-5-phosphonovaleric acid-sensitive components. Subthreshold EPSPs evoked by STh stimulation could differentially trigger pacemaker-like slow depolarization (PLSD) and low-threshold Ca2+ spike (LTS) depending on the level of baseline membrane potentials. When a subthreshold EPSP was evoked by STh stimulation during rhythmic firing, the STh-induced EPSP could shift or elevate PLSD to a more depolarized level, resulting in generation of a spike at an earlier arrhythmic timing to restart the rhythmic firing. The interspike interval after the arrhythmic spike remained almost unchanged. In contrast, when a suprathreshold EPSP for evoking spikes was produced by STh stimulation during rhythmic firing, the STh-induced spike was just interposed between two spontaneous spikes the interspike interval of which was almost the same as those seen during the preceding rhythmic firing. This ectopically induced spike did not disturb or reset rhythmic firing. It was concluded that SNc dopaminergic neurons receive monosynaptic glutamatergic inputs from STh, and subthreshold and suprathreshold EPSPs evoked by STh stimulations can induce two types of arrhythmic firing in SNc dopaminergic neurons, similar to arrhythmic occurrences of the QRS complex seen in the electrocardiogram of the atrial and ventricular arrhythmias, respectively. The former arrhythmic firing may play a crucial role in desynchronization of dopaminergic neurons.

Animals↗

PubMed1999

[Emergency stenting for iatrogenic occlusion of internal carotid artery during percutaneous transluminal angioplasty: a case report].

The authors report the case of a 68-year-old male who underwent emergency stenting for iatrogenic occlusion of the internal carotid artery while he was receiving percutaneous transluminal angioplasty. He presented with a 1-month history of transient ischemic attacks with right-sided hemiparesis. Carotid angiography revealed a 95% eccentric stenosis at the origin of the left internal carotid artery. As the stenotic lesion was higher than the level of the third cervical vertebral body, percutaneous transluminal angioplasty was performed instead of carotid endarterectomy. During the procedure, the left internal carotid artery was dissected, then obstructed completely with rapid deterioration of his neurological condition. The Palmaz-Schatz stent was successfully deployed over the site of dissection to restore normal patency through the dissected carotid artery. Following emergency stenting, his neurological signs quickly subsided. Since then, with oral administration of antiplatelet medication, he has suffered no recurrence of cerebral ischemic events. Echo-ultrasonography 4 months after stent implantation showed good patency of the stented segment. It has been reported that angioplasty is indicated for high-risk patients or surgically inaccessible lesions in stenotic disorders of the internal carotid artery. However, there are several complications reported, including intimal dissection and restenosis. The present case emphasizes the usefulness of emergency stenting when occlusion of the internal carotid artery is complicated during percutaneous transluminal angioplasty.

Aged↗

PubMed1999

[Application of RF-II pedicle screw system in lumbar spondylolisthesis].

Thirty-eight patients with isthmic(31 cases), degenerative(6 cases), and post-traumatic(1 case) spondylolisthesis were treated with RF-II pedicle screw system. Eighteen patients were classified as Degree I lesion, 14 as Degree II lesion, and 6 as Degree III lesion. Postoperatively, 34 cases(90%) obtained an anatomical improvement obviously. The percentage of the patients who got excellent and good recovery was above 90%. We conclude that RF-II pedicle screw fixation system has the strongest three-dimensional rigidity and, attains complete and satisfactory decompression. The entire intervertebral disk in the level of the lesion is suggested to be removed. The system is ingeniously and originally designed, and is easy to obtain a simple and an effective reduction of the spondylolisthesis. Additionally, the application of the system can obtain a good interbody fusion by three steps: broadening the interbody space; stuffing the space with broken bones; and compressing the involved vertebral body. The authors considered that the RF-II screw system is one of the most ideal internal fixations for the treatment of lumbar spondylolisthesis.

Adult↗

PubMed1999

[The use of ultrasound biomicroscope in the diagnosis of anterior segment intraocular foreign bodies].

PURPOSE: To evaluate the use of Ultrasound Biomicroscope(UBM) in the diagnosis of anterior segment intraocular foreign bodies(IOFBs). METHODS: Besides the rountine x-ray photography and B-scan ultrasound examination, 9 cases with difinite IOFBs injury history were examined by UBM. RESULTS: Nine cases of IOFBs were shown in UBM image, among them 4 cases were invisible in x-ray photography and B-scan ultrasound examination. The IOFBs located in anterior chamber(1 case), in posterior chamber (2 cases), in pars plana (5 cases) and in iris root(1 case). The size of IOFBs varied from 0.1 mm x 0.25 mm to 1 mm x 3 mm. The IOFBs were extracted by combined intraocular surgery or direct magnetize extraction. CONCLUSION: Although most IOFBs could be localized by X-ray and B-scan ultrasound, some located in the posterior chamber or ciliary body are hard to detect, especially when they are nonmetal and extremely small. The high resolution image of the anterior segment of the eye created by UBM can help to localize the IOFBs and show the relation between IOFBs and surrounding tissue, which is important in the diagnosis and the choice of surgery method.

Adult↗

PubMed1999

Corneal topographical changes following strabismus surgery.

PURPOSE: To study corneal topographical changes after strabismus surgery. METHODS: Computer-aided corneal topography was used in 43 strabismus patients (45 eyes) one or two days prior to and six or seven days after strabismus surgery. The spherical and cylindrical equivalents were calculated based on the simulated keratometry. RESULTS: After the surgery, only the changes at 3 mm in the inferior quadrant were statistically significant. The changes at 3 mm in the rest quadrants and the changes at 7 mm were not significant. Significant changes in spherical equivalent were found post-operatively. Neither the horizontal nor the vertical meridional equivalent showed significant changes after surgery. CONCLUSIONS: The results of corneal topographical changes following strabismus surgery in our preliminary study indicated the little effect of strabismus surgery on corneal curvature and corneal astigmatism.

Adolescent↗

PubMed1998

Two types of parasympathetic preganglionic neurones in the superior salivatory nucleus characterized electrophysiologically in slice preparations of neonatal rats.

1. The electrophysiological properties of parasympathetic preganglionic neurones in the superior salivatory nucleus were studied in thin- and thick-slice preparations of rats aged 1 and 2 weeks using the whole-cell patch-clamp technique. 2. The superior salivatory neurones were identified by a retrograde tracing method with dextran-tetramethylrhodamine-lysine. The injection of the tracer into the chorda-lingual nerve labelled the neurones innervating the submandibular ganglia and those innervating the intra-lingual ganglia, while the injection into the tip of the tongue labelled the latter group of neurones. 3. Firing characteristics were investigated mainly in the neurones of 6-8 days postnatal rats. In response to an injection of long depolarizing current pulses at hyperpolarized membrane potentials (< -80 mV) under a current clamp, the neurones labelled from the nerve displayed a train of action potentials with either a long silent period preceding the first spike (late spiking pattern) or a long silent period interposed between the first and second spikes (interrupted spiking pattern). The neurones labelled from the tongue invariably displayed the interrupted spiking pattern. 4. Under a voltage clamp, among the neurones from 6-8 days postnatal rats, those labelled from the nerve expressed either a fast or a slow transient outward current (A-current), while those labelled from the tongue invariably showed a slow transient outward current. Both the fast and slow A-currents were largely depressed by 1 mM 4-aminopyridine. 5. Similar fast and slow A-currents were observed in the neurones of rats aged 14-15 days. Both the time to peak and decay time constant of these A-currents were accelerated, suggesting a developmental trend of maturation in the activation and inactivation kinetics between 6 and 15 days postnatal. 6. Based on the differences in the firing pattern and outward current, the superior salivatory neurones can be separated into two distinct types. We discuss the functional aspects of these two types of neurones with reference to their target organs.

Algorithms↗

PubMed1998

Comparison of humoral immune responses elicited by DNA and protein vaccines based on merozoite surface protein-1 from Plasmodium yoelii, a rodent malaria parasite.

Immunization with DNA vaccines encoding relevant Ags can induce not only cell-mediated immune response but also humoral immune responses against pathogenic microorganisms in several animal models. Our previous results demonstrated that, when the C terminus (PyC2) of Plasmodium yoelii merozoite surface protein-1 (MSP-1), a leading vaccine candidate against erythrocytic stages of malaria, was expressed as a fusion protein (GST-PyC2) with glutathione S-transferase (GST), it elicited Ab-mediated protective immune responses in BALB/c mice. In our present study, we wished to examine the humoral responses to a DNA vaccine (V3) encoding GST-PyC2. The GST-PyC2 expressed in V3-transfected Cos 7 cells was recognized by a protective monoclonal Ab to PyC2 (mAb302), although the secreted product had undergone N-linked glycosylation. When BALB/c mice were immunized with V3 plasmid, anti-PyC2 Abs were successfully induced. These Abs immunoprecipitated native PyMSP-1 protein and competed with mAb302 for binding to its epitope at a level similar to those elicited by GST-PyC2 protein immunization. However, these Abs had significantly lower titers and avidities, and different isotype profiles and protective capacities against a lethal erythrocytic stage challenge, than those resulting from immunization with GST-PyC2 protein. Most surprising was the finding that, in contrast to protein immunization, there was no significant increase in the avidity of either GST-specific or PyC2-specific IgG Abs during the course of DNA immunization. This suggests that there may be little or no affinity maturation of specific Abs during DNA immunization in this system.

Animals↗

PubMed1998

Chromatographic removal of endotoxin from hemoglobin preparations. Effects of solution conditions on endotoxin removal efficiency and protein recovery.

In this work, affinity chromatography was used to remove endotoxin from human hemoglobin preparations with a Sterogene Acticlean Etox column. The effects of solution conditions on endotoxin removal efficiency and protein recovery have been investigated. It has been found that cations Na(+) or Ca(2+) reduced endotoxin removal efficiency from 73% (sample prepared with endotoxin-free water) to 31% (sample prepared with 0.15 M NaCl, ionic strength, I = 0.15 M), and from 73% sample prepared with endotoxin-free water) to 9% (sample prepared with 0.05 M CaCl2, ionic strength, I = 0.15 M). It has also been found that the protein recovery was increased from 90% to 99%, respectively.

Calcium Chloride↗

PubMed1998

Phosphorylation of mitogen-activated protein kinase by one-trial and multi-trial classical conditioning.

The pathway supporting the conditioned stimulus (CS) is one site of plasticity that has been studied extensively in conditioned Hermissenda. Several signal transduction pathways have been implicated in classical conditioning of this preparation, although the major emphasis has been on protein kinase C. Here we provide evidence for the activation and phosphorylation of a mitogen-activated protein kinase (MAPK) pathway by one-trial and multi-trial conditioning. A one-trial in vitro conditioning procedure consisting of light (CS) paired with the application of 5-HT results in the increased incorporation of 32PO4 into proteins detected with two-dimensional gel electrophoresis. Two of the phosphoproteins have molecular weights of 44 and 42 kDa, consistent with extracellular signal-regulated protein kinases (ERK1 and ERK2). Phosphorylation of the 44 and 42 kDa proteins by one-trial conditioning was inhibited by pretreatment with PD098059, A MEK1 (ERK-Activating kinase) inhibitor. Assays of ERK activity with brain myelin basic protein as a substrate revealed greater ERK activity for the group that received one-trial conditioning compared with an unpaired control group. Western blot analysis of phosphorylated ERK using antibodies recognizing the dually phosphorylated forms of ERK1 and ERK2 showed an increase in phosphorylation after one-trial conditioning compared with unpaired controls. The increased phosphorylation of ERK after one-trial conditioning was blocked by pretreatment with PD098059. Hermissenda that received 10 or 15 conditioning trials showed significant behavioral suppression compared with pseudo-random controls. After conditioning and behavioral testing, the conditioned animals showed significantly greater phosphorylation of ERK compared with the pseudo-random controls. These results show that the ERK-MAPK signaling pathway is activated in Pavlovian conditioning of Hermissenda.

Animals↗

PubMed1998

Mitogenic signaling from P1 and P2 purinergic receptors to mitogen-activated protein kinase in human fetal astrocyte cultures.

To investigate potential trophic actions of extracellular ATP in human astrocytes, we have examined mitogenic signaling by purinergic receptors in cultures prepared from first trimester rostral central nervous system tissue. We found that ATP and ATPgammaS, a hydrolysis-resistant analog, stimulated DNA synthesis, thereby indicating that P2 purinergic receptors can stimulate mitogenic signaling in these cells. In addition, ATP activated a mitogen-activated protein kinase (MAPK) termed ERK (extracellular signal-regulated protein kinase), a key component of signal transduction pathways involved in cellular proliferation and differentiation. The activation of MAPK was mediated at least in part by P2 purinergic receptors, because a P2 purinoceptor antagonist, suramin, inhibited the ATP-evoked stimulation by 50%, whereas a P1 purinergic-receptor antagonist, 8-(para-sulfonphenyl)-theophylline, was without effect. In contrast to rat astrocytes, adenosine/P1 purinergic-receptor agonists, 2-chloroadenosine and 5'-N-ethylcarboxyamidoadenosine, stimulated MAPK activity and DNA synthesis in human astrocytes. A selective inhibitor of protein kinase C, Ro 31-8220, blocked the ability of ATP and adenosine analogs to stimulate MAPK, thereby indicating that protein kinase C is upstream of MAPK in both P2- and P1-receptor signaling pathways. An inhibitor of the MAPK activator MEK, PD 098059, effectively blocked ATP- and 2-chloroadenosine-induced DNA synthesis, thereby indicating that the ERK/MAPK cascade mediates mitogenic signaling by P2 and P1 purinergic receptors in human fetal astrocytes. These findings suggest a role for P1 and P2 purinergic receptors in the proliferation of human fetal astrocytes.

Adenosine Triphosphate↗

PubMed1998

An ongoing immune response to HIV envelope gp120 in human CD4-transgenic mice contributes to T cell decline upon intravenous administration of gp120.

The mechanisms accounting for T cell depletion in AIDS patients are not yet fully understood, nor are the roles of host factors in HIV pathogenesis. We show here that an ongoing humoral immune response to HIV gp120 can sensitize non-infected cells towards apoptosis. Thus, i.v. injection of 1 microg recombinant(r) gp120 into gp120-immunized human CD4-transgenic mice (huCD4 Tg), which express huCD4 on both T and B cells, results in T and B cell depletion in peripheral blood and lymphoid tissues. On day 6 after a bolus injection of gp120, the numbers of peripheral T cells and B cells in gp120-immunized huCD4 Tg decreased sevenfold and two- to threefold, respectively. Annexin V staining revealed a higher percentage of early apoptotic cells on day 1 of gp120 i.v. injection from gp120-primed huCD4 Tg spleens compared to gp120-primed controls. Boosting the primed huCD4 Tg mice with soluble gp120 and hen egg-white lysozyme led to lower secondary titers to both antigens than found in controls. Furthermore, splenocytes from gp120-pretreated immunized huCD4 Tg had a lower level of stimulation in response to anti-CD3 treatment. These in vivo results are consistent with in vitro data demonstrating that cross-linking CD4 on splenocytes of huCD4 Tg by rgp120SF2 and anti-gp120 not only sensitizes T cells for apoptosis, but also induces apoptosis per se, and suggest that anti-gp120 responsiveness can contribute to T cell depletion in AIDS.

Animals↗

PubMed1998

Cultured human ankle and knee cartilage differ in susceptibility to damage mediated by fibronectin fragments.

According to numerous cadaveric, radiographic, and clinical studies, ankle and knee joints differ in susceptibility to osteoarthritis. To test for biochemical differences in susceptibility to damage, a chondrocytic chondrolysis system has been utilized. In this system, fibronectin fragments are added to cultured cartilage explants, resulting in enhanced release of catabolic cytokines, induction of matrix metalloproteinases, temporary suppression of proteoglycan synthesis, and consequently, severe loss of cartilage proteoglycan. We found that the addition of an amino-terminal thrombin-generated 29-kDa fibronectin fragment to cultured knee cartilage from 14 donors (average age: 53 years) usually caused a 30-50% decrease in proteoglycan content by day 7. However, of the ankle cartilage specimens examined from 21 donors (average age: 50 years), only three showed damage by day 7, one by day 14, and six by day 21, and 11 were not damaged until day 28. For eight of the donors (average age: 44 years), both knee and ankle cartilages were obtained: this allowed comparison between tissues from the same donor. The analysis showed that the ankle cartilage was much more refractory to damage than was the knee cartilage from the same donor. These data clearly show differences between ankle and knee cartilage in susceptibility to the fibronectin fragments and suggest the feasibility of use of these fragments for discerning differences in homeostasis of the ankle and knee cartilage.

Adolescent↗

PubMed1998

The importance of DT-diaphorase in mitomycin C resistance in human colon cancer cell lines.

BACKGROUND: Prior studies have suggested the multifactorial nature of mitomycin C (MMC) resistance. However, the relative importance of the different resistance mechanisms is unknown. MATERIALS AND METHODS: A panel of colon cancer cell lines with levels of MMC resistance from 2- to 15-fold compared to the parent line HT-29 was produced by repeated MMC exposure. Cell survival was measured using clonogenic assay. Glutathione and related enzymes and DT-diaphorase were measured using biochemical assays. P-glycoprotein expression was measured using flow cytometry. Topoisomerase II activity was measured using the pBR322 DNA relaxation assay. RESULTS: Multiple drug resistance mechanisms were altered in the resistant cell lines (glutathione reductase, glutathione peroxidase, topoisomerase II). However, the level of DT-diaphorase correlated best with the degree of MMC resistance. The importance of DT-diaphorase was confirmed by using BMY 25282, an MMC analogue which is less dependent on DT-diaphorase for activation. Resistance in the HT-29R54 cell line was 15-fold with MMC compared to 5-fold with BMY 25282. P-glycoprotein-mediated resistance does not appear important in this model. CONCLUSIONS: Although MMC resistance appears to be multifactorial, the results of this study strongly suggest that DT-diaphorase is the major contributor to MMC resistance under aerobic conditions. Strategies to enhance drug activation may therefore be useful for reversing MMC resistance.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1998

Molecular characterization of phenylketonuria in Japanese patients.

We characterized phenylalanine hydroxylase (PAH) genotypes of Japanese patients with phenylketonuria (PKU) and hyperphenylalaninemia (HPA). PKU and HPA mutations in 41 Japanese patients were identified by denaturing gradient gel electrophoresis and direct sequencing, followed by restriction fragment length polymorphism analysis to find a large deletion involving exons 5 and 6. Of 82 mutant alleles, 76 (92%) were genotyped showing 21 mutations. The major mutations were R413P (30.5%), R243Q (7.3%), R241 C (7.3%), IVS4nt-1 (7.3%), T2781 (7.3%), E6nt-96A-->g (6.1%), Y356X (4.9%), R111X (3.7%), and 442-706delE5/6 (2.4%). Eight new mutations (L52 S, delS70, S70P, Y77X, IVS3nt-1, A132 V, W187 C, and C265Y) and a polymorphism of IVS10nt-14 were detected. In vitro PAH activities of mutant PAH cDNA constructs were determined by a COS cell expression system. Six mutations, viz., R408Q, L52 S, R241 C, S70P, V388 M, and R243Q, had 55%, 27%, 25%, 20%, 16% and 10% of the in vitro PAH activity of normal constructs, respectively. The mean pretreatment phenylalanine concentration (0.83+/-0.21 mmol/l) of patients carrying the R408Q, R241 C, or L52 S mutation and a null mutation was significantly lower (P<0.0005) than that (1.99+/-0.65 mmol/l) of patients with both alleles carrying mutations associated with a severe genotype. Simple linear regression analysis showed a correlation between pretreatment phenylalanine concentrations and predicted PAH activity in 29 Japanese PKU patients (y=31.9-1.03x, r=0.59, P<0.0001). Genotype determination is useful in the prediction of biochemical and clinical phenotypes in PKU and can be of particular help in managing patients with this disorder.

Animals↗

PubMed1998

A phenytoin-sensitive cationic current participates in generating the afterdepolarization and burst afterdischarge in rat neocortical pyramidal cells.

We report here on the ionic mechanisms underlying the depolarizing afterpotential (DAP) in neocortical pyramidal cells, with special interest in those underlying the burst afterdischarge. Injections of short depolarizing current pulses under whole-cell current clamp with a CsCl-based internal medium generated, in most pyramidal cells, a single action potential with a plateau phase (plateau-AP), followed by a slowly decaying DAP both in the absence and presence of TTX. Under voltage-clamp, the same cells displayed a slow tail current (tail-I) at the offset of depolarization. When intracellular free Ca2+ was chelated with 10 mM BAPTA or when extracellular Ca2+ was replaced with equimolar Ba2+, neither the slow DAP nor the slow tail-I was observed. Extracellular application of Co2+ or Cd2+ reduced Ca2+ currents and the slow tail-I. Cation substitution experiments revealed that the channel generating the slow tail-I was permeable to K+ and Cs+ more than to Na+ (PK is approximately equal to PCs > PNa > PNMDG is approximately equal to PTEA). The cationic slow tail-I was not reduced by applying antagonists of the metabotropic glutamate receptor (MCPG, 1 mM) and the muscarinic receptor (atropine, 1-10 microM). Thus, the slow DAP was produced by activation of the cationic channel whose gating is solely dependent on [Ca2+]i. An increase in [K+]o from 3 to 6 or 9 mM enhanced the slow DAP, and resulted in a generation of burst afterdischarges. An anticonvulsant, phenytoin (PT; 1-10 microM) suppressed the slow DAP while enhancing the plateau-AP in the presence of TTX, most likely by blocking the cationic channel.

Animals↗

PubMed1998

An intronic enhancer is required for deflagellation-induced transcriptional regulation of a Chlamydomonas reinhardtii dynein gene.

Chlamydomonas reinhardtii flagellar regeneration is accompanied by rapid induction of genes encoding a large set of flagellar structural components and provides a model system to study coordinate gene regulation and organelle assembly. After deflagellation, the abundance of a 70-kDa flagellar dynein intermediate chain (IC70, encoded by ODA6) mRNA increases approximately fourfold within 40 min and returns to predeflagellation levels by approximately 90 min. We show by nuclear run-on that this increase results, in part, from increased rates of transcription. To localize cis induction elements, we created an IC70 minigene and measured accumulation, in C. reinhardtii, of transcripts from the endogenous gene and from introduced promoter deletion constructs. Clones containing 416 base pairs (bp) of 5'- and 2 kilobases (kb) of 3'-flanking region retained all sequences necessary for a normal pattern of mRNA abundance change after deflagellation. Extensive 5'- and 3'- flanking region deletions, which removed multiple copies of a proposed deflagellation-response element (the tub box), did not eliminate induction, and the IC70 5'-flanking region alone did not confer deflagellation responsiveness to a promoterless arylsulfatase (ARS) gene. Instead, an intron in the IC70 gene 5'-untranslated region was found to contain the deflagellation response element. These results suggest that the tub box does not play an essential role in deflagellation-induced transcriptional regulation of this dynein gene.

Animals↗

PubMed1998

Characterization of genes involved in biosynthesis of a novel antibiotic from Burkholderia cepacia BC11 and their role in biological control of Rhizoctonia solani.

Genetic manipulation of fluorescent pseudomonads has provided major insight into their production of antifungal molecules and their role in biological control of plant disease. Burkholderia cepacia also produces antifungal activities, but its biological control activity is much less well characterized, in part due to difficulties in applying genetic tools. Here we report genetic and biochemical characterization of a soil isolate of B. cepacia relating to its production of an unusual antibiotic that is very active against a variety of soil fungi. Purification and preliminary structural analyses suggest that this antibiotic (called AFC-BC11) is a novel lipopeptide associated largely with the cell membrane. Analysis of conditions for optimal production of AFC-BC11 indicated stringent environmental regulation of its synthesis. Furthermore, we show that production of AFC-BC11 is largely responsible for the ability of B. cepacia BC11 to effectively control the damping-off of cotton caused by the fungal pathogen Rhizoctonia solani in a gnotobiotic system. Using Tn5 mutagenesis, we identified, cloned, and characterized a region of the genome of strain BC11 that is required for production of this antifungal metabolite. DNA sequence analysis suggested that this region encodes proteins directly involved in the production of a nonribosomally synthesized lipopeptide.

Amino Acid Sequence↗

PubMed1998

Identification and functional characterization of a high-affinity Bel-1 DNA binding site located in the human foamy virus internal promoter.

The transcription of genes carried by primate foamy viruses is dependent on two distinct promoter elements. These are the long terminal repeat (LTR) promoter, which regulates expression of the viral structural proteins, and a second internal promoter, located towards the 3' end of the env gene, that directs expression of the viral auxiliary proteins. One of these auxiliary proteins is a potent transcriptional transactivator, termed Bel-1 in human foamy virus (HFV) and Tas or Taf in the related simian foamy viruses, that is critical for foamy virus replication. Previously, it has been demonstrated that the LTR promoter element of HFV contains a DNA binding site for Bel-1 that is critical for transcriptional activation (F. He, W. S. Blair, J. Fukushima, and B. R. Cullen, J. Virol. 70:3902-3908, 1996). Here, we extended this earlier work by using methylation interference analysis to identify and characterize the Bel-1 DNA binding sites located in the HFV LTR and internal promoter elements. Based on these data, we propose a minimal, 25-bp DNA binding site for Bel-1, derived from the HFV internal promoter element, and show that this short DNA sequence mediates efficient Bel-1 binding both in vitro and in vivo. We further demonstrate that, as determined by both in vitro and in vivo assays, the Bel-1 target site located within the HFV internal promoter binds Bel-1 with a significantly higher affinity than the cap-proximal Bel-1 target site located in the LTR promoter. This result may provide a mechanistic explanation for the observation that the internal promoter is activated significantly earlier than the LTR promoter during the foamy virus life cycle.

Base Sequence↗