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Biomedical subjects

S L Tan

Publications and source records attributed to S L Tan.

At least 91 records · Page 5Linked to original sources
PubMed1998

Control of PKR protein kinase by hepatitis C virus nonstructural 5A protein: molecular mechanisms of kinase regulation.

The PKR protein kinase is a critical component of the cellular antiviral and antiproliferative responses induced by interferons. Recent evidence indicates that the nonstructural 5A (NS5A) protein of hepatitis C virus (HCV) can repress PKR function in vivo, possibly allowing HCV to escape the antiviral effects of interferon. NS5A presents a unique tool by which to study the molecular mechanisms of PKR regulation in that mutations within a region of NS5A, termed the interferon sensitivity-determining region (ISDR), are associated with sensitivity of HCV to the antiviral effects of interferon. In this study, we investigated the mechanisms of NS5A-mediated PKR regulation and the effect of ISDR mutations on this regulatory process. We observed that the NS5A ISDR, though necessary, was not sufficient for PKR interactions; we found that an additional 26 amino acids (aa) carboxyl to the ISDR were required for NS5A-PKR complex formation. Conversely, we localized NS5A binding to within PKR aa 244 to 296, recently recognized as a PKR dimerization domain. Consistent with this observation, we found that NS5A from interferon-resistant HCV genotype 1b disrupted kinase dimerization in vivo. NS5A-mediated disruption of PKR dimerization resulted in repression of PKR function and inhibition of PKR-mediated eIF-2alpha phosphorylation. Introduction of multiple ISDR mutations abrogated the ability of NS5A to bind to PKR in mammalian cells and to inhibit PKR in a yeast functional assay. These results indicate that mutations within the PKR-binding region of NS5A, including those within the ISDR, can disrupt the NS5A-PKR interaction, possibly rendering HCV sensitive to the antiviral effects of interferon. We propose a model of PKR regulation by NS5A which may have implications for therapeutic strategies against HCV.

Animals↗
PubMed1998

Cummulative pregnancy and live birth rates after assisted conception.

Within the last 15 years in vitro fertilization (IVF) has become an established treatment modality. Its results compare favorably with that of spontaneous conception in the natural menstrual cycle in fertile women. The possibility of a success for any couple undergoing treatment will, however, depend on their characteristics. Over the past several years, the importance of these individual factors has been subjected to an extensive research. Our group has performed a number of studies that relate the cumulative conception rate (CCR) and cumulative live birth rate (CLR) with the age of the patient, the cause of infertility, and the type of ovarian stimulation used. We have also analyzed the effect of previously successful in vitro fertilization treatment upon the results of a subsequent treatment course, and the obstetric outcome of in vitro fertilization pregnancies when compared to those that are naturally conceived. In this review article, the importance of the above mentioned factors will be discussed in view of our research results and other recently available evidence.

Birth Rate↗
PubMed1997

Evidence that hepatitis C virus resistance to interferon is mediated through repression of the PKR protein kinase by the nonstructural 5A protein.

Hepatitis C virus (HCV) is the major cause of non-A non-B hepatitis and a leading cause of liver dysfunction worldwide. While the current therapy for chronic HCV infection is parenteral administration of type 1 interferon (IFN), only a fraction of HCV-infected individuals completely respond to treatment. Previous studies have correlated the IFN sensitivity of strain HCV-1b with mutations within a discrete region of the viral nonstructural 5A protein (NS5A), termed the interferon sensitivity determining region (ISDR), suggesting that NS5A may contribute to the IFN-resistant phenotype of HCV. To determine the importance of HCV NS5A and the NS5A ISDR in mediating HCV IFN resistance, we tested whether the NS5A protein could regulate the IFN-induced protein kinase, PKR, a mediator of IFN-induced antiviral resistance and a target of viral and cellular inhibitors. Using multiple approaches, including biochemical, transfection, and yeast genetics analyses, we can now report that NS5A represses PKR through a direct interaction with the protein kinase catalytic domain and that both PKR repression and interaction requires the ISDR. Thus, inactivation of PKR may be one mechanism by which HCV avoids the antiviral effects of IFN. Finally the inhibition of the PKR protein kinase, by NS5A is the first described function for this HCV protein.

Antiviral Agents↗
PubMed1997

Prospective randomized trial of the effect of two flushing media on oocyte collection and fertilization rates after in vitro fertilization.

OBJECTIVE: To assess the value of heparinized saline as a flushing medium for oocyte recovery. DESIGN: Prospective randomized study. SETTING: Academic tertiary referral center for fertility treatment. PATIENT(S): Thirty-five patients, with both ovaries intact having IVF-ET. INTERVENTION(S): Patients were randomized either to have the follicles of the left or right ovary flushed with heparinized normal saline at the time of oocyte recovery for IVF-ET. The contralateral ovary was flushed with heparinized culture medium. Oocytes obtained from each side were cultured separately and assessed for fertilization 18-21 hours after insemination. MAIN OUTCOME MEASURE(S): Collection and fertilization rates. RESULT(S): A total of 481 follicles were aspirated yielding 366 oocytes. Of these, 240 fertilized. From the side flushed with saline 185 oocytes were collected from 237 follicles, which was not significantly different from 181 oocytes collected from 244 follicles on the side flushed with culture medium (odds ratio = 1.23; 95% confidence interval = 0.79-1.92). Similarly, there was no significant difference observed in fertilization rates between oocytes obtained after saline (median 71.4%) and culture medium flush (median 75.0%) (odds ratio = 1.08; 95% confidence interval = 0.68-1.72). CONCLUSION(S): Heparinized normal saline is an equally good but cheaper and more convenient medium than standard heparinized culture medium and could replace it for flushing follicles during oocyte recovery for IVF-ET procedures.

Adult↗
PubMed1997

Inhibitory effect of human hydrosalpingeal fluid on mouse preimplantation embryonic development is significantly reduced by the addition of lactate.

Implantation and pregnancy rates following in-vitro fertilization-embryo transfer are reduced in the presence of hydrosalpinges, but the basis of the inhibition is unknown. We examined the effect of hydrosalpingeal fluid on preimplantation development of mouse embryos. Embryos cultured in 100% hydrosalpingeal fluid were significantly inhibited developmentally as compared to embryos cultured in 100% defined medium, which served as controls. In contrast, embryos cultured in 50% hydrosalpingeal fluid/50% defined medium reached the blastocyst stage at the same frequency as the controls. When lactate (final concentration 10 mM) was added to 100% hydrosalpingeal fluid, the percentage of cultured embryos that reached the blastocyst stage was significantly increased as compared to 100% hydrosalpingeal fluid, although the percentage was slightly lower than that observed for embryos cultured in the 100% defined medium. A similar but less pronounced effect occurred when pyruvate was added to hydrosalpingeal fluid. These results do not support the concept that a potent embryotoxic agent is commonly present in hydrosalpingeal fluid. Rather, they are consistent with the notion that the inhibitory effect of hydrosalpingeal fluid on embryonic development is due to the absence of essential factors, and that this deficiency can be largely corrected by the addition of energy sources.

Animals↗
PubMed1997

The NUH Memory Clinic. National University Hospital, Singapore.

OBJECTIVE: To assess the cognitive performance of elderly patients referred to the memory clinic. DESIGN: The patients were interviewed using the computerised mental state programme, GMS-AGECAT, and assessed on the Cognitive Assessment Scale (CAS) which includes the Elderly Cognitive Assessment Questionnaire (ECAQ) and the Cambridge Memory Test (CMT), modified for Chinese elderly. SUBJECTS: There were 72 Chinese elderly subjects aged 65 years and above, referred to the NUH Memory Clinic in the first year. RESULTS: Only 45 (62.5%) of the 72 subjects were diagnosed to have dementia using DSM III R criteria. There were 25 cases of Alzheimer's Disease and 20 multi-infarct dementia. In the non-demented group, 15 (20.8%) had depression or anxiety disorders and 12 (16.7%), had no mental disorders but had physical illness which could affect memory. There was a highly significant difference in the ECAQ, CMT and CAS scores of demented and non-demented elderly. There was also a significant difference in the cognitive performance of elderly with mild dementia and those with no mental disorder. CONCLUSION: The memory clinic is a useful facility for the diagnosis and management of dementia. The Cognitive Assessment Scale provides a valid and reliable battery of tests for dementia.

Aged↗
PubMed1996

Elective cryopreservation of all embryos in women at risk of developing ovarian hyperstimulation syndrome may not prevent the condition but reduces the live birth rate.

OBJECTIVE: Our goal was to evaluate the use of elective cryopreservation of all embryos to prevent the development of ovarian hyperstimulation syndrome in patients at risk while undergoing in vitro fertilization treatment. DESIGN: We analyzed 117 treatment cycles in which the serum E2 concentration on the day of hCG administration was > 10,000 pM and in whom > or = 15 oocytes were retrieved at ultrasound-directed follicle aspiration. The incidence of ovarian hyperstimulation syndrome, pregnancy, and live birth in 65 patients who had elective cryopreservation of all embryos and 52 patients who had fresh embryo transfer were compared. Independent t test and chi-square test (with Yates' correction) was used as appropriate. RESULTS: The clinical pregnancy (35 vs 17%; P < 0.03) and the live birth (27 vs 12%; P < 0.05) rates in patients receiving fresh embryo transfer was significantly higher than in those who had elective cryopreservation of all embryos. The incidence of moderate and severe ovarian hyperstimulation syndrome was similar in both groups (3.8 and 6.2%). CONCLUSIONS: Elective cryopreservation of all embryos does not reliably protect against the development of ovarian hyperstimulation syndrome but may reduce the clinical pregnancy and live birth rate.

Adult↗
PubMed1996

Outcome of frozen embryo replacement cycles following elective cryopreservation of all embryos in women at risk of developing ovarian hyperstimulation syndrome.

AIM: Our aim was to compare the outcome in subsequent frozen embryo replacement cycles in four groups of patients who had elective cryopreservation of all their embryos because they were considered to be at increased risk of developing severe ovarian hyperstimulation syndrome. DESIGN: Sixty-two (91%) of 68 IVF cycles (68 patients) in which elective cryopreservation of all embryos was performed were analyzed. All patients continued on the GnRH agonist, buserelin, after oocyte recovery until the onset of vaginal bleeding. Frozen embryo replacement occurred in a hormone replacement cycle that started either on day 3 of the withdrawal bleed (group I; N = 15) or after serum estradiol levels had fallen to < 100 pmol/L (group II; N = 16). The other patients commenced a frozen embryo replacement cycle several months later in either a hormone replacement (group III; N = 15) or a natural (group IV; N = 16) cycle. RESULTS: Two patients developed severe ovarian hyperstimulation syndrome. There were no significant differences among the four groups regarding demographic variables, the dose of hMG used, and the clinical outcome. There was a higher but not significantly different clinical pregnancy rate in group I (26.7%), compared to group II (12.5%), group III (13.3%), and group IV (18.8%). CONCLUSIONS: Several options exist for the timing and protocol used for frozen embryo replacement in patients who had elective cryopreservation for the prevention of ovarian hyperstimulation syndrome, none of which was found to be clearly superior in this observational report.

Adult↗
PubMed1996

Success of intrauterine insemination using cryopreserved donor sperm is related to the age of the woman and the number of preovulatory follicles.

OBJECTIVE: Our objective was to assess parameters associated with a successful outcome of intrauterine insemination (IUI) using cryopreserved donor sperm. DESIGN: We analyzed 750 consecutive donor IUI cycles undertaken by 363 women in an assisted conception clinic. The main outcome measure was clinical pregnancy. RESULTS: IUI was performed in 94.7% of the 750 IUI treatment cycles commenced and 180 clinical pregnancies occurred. The clinical pregnancy rate per cycle was 26.4%. The rate was significantly related to the patient's age (30.5% for age < or = 35 years and 18.1% for age > 35 years; P < 0.006) and whether there was one or more than one preovulatory follicles [20.9, 34.4, and 31.5% for one, two, and three or four follicles with a mean diameter of 14 or more mm at the time of human chorionic gonadotropin (hCG) administration; P = 0.006]. Two to four preovulatory follicles were present in 12.6% of the natural cycles, 43.6% of clomiphene citrate or tamoxifen, and 59.9% of gonadotropin stimulated cycles. The difference in the number of preovulatory follicles between stimulated and unstimulated cycles was highly significant (P < 0.0001). Pregnancy rates were 29.9% in gonadotropin-stimulated cycles, 23.6% in clomiphene citrate- or tamoxifen-stimulated cycles, 23.6% in clomiphene and 20.1% in unstimulated cycles. The difference in pregnancy rates between gonadotropin-stimulated and natural cycles was significant (P = 0.038). Cycle fecundity rates were not significantly affected by the number of previous treatment cycles, duration of infertility, gravidity and parity of the patient, presence of a spontaneous luteinizing hormone (LH) surge before the administration of hCG, or number of motile sperm in the insemination specimen. CONCLUSIONS: Success of IUI using cryopreserved donor sperm is related to the age of the women and whether there is one or more than one preovulatory follicles.

Cryopreservation↗
PubMed1996

Induction of ovulation in clomiphene-resistant polycystic ovary syndrome with pulsatile GnRH.

OBJECTIVE: To determine the efficacy of pulsatile GnRH alone and in combination with clomiphene citrate or gonadotropins in a stepwise approach for inducing ovulation in women with clomiphene-resistant polycystic ovary syndrome (PCOS). METHODS: Eighty women with clomiphene-resistant anovulatory infertility and PCOS were given subcutaneous pulsatile GnRH (15 micrograms every 90 minutes) using a portable infusion pump. If no follicular development was seen, clomiphene citrate (100 mg/day for 5 days) was given concurrently with the hormone in the next cycle of treatment. Those who still failed to ovulate regularly were treated with combined pulsatile GnRH with intramuscular gonadotropins (one ampule per day for 5-7 days). RESULTS. Sixty-six of 131 (50%) pulsatile GnRH cycles, 94 of 142 (66%) pulsatile GnRH with clomiphene cycles, and 48 of 69 (70%) pulsatile GnRH with gonadotropin cycles were ovulatory. Monofollicular response (one follicle at least 14 mm on the day of ovulation) occurred in 80.6, 83.9, and 53.6% of cycles, and multifollicular response occurred in 4.8, 3.1, and 21.6% of cycles in the three groups, respectively. Mild ovarian hyperstimulation occurred in one of the 342 cycles. The cumulative conception rate was 30% after three cycles, 60% after six cycles, and 73% after nine cycles. The miscarriage rate was 22% (ten of 45 pregnancies), and 35 women (78%) had live births (33 singletons and two sets of twins). CONCLUSION: The use of subcutaneous pulsatile GnRH alone and in combination with clomiphene citrate or gonadotropins for induction of ovulation in clomiphene-resistant PCOS in a stepwise approach produces a high cumulative conception rate associated with a low rate of multiple pregnancy and ovarian hyperstimulation syndrome.

Adult↗
PubMed1996

Relationship of ovarian stromal blood flow at the baseline ultrasound scan to subsequent follicular response in an in vitro fertilization program.

OBJECTIVE: To assess whether Doppler indices of intraovarian blood flow are related to the subsequent follicular response in an in vitro fertilization (IVF) program. METHODS: One hundred five women underwent ultrasonographic assessment of ovarian morphology, transvaginal color Doppler measurement of intraovarian blood flow and immunoassay of serum FSH during the early follicular phase of an IVF cycle. The subsequent follicular response was related to the Doppler data. RESULTS: Mean ovarian stromal peak systolic blood flow velocity was significantly correlated with the follicular response (P = .001), even after adjusting for the age of patient, type of ovary (polycystic or normal), total number of human menopausal gonadotropin ampules used, and serum FSH. Mean ovarian stromal pulsatility index was not related to the follicular response. Mean ovarian stromal peak systolic blood flow velocity was significantly lower (P = .007) in the poor-response group. The adjusted odds of a poor response increased significantly by an estimated 22% per cm/second decrease in velocity (P = .02). CONCLUSION: Ovarian stromal blood flow at the baseline ultrasound scan is correlated with subsequent follicular response and may be a new indicator for predicting ovarian responsiveness in an IVF program.

Adult↗
PubMed1996

Assessment of uterine artery blood flow on the day of human chorionic gonadotropin administration by transvaginal color Doppler ultrasound in an in vitro fertilization program.

OBJECTIVE: To assess whether measurement of uterine artery blood flow impedance (the pulsatility index) as determined by transvaginal color Doppler ultrasound on the day of hCG administration in patients undergoing IVF can predict pregnancy and implantation rates. DESIGN: Prospective observational study of women undergoing IVF. SETTING: A tertiary referral center for assisted reproduction. PATIENTS: One hundred thirty-five patients undergoing 139 IVF cycles. INTERVENTION: Transvaginal color Doppler assessment of uterine artery pulsatility index on the day of administration of hCG. MAIN OUTCOME MEASURES: Mean pulsatility index of the left and right uterine arteries, pregnancy rate, and embryo implantation rate. RESULTS: The patients were grouped into pregnant and nonpregnant groups and according to whether the pulsatility index was low (1.00 to 1.99), medium (2.00 to 2.99), or high (> or = 3.00). The pregnancy rates were 13.8%, 34.7%, and 14.3% for the low, medium, and high pulsatility index groups, respectively, and were not significantly different. The implantation rates for the same groups were 10.7%, 16.3%, and 5.4%, respectively. The implantation rate for all the patients with pulsatility index < 3.00 (and especially 2.00 to 2.99) was significantly higher than the high pulsatility index group. CONCLUSIONS: The study suggests that the measurement of uterine artery pulsatility index on the day of hCG predicts subsequent implantation rates. It may allow the administration of hCG to be deferred until uterine artery pulsatility index falls to < 3.00, which may result in improved implantation rates.

Adult↗
PubMed1996

Comparison of intravenous albumin and transfer of fresh embryos with cryopreservation of all embryos for subsequent transfer in prevention of ovarian hyperstimulation syndrome.

OBJECTIVE: To compare the efficacy of administration i.v. albumin to prevent severe ovarian hyperstimulation syndrome (OHSS) in patients undergoing ovarian stimulation for IVF with a standard policy of cryopreserving all embryos and to assess the impact of the two methods of treatment on pregnancy rates (PRs). DESIGN: Prospective randomized study. SETTING: A tertiary referral center for assisted conception. PATIENTS: Twenty-six patients undergoing IVF treatment cycles who were considered to be at high risk of developing severe OHSS on the basis of their serum E2 concentrations on the day of hCG administration and the number of oocytes collected. INTERVENTION: In group 1 (n = 13) all the generated embryos were cryopreserved to be transferred subsequently in hormonally manipulated cycles. In group 2 (n = 13) patients received IV infusions of albumin on the day of oocyte retrieval and 5 days later. Patients in group 2 had transfers of fresh embryos. MAIN OUTCOME MEASURES: The total dosage of hMG used, total number of follicles developed, number of follicles > 14 mm in diameter, serum E2 concentrations and endometrial thickness on day of hCG administration, number of oocytes retrieved, number and quality of embryos generated, PRs per cycle commenced, and onset and degree of any OHSS developed. RESULTS: There were no significant differences in the above parameters between the two groups, except for PRs that were significantly higher in patients who had all embryos cryopreserved (38.6% versus 0%). CONCLUSION: The policy of cryopreserving all generated embryos appears as effective as the administration of i.v. albumin in preventing OHSS in high-risk patients and produces significantly higher PRs.

Adult↗
PubMed1996

Transvaginal three-dimensional ultrasound: reproducibility of ovarian and endometrial volume measurements.

OBJECTIVE: To assess the reproducibility of ovarian and endometrial volume measurements obtained using transvaginal three-dimensional (3D) ultrasound (US). DESIGN: Prospective clinical study. SETTING: A tertiary referral center for assisted reproduction. PATIENTS: Forty women undergoing ovarian stimulation for IVF-ET using the long protocol of GnRH agonist. INTERVENTION: Three observers independently measured 20 stored ovarian scanned volumes and 20 endometrial volumes. Also, ovarian volume was calculated from three diameters obtained by two-dimensional (2D) US. MAIN OUTCOME MEASURE: Analysis of variance, the paired Student's t-test, and calculation of intraclass and interclass correlation coefficients were used for statistical analysis. RESULTS: Three-dimensional ovarian volume measurements were not significantly different from ovarian volume calculated from three diameters (7.98 versus 7.58 mL). The mean endometrial volume measurement was 3.56 mL. The intraobserver coefficient of variation for both ovarian and endometrial volume was 8%. The interobserver coefficient of variation was 9% for ovarian volume and 11% for endometrial volume. Intraclass and interclass correlation coefficients were 0.95 and 0.95 for ovarian volume and 0.90 and 0.82, respectively, for endometrial volume. CONCLUSION: Transvaginal 3D US produces highly reproducible ovarian and endometrial volume measurements.

Embryo Transfer↗
PubMed1996

Blood flow changes in the intraovarian arteries during the periovulatory period: relationship to the time of day.

Intraovarian blood flow was assessed by transvaginal color and pulsed Doppler ultrasound prospectively in six women during the periovulatory period. All patients had regular ovulatory menstrual cycles and a mid-luteal serum progesterone level consistent with spontaneous ovulation in the preceding cycle. Each patient underwent serial transvaginal ultrasound examination and Doppler assessment of blood flow in the ovarian stroma and in the wall of the dominant follicle or corpus luteum. When the mean follicular diameter was > 16 mm or day -2 from the estimated day of ovulation was reached, patients were scanned at 6-h intervals at 06.00, 12.00, 18.00 and 24.00 until follicular rupture, to investigate the presence of circadian rhythms. The pulsatility index (PI) and the maximum peak systolic blood flow velocity (Vmax) were calculated as Doppler indices of impedance to blood flow and velocity, respectively. A venous blood sample was taken at each visit for subsequent hormonal analysis. There was no significant circadian fluctuation in either mean follicular Vmax or mean follicular PI before or after the luteinizing hormone (LH) surge. Similarly, mean Vmax and mean PI in the non-dominant ovarian stroma did not show any significant fluctuation over the same periods. The mean PI in the dominant ovarian stroma showed daily fluctuations with the highest values occurring most commonly at 06.00 and the nadir in the afternoon and late evening. This fluctuation was the reverse of the circadian variation previously demonstrated in the uterine artery. However, in contrast to the uterine artery, comparison between the mean PI values in the dominant ovarian stroma at 06.00 and 18.00 only approached significance after the LH surge. The dominant ovarian stroma Vmax tended to rise during the day, although the differences between 06.00 and 18.00 were also not significant. Although there are fluctuations in Doppler blood flow indices in the dominant ovarian stroma, further studies are necessary to demonstrate whether this represents a significant circadian rhythm.

Adult↗
PubMed1996

Blood flow changes in the ovarian and uterine arteries during the normal menstrual cycle.

OBJECTIVE: Our purpose was to study the hemodynamic changes in the uterine and intraovarian vessels during the normal menstrual cycle and to relate the vascular changes to hormonal index values. STUDY DESIGN: Seven women who had bilateral tubal patency, a normal pelvis on laparoscopy, and regular ovulatory cycles underwent serial transvaginal ultrasonography on menstrual cycle day 2, daily from estimated day of ovulation-6, hourly from estimated day of ovulation-2, or when the mean follicular diameter was > 16 mm (whichever was earlier) until 6 hours after follicular rupture and once 7 days after follicular rupture. At each scan uterine and intraovarian blood flow was assessed with transvaginal color Doppler ultrasonography and serum concentrations of follicle-stimulating hormone, luteinizing hormone, estradiol, and progesterone assessed by fluoroimmunoassay. RESULTS: In one case there was evidence of a luteinized unruptured follicle and the patient was therefore excluded from analysis. In the other six women there was spontaneous ovulation at a mean of 39 hours after the onset of the luteinizing hormone surge. On the side with the dominant follicle, follicular and ovarian stromal peak systolic blood flow velocity rose significantly during the menstrual cycle with no significant change in pulsatility index. The changes in blood flow velocity correlated significantly with changes in serum follicle-stimulating hormone, luteinizing hormone and progesterone concentrations. There were no significant changes in either blood flow velocity or pulsatility index in the contralateral ovary. Uterine artery time-averaged maximum velocity on the side of the developing ovarian follicle increased during the menstrual cycle with no significant change in the contralateral vessel. Uterine artery pulsatility index on the side of the developing follicle declined during the midluteal phase and was significantly lower than on the contralateral side. The changes in time-averaged maximum velocity correlated with the changes in serum estradiol and progesterone concentrations. CONCLUSION: The vascular changes in the wall of the dominant ovarian follicle and ovarian stroma during the menstrual cycle are consistent with activity of angiogenic-like factors. The decline in uterine artery resistance during the midluteal phase may reflect optimal vascularity for implantation of the blastocyst.

Adult↗
PubMed1996

Interaction of the interferon-induced PKR protein kinase with inhibitory proteins P58IPK and vaccinia virus K3L is mediated by unique domains: implications for kinase regulation.

Expression of the double-stranded RNA-activated protein kinase (PKR) is induced by interferons, with PKR activity playing a pivotal role in establishing the interferon-induced antiviral and antiproliferative states. PKR is directly regulated by physical association with the specific inhibitor, P58IPK, a cellular protein of the tetratricopeptide repeat (TPR) family, and K3L, the product of the corresponding vaccinia virus gene. P58IPK and K3L repress PKR activation and activity. To investigate the mechanism of P58IPK- and K3L-mediated PKR inhibition, we have used a combination of in vitro and in vivo binding assays to identify the interactive regions of these proteins. The P58IPK-interacting site of PKR was mapped to a 52-amino-acid aa segment (aa 244 to 296) spanning the ATP-binding region of the protein kinase catalytic domain. The interaction with PKR did not require the C-terminal DNA-J homology region of P58IPK but was dependent on the presence of the eukaryotic initiation factor 2-alpha homology region, mapping to the 34 aa within the sixth P58IPK TPR motif. Consistent with other TPR proteins, P58IPK formed multimers in vivo: the N-terminal 166 aa were both necessary and sufficient for complex formation. A parallel in vivo analysis to map the K3L-binding region of PKR revealed that like P58IPK , K3L interacted exclusively with the PKR protein kinase catalytic domain. In contrast, however, the K3L-binding region of PKR was localized to within aa 367 to 551, demonstrating that each inhibitor bound PKR in unique, nonoverlapping domains. These data, taken together, suggest that P58IPK and K3L may mediate PKR inhibition by distinct mechanisms. Finally, we will propose a model of PKR inhibition in which P58IPK or a P58IPK complex binds PKR and interferes with nucleotide binding and autoregulation, while formation of a PKR-K3L complex interferes with active-site function and/or substrate association.

Base Sequence↗