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Biomedical subjects

R W King

Publications and source records attributed to R W King.

At least 37 records · Page 2Linked to original sources
PubMed1998

Serum creatine kinase is an unreliable predictor of ectopic pregnancy.

OBJECTIVE: To compare serum creatine kinase (CK) values in patients with ectopic pregnancy vs patients with threatened miscarriage or normal pregnancy. METHODS: An observational case-control study was performed at an urban teaching hospital. Pregnant women with a quantitative beta-hCG obtained for suspicion of ectopic pregnancy were evaluated. Excluded were cases with recent trauma, i.m. injections, surgery, or history of heart, liver, or muscle disease. The serum beta-hCG and CK values were recorded and compared between groups with 1-way ANOVA and Tukey's multiple comparison procedure at the overall 0.05 level. RESULTS: The 15 ectopic, 28 threatened miscarriage, and 21 normal pregnancy cases were of similar gestational ages (p = 0.2), ranging from 3 to 12 weeks. Although the CK values for ectopic pregnancy (88.8 +/- 33.6 IU/L) exceeded those for threatened miscarriage (65.9 +/- 59.0 IU/L) and normal pregnancy (56.0 +/- 38.1 U/L) (p = 0.02), there was significant overlap between groups. CK values were at or above a cutoff of 74 IU/L in 80% (95% confidence interval: 52-96%) of ectopic pregnancies, 25% (11-45%) of threatened miscarriages, and 14% (3-36%) of normal pregnancies. CONCLUSIONS: Although the ectopic pregnancy population is characterized by a higher mean CK than are patients with threatened miscarriage or a normal pregnancy, a significant overlap in CK values makes use of this serum marker unreliable for detecting ectopic pregnancy.

Abortion, Threatened↗
PubMed1998

Inhibition of human hepatitis B virus replication by AT-61, a phenylpropenamide derivative, alone and in combination with (-)beta-L-2',3'-dideoxy-3'-thiacytidine.

AT-61, a member of a novel class of phenylpropenamide derivatives, was found to be a highly selective and potent inhibitor of human hepatitis B virus (HBV) replication in four different human hepatoblastoma cell lines which support the replication of HBV (i.e., HepAD38, HepAD79, 2.2.15, and transiently transfected HepG2 cells). This compound was equally effective at inhibiting both the formation of intracellular immature core particles and the release of extracellular virions, with 50% effective concentrations ranging from 0.6 to 5.7 microM. AT-61 (27 microM) was able to reduce the amount of HBV covalently closed circular DNA found in the nuclei of HepAD38 cells by >99%. AT-61 at concentrations of >27 microM had little effect on the amount of viral RNA found within the cytoplasms of induced HepAD38 cells but reduced the number of immature virions which contained pregenomic RNA by >99%. The potency of AT-61 was not affected by one of the mutations responsible for (-)-beta-L-2', 3'-dideoxy-3' thiacytidine (3TC) resistance in HBV, and AT-61 acted synergistic with 3TC to inhibit HBV replication. AT-61 (81 microM) was not cytotoxic or antiproliferative to several cell lines and had no antiviral effect on woodchuck or duck HBV, human immunodeficiency virus type 1, herpes simplex virus type 1, vesicular stomatitis virus, or Newcastle disease virus. Therefore, we concluded that the antiviral activity of AT-61 is specific for HBV replication and most likely occurs at one of the steps between the synthesis of viral RNA and the packaging of pregenomic RNA into immature core particles.

Amides↗
PubMed1998

The M539V polymerase variant of human hepatitis B virus demonstrates resistance to 2'-deoxy-3'-thiacytidine and a reduced ability to synthesize viral DNA.

The cytosine analog 2'-deoxy-3'-thiacytidine (3TC) has been shown to be an effective treatment for chronic hepatitis B virus (HBV) infection. However, several liver transplant patients who were undergoing treatment with 3TC for HBV infection experienced a breakthrough of virus while on 3TC. The predominant virus found in these patients' sera contained either a valine or isoleucine for the methionine in the highly conserved YMDD nucleotide binding site in the HBV polymerase. To determine the biological relevance of the Met-to-Val substitution, we mutated a plasmid that contained a cDNA copy of the HBV pregenomic RNA such that when virus replication occurred during transient transfection of HepG2 cells, an M539V polymerase variant was produced. We found that in transiently transfected cells, this variant was approximately 330-fold less sensitive to the antiviral effects of 3TC and produced 7-fold less viral DNA than the wild type.

Antiviral Agents↗
PubMed1998

The hepatitis B virus M539V polymerase variation responsible for 3TC resistance also confers cross-resistance to other nucleoside analogues.

A variant of hepatitis B virus (HBV) containing a Met-to-Val substitution (M539V) in the YMDD motif of the polymerase nucleoside-binding domain exhibited resistance to the cytosine analogue lamivudine (3TC). To determine if the mutation responsible for the M539V polymerase variant affected the sensitivity of the virus to other nucleoside analogues, we constructed a tetracycline-responsive cell line, HepAD79. This cell line is stably transfected with a cDNA copy of the pregenomic RNA of an HBV genome containing an A-to-G mutation in the first position of the polymerase gene codon 539. This mutation results in a Met-to-Val substitution at amino acid 539 of the polymerase. When grown under the proper conditions, HepAD79 cells produced HBV RNA, contained HBV DNA associated with immature core particles and released core-associated HBV DNA into the medium. The M539V polymerase variant produced in this cell line was approximately 26-fold less sensitive to the antiviral effects of 3TC than wild-type virus. In addition, this variant demonstrated decreased sensitivity to the cytosine analogues FTC and ddC, as well as the thymidine analogue AZT.

Antiviral Agents↗
PubMed1997

Systematic identification of mitotic phosphoproteins.

BACKGROUND: Cyclin-dependent kinases (CDKs) are thought to initiate and coordinate cell division processes by sequentially phosphorylating key targets; in most cases these substrates remain unidentified. RESULTS: Using a screen that scores for phosphorylation of proteins, which were translated from pools of cDNA plasmids in vitro, by either phosphoepitope antibody recognition or electrophoretic mobility shifts, we have identified 20 mitotically phosphorylated proteins from Xenopus embryos, 15 of which have sequence similarity to other proteins. Of these proteins, five have previously been shown to be phosphorylated during mitosis (epithelial-microtubule associated protein-115, Oct91, Elongation factor 1gamma, BRG1 and Ribosomal protein L18A), five are related to proteins postulated to have roles in mitosis (epithelial-microtubule associated protein-115, Schizosaccharomyces pombe Cdc5, innercentrosome protein, BRG1 and the RNA helicase WM6), and nine are related to transcription factors (BRG1, negative co-factor 2alpha, Oct91, S. pombe Cdc5, HoxD1, Sox3, Vent2, and two isoforms of Xbr1b). Of 16 substrates tested, 14 can be directly phosphorylated in vitro by the mitotic CDK, cyclin B-Cdc2, although three of these may be physiological substrates of other kinases activated during mitosis. CONCLUSIONS: Examination of this broad set of mitotic phosphoproteins has allowed us to draw three conclusions about how the activation of CDKs regulates cell-cycle events. First, Cdc2 itself appears to directly phosphorylate most of the mitotic phosphoproteins. Second, during mitosis most of the substrates are phosphorylated more than once and a number may be targets of multiple kinases, suggesting combinatorial regulation. Third, the large fraction of mitotic phosphoproteins that are presumptive transcription factors, two of which have been previously shown to dissociate from DNA during mitosis, suggests that an important function of mitotic phosphorylation is to strip the chromatin of proteins associated with gene expression.

Animals↗
PubMed1997

Inducible expression of human hepatitis B virus (HBV) in stably transfected hepatoblastoma cells: a novel system for screening potential inhibitors of HBV replication.

We report the development and isolation of a cell line, termed HepAD38, that replicates human hepatitis B virus (HBV) under conditions that can be regulated with tetracycline. In the presence of the antibiotic, this cell line is free of virus due to the repression of pregenomic (pg) RNA synthesis. Upon removal of tetracycline from the culture medium, the cells express viral pg RNA, accumulate subviral particles in the cytoplasm that contain DNA intermediates characteristic of viral replication, and secrete virus-like particles into the supernatant. Since the HepAD38 cell line can produce high levels of HBV DNA, it should be useful for analyses of the viral replication cycle that depend upon viral DNA synthesis in a synchronized fashion. In addition, this cell line has been formatted into a high-throughput, cell-based assay that permits the large-scale screening of diverse compound libraries for new classes of inhibitors of HBV replication.

Cell Line↗
PubMed1996

How proteolysis drives the cell cycle.

Oscillations in the activity of cyclin-dependent kinases (CDKs) promote progression through the eukaryotic cell cycle. This review examines how proteolysis regulates CDK activity-by degrading CDK activators or inhibitors-and also how proteolysis may directly trigger the transition from metaphase to anaphase. Proteolysis during the cell cycle is mediated by two distinct ubiquitin-conjugation pathways. One pathway, requiring CDC34, initiates DNA replication by degrading a CDK inhibitor. The second pathway, involving a large protein complex called the anaphase-promoting complex or cyclosome, initiates chromosome segregation and exit from mitosis by degrading anaphase inhibitors and mitotic cyclins. Proteolysis therefore drives cell cycle progression not only by regulating CDK activity, but by directly influencing chromosome and spindle dynamics.

Anaphase↗
PubMed1996

Identification of BIME as a subunit of the anaphase-promoting complex.

The initiation of anaphase and exit from mitosis require the activation of a proteolytic system that ubiquitinates and degrades cyclin B. The regulated component of this system is a large ubiquitin ligase complex, termed the anaphase-promoting complex (APC) or cyclosome. Purified Xenopus laevis APC was found to be composed of eight major subunits, at least four of which became phosphorylated in mitosis. In addition to CDC27, CDC16, and CDC23, APC contained a homolog of Aspergillus nidulans BIME, a protein essential for anaphase. Because mutation of bimE can bypass the interphase arrest induced by either nimA mutation or unreplicated DNA, it appears that ubiquitination catalyzed by APC may also negatively regulate entry into mitosis.

Amino Acid Sequence↗
PubMed1996

Identification of a novel ubiquitin-conjugating enzyme involved in mitotic cyclin degradation.

BACKGROUND: The destruction of cyclin B is required for exit from mitosis, and is mediated by the ubiquitin pathway. Recently, a 20S complex, termed the anaphase-promoting complex (APC) or the cyclosome, has been genetically and biochemically identified as the cyclin-specific ubiquitin ligase (E3). In addition, a ubiquitin-conjugating enzyme (E2), UBC4, was shown to be involved in cyclin ubiquitination in Xenopus egg extracts. Another E2 activity, designated UBCx, can independently support cyclin ubiquitination in Xenopus. A similar activity (E2-C) has also been observed in clams. However, the molecular identity of Xenopus UBCx or clam E2-C has not been established. RESULTS: We have purified and cloned Xenopus UBCx. Sequence comparisons with known E2s reveal that UBCx is a novel ubiquitin-conjugating enzyme. Purified recombinant UBCx is sufficient to complement purified APC and E1 in destruction box-dependent cyclin ubiquitination. UBCx and UBC4 are active in a similar concentration range and with similar kinetics. At saturating enzyme concentrations, UBCx converts twice as much substrate into ubiquitin conjugates, but generates conjugates of lower molecular mass than UBC4. CONCLUSIONS: UBCx is a novel ubiquitin-conjugating enzyme involved in cyclin ubiquitination in Xenopus. Like UBC4, ubiquitination catalyzed by UBCx is dependent on both the destruction box and the APC, suggesting that these E2s function through a similar mechanism. However, as the patterns of conjugates generated by these E2s are distinct, these enzymes may play different roles in promoting cyclin proteolysis in mitosis.

Amino Acid Sequence↗
PubMed1996

Blue-light promotion of flowering is absent in hy4 mutants of Arabidopsis.

Loss of a blue-light photoreceptor in the hy4 mutants of Arabidopsis thaliana (L.) Heynh substantially delayed flowering (> 100 d to flower vs. 40-50 d), especially with blue light exposure from lamps lacking much red (R) and/or far-red (FR) light. Red night breaks were promotory but flowering was still later for the hy4-101 mutant. However, with exposure to light from FR-rich lamps, flowering of all mutants was early and no different from the wild type. Thus, flowering of Arabidopsis involves a blue-light photoreceptor and other, often more effective photoreceptors. The latter may involve phytochrome photoresponses to R and FR, but with little or no phytochrome response to blue wave-lengths.

Arabidopsis↗
PubMed1996

Mutagenic analysis of the destruction signal of mitotic cyclins and structural characterization of ubiquitinated intermediates.

Mitotic cyclins are abruptly degraded at the end of mitosis by a cell-cycle-regulated ubiquitin-dependent proteolytic system. To understand how cyclin is recognized for ubiquitin conjugation, we have performed a mutagenic analysis of the destruction signal of mitotic cyclins. We demonstrate that an N-terminal cyclin B segment as short as 27 residues, containing the 9-amino-acid destruction box, is sufficient to destabilize a heterologous protein in mitotic Xenopus extracts. Each of the three highly conserved residues of the cyclin B destruction box is essential for ubiquitination and subsequent degradation. Although an intact destruction box is essential for the degradation of both A- and B-type cyclins, we find that the Xenopus cyclin A1 destruction box cannot functionally substitute for its B-type counterpart, because it does not contain the highly conserved asparagine necessary for cyclin B proteolysis. Physical analysis of ubiquitinated cyclin B intermediates demonstrates that multiple lysine residues function as ubiquitin acceptor sites, and mutagenic studies indicate that no single lysine residue is essential for cyclin B degradation. This study defines the key residues of the destruction box that target cyclin for ubiquitination and suggests there are important differences in the way in which A- and B-type cyclins are recognized by the cyclin ubiquitination machinery.

Amino Acid Sequence↗
PubMed1996

Emergency physician use of ultrasonography in blunt abdominal trauma.

OBJECTIVE: To evaluate the diagnostic utility of abdominal diagnostic ultrasonography (DUS) performed by emergency physicians for intraperitoneal fluid caused by blunt abdominal trauma (BAT). METHODS: The design was a prospective, blind, observational study. During a 15-month period, a convenience sample of patients presenting to the ED with BAT necessitating CT scan of the abdomen, diagnostic peritoneal lavage (DPL), or laparotomy was studied. Scans were performed by an emergency medicine (EM) attending, or a resident supervised by an attending, using a real-time sector ultrasound scanner with a 3.5-MHz probe. Training in DUS included a 1-hour didactic session and 1 hour of practice on human volunteers. Free intraperitoneal fluid was defined as an anechoic stripe in the hepatorenal, bladder-rectal, or splenorenal space, and constituted a positive DUS study. Free intraperitoneal fluid detected on abdominal CT scan, DPL, and/or laparotomy was the criterion standard. RESULTS: Of 110 patients scanned, 13 were excluded secondary to technical difficulty or lack of diagnostic follow-up modalities. Of the remaining 97 patients, there were 24 females and 73 males, ranging from ages 2 to 78 years. DUS detected intraperitoneal fluid in 21 subjects, including 3 false positives. There were 6 false-negative DUS examinations. DUS had a sensitivity of 75% (95% CI 53-90%), a specificity 96% of (95% CI 89-99%), and an accuracy of 91% (95% CI 83-96%). No false-positive or false-negative DUS study occurred after the first 67 cases. The mean interval for a DUS scan was 4.9 +/- 2.9 minutes, ranging from 0.5 to 16 minutes, and the mean intervals were not different between the positive and the negative studies. The accuracies of DUS were similar in the pediatric patients, 97% (95% CI 83-100%), and in the adults, 88% (95% CI 78-95%). The hepatorenal view provided the highest sensitivity as well as the least number of uninterpretable scans of the 3 DUS views. CONCLUSION: Emergency physicians with minimal training can use DUS with fair sensitivity and good specificity and accuracy to detect free intraperitoneal fluid in both pediatric and adult BAT victims. The hepatorenal view provides the highest sensitivity for intraperitoneal fluid, although the 3-view series (with hepatorenal, bladder-rectal, and splenorenal spaces) can typically be performed within 5 minutes and may increase the specificity and accuracy.

Abdominal Injuries↗
PubMed1996

Shock index as a marker for significant injury in trauma patients.

OBJECTIVE: To determine whether the shock index (SI), defined as the ratio of heart rate (HR) to systolic blood pressure (SBP), is a useful marker for significant injury in trauma patients. METHODS: A retrospective database analysis was used to relate the SI to the clinical measures: death within 24 hours, injury severity score (ISS) > or = 16, intensive care unit (ICU) stay > or = 1 day, and amount of blood transfused (BT) > or = 2 units. Consecutive trauma patients seen at one level I trauma center over a 24-month period were reviewed; excluded were patients not requiring trauma team consultation, or those with either incomplete records, severe head injury (Glasgow Coma Scale score < or = 8), or age < 14 years. The SI was calculated from ED admission vital signs. Receiver operating characteristic (ROC) curves were used to find the value of the SI that maximized the sum of sensitivity and specificity for predicting each measure, separately; a separate analysis was done to determine the optimal SI threshold for predicting any of the severity measures. RESULTS: 1,101 cases met study criteria. The optimal SI values (by ROC analysis) for predicting the severity measures were: 1.10 for death < 24 hours, 0.71 for ISS > or = 16, 0.77 for ICU > or = 1 day, and 0.85 for BT > or = 2 units. The optimal SI value (by ROC analysis) for any of the above measures was 0.83; use of this SI cutoff provided a sensitivity of 37% (95% CI 32-42%), a specificity of 83% (95% CI 80-87%), and a negative predictive value of 58% (95% CI 54-61%) for any measure. This SI threshold predicted between 24% fewer cases and 4% more cases of poor outcome than did the optimal thresholds HR and SBP, respectively. CONCLUSION: The optimal SI threshold performed similarly to the optimal threshold HR or SBP for prediction of injury severity.

Adolescent↗
PubMed1995

A 20S complex containing CDC27 and CDC16 catalyzes the mitosis-specific conjugation of ubiquitin to cyclin B.

Cyclin B is degraded at the onset of anaphase by a ubiquitin-dependent proteolytic system. We have fractionated mitotic Xenopus egg extracts to identify components required for this process. We find that UBC4 and at least one other ubiquitin-conjugating enzyme can support cyclin B ubiquitination. The mitotic specificity of cyclin ubiquitination is determined by a 20S complex that contains homologs of budding yeast CDC16 and CDC27. Because these proteins are required for anaphase in yeast and mammalian cells, we refer to this complex as the anaphase-promoting complex (APC). CDC27 antibodies deplete APC activity, while immunopurified CDC27 complexes are sufficient to complement either interphase extracts or a mixture of recombinant UBC4 and the ubiquitin-activating enzyme E1. These results suggest that APC functions as a regulated ubiquitin-protein ligase that targets cyclin B for destruction in mitosis.

Anaphase↗
PubMed1995

Identification of a clinical isolate of HIV-1 with an isoleucine at position 82 of the protease which retains susceptibility to protease inhibitors.

The HIV-1 protease (PR) is essential for the production of mature virions. As such, it has become a target for the development of anti-HIV chemotherapeutics. Multiple passages of virus in cell culture in the presence of PR inhibitors have resulted in the selection of variants with decreased sensitivity to inhibitors of the PR. The most common alteration observed is a single amino acid change at position 82. This particular position has been well characterized by several laboratories as being important for the susceptibility of the virus to inhibitors of PR function. Mutations which result in the substitution of the wild-type valine with alanine, phenylalanine, threonine or isoleucine at position 82 of the PR have been associated with decreased sensitivity to several PR inhibitors. We describe here a clinical strain of HIV-1 that contains an isoleucine at position 82 of the PR instead of the usual valine. This strain is unique in that it was isolated from a patient that was anti-retroviral naive, and in the past, variants at position 82 of the PR have only been found after treatment of patients or cell culture with PR inhibitors. Moreover, this virus remains sensitive to PR inhibitors of the cyclic urea and C-2 symmetrical diol classes.

Amino Acid Sequence↗
PubMed1995

Flowering responses to altered expression of phytochrome in mutants and transgenic lines of Arabidopsis thaliana (L.) Heynh.

The long-day plant Arabidopsis thaliana (L.) Heynh. flowers early in response to brief end-of-day (EOD) exposures to far-red light (FR) following a fluorescent short day of 8 h. FR promotion of flowering was nullified by subsequent brief red light (R) EOD exposure, indicating phytochrome involvement. The EOD response to R or FR is a robust measure of phytochrome action. Along with their wild-type (WT) parents, mutants deficient in either phytochrome A or B responded similarly to the EOD treatments. Thus, neither phytochrome A nor B exclusively regulated flowering, although phytochrome B controlled hypocotyl elongation. Perhaps a third phytochrome species is important for the EOD responses of the mutants and/or their flowering is regulated by the amount of the FR-absorbing form of phytochrome, irrespective of the phytochrome species. Overexpression of phytochrome A or phytochrome B resulted in differing photoperiod and EOD responses among the genotypes. The day-neutral overexpressor of phytochrome A had an EOD response similar to all of the mutants and WTs, whereas R EOD exposure promoted flowering in the overexpressor of phytochrome B and FR EOD exposure inhibited this promotion. The comparisons between relative flowering times and leaf numbers at flowering of the over-expressors and their WTs were not consistent across photoperiods and light treatments, although both phytochromes A and B contributed to regulating flowering of the transgenic plants.

Arabidopsis↗