How U.S. health plans can be more proactive about identifying and treating CRI.
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Humans↗
Biomedical subjects
R London
Publications and source records attributed to R London.
Spontaneous hydrolysis of 4-trifluoromethylphenol to a quinone methide and subsequent protein alkylation.
4-Trifluoromethylphenol (4-TFMP) was cytotoxic to precision-cut rat liver slices as indicated by loss of intracellular potassium. Intracellular glutathione levels decreased and fluoride ion levels increased in a time and concentration-dependent manner. The cytotoxicity of 4-TFMP did not appear to be due to the release of fluoride, however, since equimolar concentrations of sodium fluoride or potassium fluoride were not toxic. The ortho isomer (2-TFMP), which had a threefold slower rate of fluoride release, was much less toxic to liver slices. In incubations without slices, 4-TFMP spontaneously hydrolyzed in aqueous buffer at physiological pH to form 4-hydroxybenzoic acid via a quinone methide intermediate. The quinone methide was trapped by the addition of glutathione. Analysis of the glutathione adduct indicated that all of the fluorine atoms were lost during the hydrolysis, yielding a cresol derivative with the glutathione moiety attached to a benzylic carbonyl group. The glutathione conjugate was the primary product formed at low alkylphenol/glutathione ratios; however, at higher 4-TFMP concentrations additional unidentified products were observed. 4-TFMP also inhibited the in vitro enzyme activity of purlfied glyceraldehyde-3-phosphate dehydrogenase, a sulfhydryl-dependent enzyme, in a time and concentration-dependentmanner. Loss of thiol residues closely paralleled the loss in enzyme activity. The coaddition of glutathione prevented 4-TFMP-induced loss of enzyme activity. The cytotoxicity of 4-TFMP therefore appears to be due to spontaneous quinone methide formation and subsequent alkylation of cellular macromolecules.
Alkylation↗
Characterization of human and murine PMP20 peroxisomal proteins that exhibit antioxidant activity in vitro.
We have isolated the cDNAs encoding human and mouse homologues of a yeast protein, termed peroxisomal membrane protein 20 (PMP20). Comparison of the amino acid sequences of human (HsPMP20) and mouse (MmPMP20) PMP20 proteins revealed a high degree of identity (93%), whereas resemblance to the yeast Candida boidinii PMP20A and PMP20B (CbPMP20A and CbPMP20B) was less (30% identity). Both HsPMP20 and MmPMP20 lack transmembrane regions, as do CbPMP20A and CbPMP20B. HsPMP20 mRNA expression was low in human fetal tissues, especially in the brain. In adult tissues, HsPMP20 mRNA was expressed in the majority of tissues tested. HsPMP20 and MmPMP20 contained the C-terminal tripeptide sequence Ser-Gln-Leu (SQL), which is similar to the peroxisomal targeting signal 1 utilized for protein import into peroxisomes. HsPMP20 bound directly to the human peroxisomal targeting signal 1 receptor, HsPEX5. Mutagenesis analysis showed that the C-terminal tripeptide sequence, SQL, of HsPMP20 is necessary for its binding to HsPEX5. Subcellular fractionation of HeLa cells, expressing epitope-tagged PMP20, revealed that HsPMP20 is localized in the cytoplasm and in a particulate fraction containing peroxisomes. Double-staining immunofluorescence studies showed colocalization of HsPMP20 and thiolase, a bona fide peroxisomal protein. The amino acid sequence alignment of HsPMP20, MmPMP20, CbPMP20A, and CbPMP20B displayed high similarity to thiol-specific antioxidant proteins. HsPMP20 exerted an inhibitory effect on the inactivation of glutamine synthetase in the thiol metal-catalyzed oxidation system but not in the nonthiol metal-catalyzed oxidation system, suggesting that HsPMP20 possesses thiol-specific antioxidant activity. In addition, HsPMP20 removed hydrogen peroxide by its thiol-peroxidase activity. These results indicate that HsPMP20 is imported into the peroxisomal matrix via PEX5p and may work to protect peroxisomal proteins against oxidative stress. Because some portion of PMP20 might also be present in the cytosol, HsPMP20 may also have a protective effect in the cytoplasm.
Amino Acid Sequence↗
The receptor protein tyrosine phosphatase, PTP-RO, is upregulated during megakaryocyte differentiation and Is associated with the c-Kit receptor.
We have recently isolated a cDNA encoding a novel human receptor-type tyrosine phosphatase, termed PTP-RO (for a protein tyrosine phosphatase receptor omicron), from 5-fluorouracil-treated murine bone marrow cells. PTP-RO is a human homologue of murine PTPlambda and is related to the homotypically adhering kappa and mu receptor-type tyrosine phosphatases. PTP-RO is expressed in human megakaryocytic cell lines, primary bone marrow megakaryocytes, and stem cells. PTP-RO mRNA and protein expression are upregulated upon phorbol 12-myristate 13-acetate (PMA) treatment of the megakaryocytic cell lines CMS, CMK, and Dami. To elucidate the function of PTP-RO in megakaryocytic cells and its potential involvement in the stem cell factor (SCF)/c-Kit receptor pathway, COS-7 and 293 cells were cotransfected with the cDNAs of both the c-Kit tyrosine kinase receptor and PTP-RO. PTP-RO was found to be associated with the c-Kit receptor in these transfected cells and the SCF/Kit ligand induced a rapid tyrosine phosphorylation of PTP-RO. Interestingly, these transfected cells demonstrated a decrease in their proliferative response to the SCF/Kit ligand. In addition, we assessed the association of PTP-RO with c-Kit in vivo. The results demonstrated that PTP-RO associates with c-Kit but not with the tyrosine kinase receptor FGF-R and that PTP-RO is tyrosine-phosphorylated after SCF stimulation of Mo7e and CMK cells. Antisense oligonucleotides directed against PTP-RO mRNA sequences significantly inhibited megakaryocyte progenitor proliferation. Therefore, these data show that the novel tyrosine kinase phosphatase PTP-RO is involved in megakaryocytopoiesis and that its function is mediated by the SCF/c-Kit pathway.
Adolescent↗
Renal disease in the managed care setting: selection and monitoring of outcome criteria.
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Humans↗
Payer cost savings with endometrial ablation therapy.
CONTEXT: Dysfunctional uterine bleeding (DUB) is a significant cost burden for payers in the US healthcare system because hysterectomy, the common curative treatment, is associated with high hospitalization costs. OBJECTIVE: To determine the potential economic benefit to payers of endometrial ablation as an alternate treatment for the benign DUB disorder. STUDY DESIGN: A retrospective analysis of healthcare claims including the total direct costs to the payer (reimbursement) and patient (copayment). The study was designed to capture all DUB-related claims costs for the entire episode of care from initial diagnosis through follow-up care for 12 months postprocedure. PATIENTS AND METHODS: Twenty-four months of claims data from premenopausal women aged 25 to 50 years enrolled in a large managed care organization were screened based on relevant diagnostic and procedural codes. Incidence and costs of hysterectomy and ablation were determined, and potential payer savings were calculated based on hypothetical hysterectomy-to-ablation conversion rates of 25% to 50%. RESULTS: By performing ablation in lieu of hysterectomy for DUB, an average per-case savings of approximately $4,300 is possible. Potential annual payer savings are approximately $515,000 and $1.03 million for a 1-million-member plan, based on the 25% and 50% conversion rates, respectively. The recently approved uterine balloon therapy ablation technique could be instrumental in overcoming current barriers to wider utilization of ablation surgery. CONCLUSION: If ablation is used in lieu of hysterectomy when medically appropriate, a payer organization could reduce the cost of treating patients with DUB who are not responsive to drug therapy or dilation and curettage alone. Our data suggest that hysterectomy is the most common surgical therapy for this disorder, even though the less invasive endometrial ablation approach is more consistent with accepted DUB treatment guidelines. Payers therefore have an economic incentive to adopt guidelines and reimbursement policies that promote ablation therapy for DUB.
Adult↗
Toward a standard of care for treating suicidal outpatients: a survey of social workers' beliefs about appropriate treatment behaviors.
This study attempts to establish what reasonable, prudent professionals, named in the standard of care, believe is appropriate treatment for suicidal outpatients. We hope to move toward establishing a standard of care based on actuarial data, rather than after the fact evaluation by experts, judges, and lawyers. Previously, Greaney (1995, doctoral dissertation, Pacific Graduate School, Palo Alto, CA) empirically examined the beliefs of psychologists in appropriately treating suicidal outpatients. Our goal is to broaden the perspective of existing empirical data by examining the beliefs of clinical social workers in treating suicidal outpatients.
Ambulatory Care↗
A nephrologist's guide to managed care contracting.
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Contract Services↗
Regulation of the Ca2+ gradient across the sarcoplasmic reticulum in perfused rabbit heart. A 19F nuclear magnetic resonance study.
Myocardial contractility depends on Ca2+ release from and uptake into the sarcoplasmic reticulum (SR). The Ca2+ gradient between the SR matrix and the cytosol (SR Ca2+ gradient) is maintained by the SR Ca2+-ATPase using the free energy available from hydrolysis of ATP. The activity of the SR Ca2+-ATPase is not only dependent on the energy state of the cell but is also kinetically regulated by SR proteins such as phospholamban. To evaluate the importance of thermodynamic and kinetic regulation of the SR Ca2+ gradient, we examined the relationship between the energy available from ATP hydrolysis (DeltaGATP) and the energy required for maintenance of the SR Ca2+ gradient (DeltaGCa2+SR) during physiological and pathological manipulations that alter DeltaGATP and the phosphorylation state of phospholamban. We used our previously developed 19F nuclear magnetic resonance method to measure the ionized [Ca2+] in the SR of Langendorff-perfused rabbit hearts. We found that addition of either pyruvate or isoproterenol resulted in an increase in left ventricular developed pressure and an increase in [Ca2+]SR. Pyruvate increased DeltaGATP, and the increase in the SR Ca2+ gradient was matched to the increase in DeltaGATP; DeltaGATP increased from 58.3+/-0.5 to 60.4+/-1.0 kJ/mol (P<0.05), and DeltaGCa2+SR increased from 47.1+/-0.3 to 48.5+/-0.1 kJ/mol (P<0.05). In contrast, the increase in the SR Ca2+ gradient in the presence of isoproterenol occurred despite a decline in DeltaGATP from 58. 3+/-0.5 to 55.8+/-0.6 kJ/mol. Thus, the data indicate that the SR Ca2+ gradient can be increased by an increase in DeltaGATP, and that the positive inotropic effect of pyruvate can be explained by improved energy-linked SR Ca2+ handling, whereas the results with isoproterenol are consistent with removal of the kinetic limitation of phospholamban on the activity of the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase, which allows the SR Ca2+ gradient to move closer to its thermodynamic limit. Ischemia decreases DeltaGATP, and this should also have an effect on SR Ca2+ handling. During 30 minutes of ischemia, DeltaGATP decreased by 12 kJ/mol, but the decrease in DeltaGCa2+SR was 16 kJ/mol, greater than would be predicted by the fall in DeltaGATP and consistent with increased SR Ca2+ release and increased SR Ca2+ cycling. Because ischemic preconditioning is reported to decrease SR Ca2+ cycling during a subsequent sustained period of ischemia, we examined whether ischemic preconditioning affects the relationship between the fall in DeltaGATP and the fall in DeltaGCa2+SR during ischemia. We found that preconditioning attenuated the fall in DeltaGCa2+SR during ischemia; the fall in DeltaGCa2+SR was of comparable magnitude to the fall in DeltaGATP, and this was associated with a significant improvement in functional recovery during reperfusion. The data suggest that there is both thermodynamic regulation of the SR Ca2+ gradient by DeltaGATP and kinetic regulation, which can alter the relationship between DeltaGATP and DeltaGCa2+SR.
Adenosine Triphosphate↗
Mouse steroid sulfotransferases: substrate specificity and preliminary X-ray crystallographic analysis.
Three mouse cytosolic sulfotransferases were expressed in Escherichia coli cells in order to study their substrate specificities toward natural as well as synthetic steroid hormones. The Km and Vmax values confirmed the high substrate specificity of estrogen and hydroxysteroid sulfotransferases toward estradiol and dehydroepiandrosterone, respectively. In sharp contrast, the synthetic estrogen diethylstilbestrol was metabolized efficiently by both enzymes to its disulfate ester. These sulfotransferases display highly stereospecific sulfotransferase activity for sulfating only the trans-isomer of diethylstilbestrol. Crystals suitable for high-resolution structure determination of estrogen sulfotransferase were grown with polyethylene glycol. The crystals belong to the orthorhombic space group P2(1)2(1)2, and diffracted to 2.5 A.
Animals↗
Characterization and chromosomal localization of PTPRO, a novel receptor protein tyrosine phosphatase, expressed in hematopoietic stem cells.
Hematopoietic stem cells (HSCs) support blood cells throughout life by utilizing their self-renewing and multilineage differentiating capabilities. Hematopoietic growth factors mediate their effects on stem cells by the tyrosine phosphorylation of proteins. Regulation of tyrosine phosphorylation is partially mediated by protein tyrosine phosphatases (PTPases). A possible mechanism by which hematopoietic stem cells maintain their self-renewing capacity and undifferentiated state is by controlling the balanced and opposing actions of protein tyrosine kinases (PTKs), receptors for growth factors, and PTPases. We have characterized the expression of PTPases in 5-fluorouracil (5-FU)-treated murine bone marrow cells, which represent a very primitive population of progenitors enriched for reconstituting stem cells, by using a consensus polymerase chain reaction (PCR) method. Several PTPases were expressed abundantly in the 5-FU-treated bone marrow stem cells. A novel PTP, termed protein tyrosine phosphatase receptor omicron (PTPRO), which is related to the homotypically adhering kappa, mu and PCP-2 receptor-type tyrosine phosphatases, was identified and characterized. We have cloned the murine and full-length human PTPRO cDNAs which share 89% homology, indicating that PTPRO is highly conserved between these species. The human PTPRO cDNA clone encodes a polypeptide of 1439 amino acids (aa) and has a calculated molecular mass of approximately 162 kDa. PTPRO consists of an extracellular segment containing a MAM domain, an immunoglobulin (Ig) domain, four fibronectin-type III (FN-III) repeats, a transmembrane segment, and two tandem intracellular PTP domains. The human PTPRO gene was assigned to human chromosome 1p35-pter using Southern blot analyses of genomic DNAs from rodent/human somatic hybrid cell lines containing human chromosome 1 or the p35-pter region of the chromosome. The mouse Ptpro gene was mapped to chromosome 4, closely linked to D4Mit16 and Elp1 (elliptocytosis-1), by using genomic DNAs from a (C57BL/6J x Mus spretus)F1 x Mus spretus backcross. In fetal tissues, PTPRO expression was observed in the brain and lung, whereas lower levels were observed in the kidney. In adult tissues, PTPRO was less restricted and was observed in the lung, heart, skeletal muscle, prostate, testis, and in various areas of the brain, indicating that PTPRO expression is developmentally regulated. Expression of PTPRO was also observed in human CD34+ bone marrow cells and 5-FU-treated murine primitive stem cells. These results suggest a potential role for PTPRO in stem cell adhesion and in mediating homophilic cell-cell interactions in other cell types.
Amino Acid Sequence↗
Tyrosine phosphorylation of the related adhesion focal tyrosine kinase in megakaryocytes upon stem cell factor and phorbol myristate acetate stimulation and its association with paxillin.
We have characterized signaling pathways involving the related adhesion focal tyrosine kinase (RAFTK, also known as PYK2 or CAK-beta) in CMK human megakaryocytic cells. Stem cell factor, which potentiates the growth of megakaryocytes and their progenitors, and phorbol myristate acetate, which causes differentiation of megakaryocytic cell lines, induced the tyrosine phosphorylation of RAFTK but not of focal adhesion kinase. Stimulation of CMK cells with stem cell factor resulted in an increase in the autophosphorylation and kinase activity of RAFTK. Phosphorylation of RAFTK under these conditions was mediated by a protein kinase C-dependent pathway. Cytochalasin D, which disrupts the cytoskeleton, abolished the phosphorylation of RAFTK upon phorbol myristate acetate and stem cell factor stimulation, indicating that RAFTK association with the actin cytoskeleton appears to be critical for its phosphorylation. In addition, we observed an association of RAFTK with paxillin, a 68-kDa cytoskeleton protein. Using in vitro binding assays, RAFTK and paxillin were shown to bind directly through the C-terminal proline-rich domain. Transient overexpression of a dominant-negative mutant of RAFTK inhibited significantly the tyrosine phosphorylation of paxillin upon phorbol myristate acetate stimulation. These observations indicate that RAFTK might play an important role in the phosphorylation of signaling pathways within the focal adhesions and that RAFTK participates in signaling events that link signals from the cell surface to the cytoskeleton. Furthermore, this study suggests that RAFTK might be involved in megakaryocyte proliferation and differentiation.
Binding Sites↗
Brief physician advice for problem alcohol drinkers. A randomized controlled trial in community-based primary care practices.
OBJECTIVE: Project TrEAT (Trial for Early Alcohol Treatment) was designed to test the efficacy of brief physician advice in reducing alcohol use and health care utilization in problem drinkers. DESIGN: Randomized controlled clinical trial with 12-month follow-up. SETTING: A total of 17 community-based primary care practices (64 physicians) located in 10 Wisconsin counties. PARTICIPANTS: Of the 17695 patients screened for problem drinking, 482 men and 292 women met inclusion criteria and were randomized into a control (n=382) or an experimental (n=392) group. A total of 723 subjects (93%) participated in the 12-month follow-up procedures. INTERVENTION: The intervention consisted of two 10- to 15-minute counseling visits delivered by physicians using a scripted workbook that included advice, education, and contracting information. MAIN OUTCOME MEASURES: Alcohol use measures, emergency department visits, and hospital days. RESULTS: There were no significant differences between groups at baseline on alcohol use, age, socioeconomic status, smoking status, rates of depression or anxiety, frequency of conduct disorders, lifetime drug use, or health care utilization. At the time of the 12-month follow-up, there were significant reductions in 7-day alcohol use (mean number of drinks in previous 7 days decreased from 19.1 at baseline to 11.5 at 12 months for the experimental group vs 18.9 at baseline to 15.5 at 12 months for controls; t=4.33; P<.001), episodes of binge drinking (mean number of binge drinking episodes during previous 30 days decreased from 5.7 at baseline to 3.1 at 12 months for the experimental group vs 5.3 at baseline to 4.2 at 12 months for controls; t=2.81; P<.001), and frequency of excessive drinking (percentage drinking excessively in previous 7 days decreased from 47.5% at baseline to 17.8% at 12 months for the experimental group vs 48.1% at baseline to 32.5% at 12 months for controls; t=4.53; P<.001). The chi2 test of independence revealed a significant relationship between group status and length of hospitalization over the study period for men (P<.01). CONCLUSIONS: This study provides the first direct evidence that physician intervention with problem drinkers decreases alcohol use and health resource utilization in the US health care system.
Adult↗
Practice guideline for arterial blood gas measurement in the intensive care unit decreases numbers and increases appropriateness of tests.
OBJECTIVE: To test the hypothesis that implementation of a practice guideline for blood gas measurement would decrease numbers and increase appropriateness of tests (according to criteria in the guideline) for up to 1 yr after introduction of the guideline. DESIGN: Numbers of tests and appropriateness of each test were measured retrospectively during each of five periods: two baseline periods 2 yrs and 1 yr before introduction of the guideline and three follow-up periods 2 to 3 months, 6 to 7 months, and 12 to 13 months after introduction of the guideline. SETTING: A ten-bed multidisciplinary intensive care unit (ICU) within a 500-bed tertiary teaching hospital. PATIENTS: A random sample of 30 patients admitted to the ICU during each of the periods specified above. INTERVENTIONS: The nominal group process was used to develop a new guideline and a multipronged educational approach was used to facilitate implementation of the guideline. MEASUREMENTS AND MAIN RESULTS: At 2 to 3 months, test numbers decreased from 4.9 +/- 1.6 to 3.1 +/- 1.8 (SD) tests/patient/day and to 2.4 +/- 1.2 tests/patient/day at 12 to 13 months. Appropriateness increased from a mean of 44% at baseline to 78% at 2 to 3 months and 79% at 12 to 13 months. There were no differences in Acute Physiology and Chronic Health Evaluation scores or ICU mortality among the patient groups and no differences in number of ventilator days or time to wean from ventilation. Cost-minimization analysis showed that the incremental cost-saving 1 yr after introduction of the guideline was $19.18 per patient per day. CONCLUSIONS: Implementation of this guideline for arterial blood gas measurement increases efficiency of test utilization without prolonging mechanical ventilation or affecting outcome.
APACHE↗
Modulation of megakaryocytopoiesis by human macrophage-stimulating protein, the ligand for the RON receptor.
We observed that human megakaryocytes expressed the heterodimeric tyrosine kinase RON, which serves as a receptor for macrophage-stimulating protein (MSP). MSP appears to be structurally related to hepatocyte growth factor (HGF), which is a pleiotropic growth factor for a broad spectrum of tissues and cell types. The effects of human rMSP and rHGF on permanent human megakaryocytic cell lines as well as on human and murine primary marrow megakaryocytes were studied. MSP enhanced the maturation of the primary bone marrow megakaryocytes and human megakaryocytic cell lines, CMK and DAMI, as assessed by an increase in ploidy content. The increase in ploidy was blocked by specific Abs for MSP and by anti-IL-6 Abs. MSP treatment of primary human marrow megakaryocytes, DAMI cells, or CMK cells resulted in enhanced secretion of IL-6. The addition of MSP to cultures of immature murine megakaryoblasts showed a significant growth response, similar to that of exogenous IL-6. This increased growth of immature murine megakaryoblasts in response to MSP was abrogated either by Abs against MSP or by neutralizing mAbs to IL-6. HGF, over a range of concentrations (10 to 100 ng/ml) alone or in combination with IL-3, granulocyte-macrophage-CSF, or IL-6, had no effect on differentiation of human or murine marrow megakaryocytes. These results indicate that megakaryocytes express a novel tyrosine kinase receptor (RON), and that its ligand, MSP, appears capable of regulating megakaryocyte maturation, possibly via an autocrine mechanism mediated by induction of the cytokine IL-6.
Animals↗
Studies on the mechanism of hepatotoxicity of 4-methylphenol (p-cresol): effects of deuterium labeling and ring substitution.
We recently observed that 4-methylphenol (p-cresol) is toxic to rat liver tissue slices. A possible mechanism involves biotransformation of 4-methylphenol to a reactive quinone methide intermediate which covalently binds to cellular macromolecules and elicits cytotoxicity. In order to obtain further evidence for this proposed mechanism, we studied the effects of deuterium-labeled 4-methylphenol (4-[alpha, alpha, alpha-d3]-methylphenol), and the presence of various ring substituents, on the metabolism and toxicity of 4-methylphenol in precision cut liver slices prepared from male Sprague-Dawley rats. Deuterium-labeled 4-methylphenol was significantly less toxic than the parent compound in rat liver slices (LC50 = 3.36 vs. 1.31 mM, respectively). In addition, the deuterium-labeled compound was metabolized to a reactive intermediate (measured as glutathione conjugate formation) at a slower rate than that of 4-methylphenol in both liver slices and liver microsomal incubations. The presence of electron withdrawing substituents (2-chloro or 2-bromo) markedly enhanced both metabolism and toxicity, with the exception of 2,6-dibromocresol, which was similar to cresol in terms of rate of metabolism and toxicity. Conversely, the presence of electron donating substituents (2-methoxy, 2-methyl or 2,6-dimethyl) diminished metabolism and toxicity. In addition, methylation of the hydroxyl group to form 4-methylanisole, greatly reduced toxicity. These results support the hypothesis that the toxicity of 4-methylphenol is dependent on the formation of a reactive quinone methide intermediate.
Animals↗
Metabolism and toxicity of 4-hydroxyphenylacetone in rat liver slices: comparison with acetaminophen.
Acetaminophen is oxidized by cytochrome P450 to a reactive quinone imine, N-acetyl-p-benzoquinone imine, which is thought to be responsible for its hepatotoxic effects. 4-Hydroxyphenylacetone (4-HPA) is a structural analog of acetaminophen in which the amine group is replaced by a methylene group. Following a similar metabolic pathway, 4-HPA would be oxidized to form a reactive quinone methide intermediate. We compared the metabolism and toxicity of 4-HPA and acetaminophen in liver microsomes and precision-cut liver slices from male Sprague-Dawley rats. Both 4-HPA and acetaminophen formed glutathione conjugates in microsomal incubations. 4-HPA formed diastereomeric glutathione conjugates, which is consistent with the formation of an intermediate quinone methide. The rate of conjugate formation with 4-HPA was 8.5-fold greater than that with acetaminophen. In rat liver slices a concentration of 5 mM 4-HPA killed approximately 50% of hepatocytes after 6 hr of incubation, whereas acetaminophen was not toxic at concentrations up to 50 mM. N-Acetylcysteine protected slices from 4-HPA-induced toxicity, whereas phenobarbital enhanced metabolism and toxicity. In summary, 4-HPA is more hepatotoxic than acetaminophen, and this may be the result of differences in the metabolic rate and/or the type of reactive intermediate formed.
Acetaminophen↗
Identification and characterization of a novel related adhesion focal tyrosine kinase (RAFTK) from megakaryocytes and brain.
We have isolated a cDNA encoding a novel human intracytoplasmic tyrosine kinase, termed RAFTK (for a related adhesion focal tyrosine kinase). In addition, we have cloned and characterized the murine homolog of the human RAFTK cDNA. Comparison of the deduced amino acid sequences of human RAFTK and murine Raftk cDNAs revealed 95% homology, indicating that RAFTK is highly conserved between these species. The RAFTK cDNA clone, encoding a polypeptide of 1009 amino acids, has closest homology (48% identity, 65% similarity) to the focal adhesion kinase (pp125FAK). Comparison of the deduced amino acid sequences also indicates that RAFTK, like pp125FAK, lacks a transmembrane region, myristylation sites, and SH2 and SH3 domains. In addition, like pp125FAK, RAFTK contains a kinase domain flanked by large N-terminal (426 residues) and C-terminal (331 residues) domains, and the C-terminal region contains a predicted proline-rich stretch of residues. In fetal tissues, RAFTK expression was abundant in brain, and low levels were observed in lung and liver. In adult tissues, it was less restricted, indicating that RAFTK expression is developmentally up-regulated. Expression of RAFTK was also observed in human CD34+ marrow cells, primary bone marrow megakaryocytes, platelets, and various areas of brain. The human RAFTK gene was assigned to human chromosome 8 using genomic DNAs from human/rodent somatic cell hybrid lines. The mouse Raftk gene was mapped to chromosome 14, closely linked to gonadotropin-releasing hormone. Using specific antibodies for RAFTK, a approximately 123-kDa protein from the human megakaryocytic CMK cell line was immunoprecipitated. Treatment of the megakaryocytic CMK cells with thrombin caused a rapid induction of tyrosine phosphorylation of RAFTK protein. The structural features of RAFTK suggest that it is a member of the focal adhesion kinase gene family and may participate in signal transduction in human megakaryocytes and brain as well as in other cell types.
Amino Acid Sequence↗