Several Bcr-Abl kinase domain mutants associated with imatinib mesylate resistance remain sensitive to imatinib

Imatinib mesylate is a selective Bcr-Abl kinase inhibitor, effective in the treatment of chronic myelogenous leukemia. Most patients in chronic phase maintain durable responses; however, many in blast crisis fail to respond, or relapse quickly. Kinase domain mutations are the most commonly identified mechanism associated with relapse. Many of these mutations decrease the sensitivity of the Abl kinase to imatinib, thus accounting for resistance to imatinib. The role of other mutations in the emergence of resistance has not been established. Using biochemical and cellular assays, we analyzed the sensitivity of several mutants (Met244Val, Phe311Leu, Phe317Leu, Glu355Gly, Phe359Val, Val379Ile, Leu387Met, and His396Pro/Arg) to imatinib mesylate to better understand their role in mediating resistance.While some Abl mutations lead to imatinib resistance, many others are significantly, and some fully, inhibited. This study highlights the need for biochemical and biologic characterization, before a resistant phenotype can be ascribed to a mutant.     

Organophosphonate-degrading PhnZ reveals an emerging family of HD domain mixed-valent diiron oxygenases

The founding members of the HD-domain protein superfamily are phosphohydrolases, and newly discovered members are generally annotated as such. However, myo-inositol oxygenase (MIOX) exemplifies a second, very different function that has evolved within the common scaffold of this superfamily. A recently discovered HD protein, PhnZ, catalyzes conversion of 2-amino-1-hydroxyethylphosphonate to glycine and phosphate, culminating a bacterial pathway for the utilization of environmentally abundant 2-aminoethylphosphonate. Using Mössbauer and EPR spectroscopies, X-ray crystallography, and activity measurements, we show here that, like MIOX, PhnZ employs a mixed-valent Fe^II/Fe^III cofactor for the O₂-dependent oxidative cleavage of its substrate. Phylogenetic analysis suggests that many more HD proteins may catalyze yet-unknown oxygenation reactions using this hitherto exceptional Fe^II/Fe^III cofactor. The results demonstrate that the catalytic repertoire of the HD superfamily extends well beyond phosphohydrolysis and suggest that the mechanism used by MIOX and PhnZ may be a common strategy for oxidative C-X bond cleavage.With the number of known protein sequences rising exponentially, assignment of functions to the ever-expanding proteome is a central, largely unmet challenge in the molecular life sciences. Assignment to a known superfamily based on sequence or structure is usually the first approach to predict the function of an uncharacterized protein and often provides valuable hints for further studies. However, nature uses divergent evolution to create new functions, leading to functional diversification within superfamilies that can be as modest as different substrate specificities or as profound as fundamentally different reaction types (1). Without any biochemical or biological information, functional predictions based solely on superfamily assignment can thus be misleading or incorrect (2).The HD domain superfamily, recognized by Aravind and Koonin in 1998 on the basis of sequence analysis (3), now contains more than 37,000 members occurring in a broad range of organisms in all three domains of life and in more than 240 distinct domain architectures (4). HD proteins possess a conserved α-helical core containing their characteristic H…HD…D sequence motif that binds a divalent metal ion (often Zn^II, Mg^II, or Mn^II) (5). HD proteins with a dinuclear metal cluster, in which two additional histidines from within the D…D region of the domain form a second metal binding site, have also been identified (6, 7). Only a limited number of HD proteins have been biochemically characterized; they almost exclusively catalyze phosphoester hydrolysis, which has led to the general assignment of HD proteins as phosphohydrolases (3, 5, 8–12). The wide phylogenetic distribution and diverse domain architectures of the HD proteins suggest, however, that additional activities might have evolved within the superfamily. Indeed, the enzyme myo-inositol oxygenase (MIOX) exemplifies a mechanistically very different catalytic function for an HD protein (13–15). In MIOX, the conserved H…HD…H…H…D motif binds two iron ions, and the enzyme uses an unprecedented mixed-valent Fe^II/Fe^III cofactor form to catalyze the four-electron (4e^–) oxidative C-C bond cleavage converting myo-inositol (MI; cyclohexan-1,2,3,5/4,6-hexa-ol) to d-glucuronate (13, 16, 17). The Fe^III site coordinates MI, serving as Lewis acid to activate the substrate, and the Fe^II site activates O₂ to produce a superoxo-Fe^III/Fe^III intermediate that initiates the reaction by abstracting a hydrogen atom from C1 of MI (6, 18).The recently discovered HD protein, PhnZ, catalyzes the 4e^– oxidative C-P bond cleavage of 2-amino-1-hydroxyethylphosphonate (OH-AEP) to glycine and phosphate (19) (Fig. 1). It is the second of two enzymes in a recently discovered pathway that degrades the most abundant environmental phosphonate compound, 2-aminoethylphosphonate (2-AEP) (20–23), and its reaction represents the third type of enzymatic C-P bond cleavage to be recognized (23–26). PhnZ was shown to require iron for activity, but the nature of the iron cofactor and its mechanism are unknown (19). The first enzyme in the pathway, the Fe^II- and α-ketoglutarate(αKG)-dependent oxygenase, PhnY, introduces the hydroxyl group on C1 of 2-AEP, activating the compound for subsequent C-P bond cleavage by PhnZ (19). The assignment of PhnZ to the HD domain superfamily, the conservation of the six residues that coordinate the cofactor in MIOX, the dependence of its catalytic activity on the presence of iron, and the analogy between the PhnZ-catalyzed oxidation and that catalyzed by MIOX all suggest that PhnZ might provide the second example of an HD protein using a mixed-valent diiron cofactor to catalyze an oxygenation (rather than a hydrolysis) reaction.Open in a separate windowFig. 1.Reaction catalyzed by PhnZ.Here, we demonstrate by Mössbauer and EPR spectroscopy that PhnZ harbors a diiron cluster and, like MIOX, markedly stabilizes the mixed-valent Fe^II/Fe^III state. The X-ray crystal structure of PhnZ confirms the presence of a diiron cofactor and shows that the substrate is bound to the site 2 Fe ion in a manner analogous to the binding mode of MI in MIOX. PhnZ is an oxygenase requiring O₂ for its reaction, as determined by activity assays and isotope-tracer experiments. Freeze-quench (FQ) EPR experiments demonstrate that the Fe^II/Fe^III state is competent to react rapidly with O₂ to effect the 4e⁻ oxidation of OH-AEP. Our results thus confirm that PhnZ is the second example of an HD protein using a Fe^II/Fe^III diiron cofactor for oxidative bond cleavage and imply that the mechanism used by MIOX and PhnZ might be a more widespread strategy for the oxidative cleavage of C-X bonds in yet unknown reactions. Indeed, phylogenetic analysis of the HD superfamily suggests that there are potentially many other HD domain mixed-valent diiron oxygenases (HD-MVDOs). These findings demonstrate that the catalytic repertoire of the HD superfamily extends well beyond phosphohydrolysis and illustrate once more how nature creates distinct catalytic functions within a common protein scaffold by divergent evolution.     

Cytotoxicity of Bile in Human Hep G2 Cells and in Primary Cultures of Rat Hepatocytes

There has been increasing interest in the development of a hepatocyte bioreactor for the treatment of acute hepatic failure; however, little is known about the effect of hepatocyte byproducts on the viability of the cells in the bioreactor environment. We investigated the effects of increasing concentrations of bile on the growth and viability of the human hepatoma cell line Hep G2 and on the cytochrome P-450 content and dependent mixed function oxidase (MFO) activities, reduced glutathione (GSH) content, and glutathione S-transferase (GST) activity of primary cultures of rat hepatocytes. Our purpose was to determine whether or not it would be necessary to pretreat the plasma from patients with acute liver failure to remove elevated bile concentrations which might be toxic to the hepatocytes in an artificial liver device. Bile was found to inhibit Hep G2 cell growth at concentrations as low as 0.1% and to decrease viability at concentrations above 0.5%. The cytochrome P-450 and GSH contents and the activities of the MFO system and of GST were decreased in the primary cultures of hepatocytes following 24 h treatment with concentrations of bile at and above 0.5%. The MFO activities associated with different cytochrome P-450 isoenzymes decreased to different extents in the presence of bile with the O-dealkylation of pentoxyresorufin being more labile than that of ethoxyresorufin. Our data indicate that elevated bile concentrations are cytotoxic to liver cells, and it may be necessary to pretreat patient plasma to decrease its bile content to protect the cells during the clinical operation of a hepatocyte bioreactor device.     

FURTHER EXPERIMENTS ON THE EFFECTS OF LONG CONTINUED INTRAPERITONEAL INJECTIONS OF PROTEINS

We have attempted to discover whether or not certain protein materials, such as albumose, casein, and albumen, when introduced parenterally (peritoneally) into experimental animals, are able to produce organic lesions. These proteins were used alone or in combination with chloroform, which was administered in oil and as an anesthetic. Our results were negative.     

Reduced Mortality Rates after Intensive Statin Therapy in Managed-Care Patients

OBJECTIVE: To evaluate whether intensive statin therapy in a managed-care setting produces greater clinical benefit than more moderate statin use. METHODS: Adults hospitalized for a coronary heart disease (CHD) event were identified from a longitudinal database of pharmaceutical and medical claims. Propensity scores representing a patient's likelihood of receiving statin therapy were calculated. Statin-treated patients were those who received statin therapy within 30 days of hospital discharge after a CHD event, had been supplied with statin therapy for at least 10 days during the follow-up period, and received statin therapy for at least 10 days before the first recurrent CHD event. Standard or intensive statin therapy was identified according to low-density lipoprotein cholesterol reductions expected with statin dose. Patients in the standard and intensive groups were matched by propensity scores to patients not receiving statin therapy after discharge. Patients in the standard statin therapy group were also matched to patients who received intensive statin therapy. Mortality rates after hospital discharge were compared in all matched groups. RESULTS: Patients treated with standard therapy experienced a 32% reduction in risk of death compared with patients not receiving statin therapy (P = 0.003). Patients who received intensive statin therapy after a CHD event experienced a 42% reduction in risk of mortality (P = 0.002) versus those not receiving statin therapy. Compared with standard therapy, intensive statin treatment further reduced the risk of death by 29% (P = 0.020). CONCLUSIONS: High risk CHD patients benefit from intensive statin therapy in a real-world, managed-care cohort, confirming the results of randomized clinical trials.     

Joint nursing appointments: a vehicle for influencing health care change

With the rapid pace of health care change nurses need to organize and respond quickly to the debate on future health care policy Nurses from practice and academia working together can capitalize on their unique knowledge and expertise to influence the development of consumer- and health-oriented policies In the past joint appointments have served to unite nurses in the education and research domains These appointments must evolve to encompass policy-focused activities that will enable nurses to make a significant contribution to the transformation of the health care delivery system     

An evaluation of program implementation strategies for a rural first-responder system

An experimental emergency first-responder system was introduced in 36 small rural Georgia communities as a means for addressing inadequate access to emergency medical services for these communities. A prospective evaluation was designed to address the most efficient and effective means to organize, implement, and administrate such a program on a regional basis. Key to the program were community-selected residents who served as Emergency Medical Coordinators (EMCs) and performed as first responders, information sources on emergency care, and system organizers. The evaluation examined the process of organizing the program through local government versus voluntary group sponsors in terms of response and participation by communities and their ability to select effective EMCs. It assessed the effectiveness of a set of criteria for selecting residents as EMCs against three sets of performance measures encompassing first-responder skills and activities which maintained public awareness and program visibility. Critical to program success was the degree of sponsor involvement in selecting EMCs.Dr. Kay is Assistant Professor in the School of Public Health, University of Michigan, Ann Arbor, MI 48109; Dr. Myrick is Associate Professor in the School of Health Systems, Georgia Institute of Technology, Atlanta, GA 30332. This project was supported by the National Center for Health Services Research, OASH, DHHS, Grant No. HS 02507. A version of this paper was presented at the American Public Health Association Annual Meeting, Los Angeles, CA, November 2, 1981.     

Grounded theory: an exploration of process and procedure

Grounded theory, as an evolving qualitative research method, is a product of its history as well as of its epistemology. Within the literature, there have been a number of discussions focusing on the differences between Glaser's (1978, 1992) and Strauss's (1987, 1990) versions of grounded theory. The purpose of this article is to add a level of depth and breadth to this discussion through specifically exploring the Glaser-Strauss debate by comparing the data analysis processes and procedures advocated by Glaser and by Strauss. To accomplish this task, the authors present the article in two sections. First, they provide relevant background information on grounded theory as a research method. Second, they pursue a more in-depth discussion of the positions of Glaser, using Glaser's work, and Strauss, using Strauss's and Strauss and Corbin's (1990) work, regarding the different phases of data analysis, specifically addressing the coding procedures, verification, and the issue of forcing versus emergence.