Purchasing oncology services. Kerr L. White Institute/American Cancer Society Task Force on Purchasing Oncology Services.

BACKGROUND: A multidisciplinary panel representing various stakeholders in the health care delivery and oncology services marketplace was convened to develop specific criteria for healthcare purchasers to consider when evaluating the structures and processes of health plans. These rank ordered criteria also can be used by oncologic service providers and health plan designers as a yardstick for the services they offer. METHODS: A multidisciplinary 31-member Task Force was assembled by the Kerr L. White Institute and the American Cancer Society in March 1997. Task Force members were selected for their ability to offer expert insight as purchasers, suppliers, policymakers, consumers, or stakeholders in the health care marketplace. A preference-weighted majority voting rule was used to identify the three most important recommendations of the 10 that were generated through a modified Delphi technique. To test the practicality of the top three recommendations, leaders of large managed care organizations (MCOs) were surveyed; the results of this survey then were compared with the results of the Task Force survey. RESULTS: The three most important recommendations from the Task Force were that health plans provide access to: 1) comprehensive cancer care, 2) preventive and screening services, and 3) second opinions and treatment options supported by scientific evidence. The difference between the responses of the Task Force and the MCOs was that MCOs placed the highest importance on evidence-based decision-making, with their next three rankings coinciding with those identified by the Task Force. CONCLUSIONS: The value of these summary recommendations will be realized through their use by both purchasers and suppliers to influence the structure and content of the delivery of oncologic services.

How to judge the cancer services benefit component of your health insurance plan.

BACKGROUND: Members of the oncology community, rich in data from years of published clinical research, epidemiology, and tumor registry information, expect businesses to evaluate the quality of cancer care using classic scientific methods. However, businesses lack sophistication in these data and tend to use significantly different criteria relating to cost, service, and access. Logistical problems also abound, such as the wide variety of cancers, small sample sizes, and the frequency with which patients change insurance plans. METHODS: Adapting an approach first proposed by Donabedian employing criteria that can be measured, easily gathered, and whose presence/absence can be interpreted as having a consequence for patient care, the author proposes a system of evaluating cancer health care benefits. CONCLUSIONS: Recognizing that classic outcomes analysis is too complex and inappropriate for business, companies and oncology providers may be able to develop obtainable measures whose presence or absence has intuitive and documented relation to the quality of cancer care.

Evidence for a central role of lysine 15 of Azotobacter vinelandii nitrogenase iron protein in nucleotide binding and protein conformational changes.

Biological nitrogen fixation catalyzed by purified nitrogenase requires the hydrolysis of a minimum of 16 MgATP for each N2 reduced. In the present study, we demonstrate a central function for Lys-15 of Azotobacter vinelandii nitrogenase iron protein (FeP) in the interaction of nucleotides with nitrogenase. Changing Lys-15 of the FeP to Arg resulted in an FeP with a dramatically reduced affinity for both MgATP and MgADP. From equilibrium column binding experiments at different nucleotide concentrations, apparent dissociation constants (Kd) for wild type FeP binding of MgADP (143 microM) and MgATP (571 microM) were determined. Over the same nucleotide concentration ranges, the K15R FeP showed no significant affinity for either nucleotide. This contrasts sharply with previous results with an FeP in which Lys-15 was changed to Gln (K15Q) where it was found that the K15Q FeP bound MgADP with the same affinity as wild type FeP and MgATP with a slightly reduced affinity. Analysis of K15R FeP by EPR, circular dichroism (CD), and microcoulometry revealed that the [4Fe-4S] cluster was unaffected by the amino acid change and that addition of either MgADP or MgATP did not result in the protein conformational changes normally detected by these techniques. These results are integrated into a model for how MgATP and MgADP bind and induce conformational changes within the FeP.

Analysis of a gene region required for dihydrogen oxidation in Azotobacter vinelandii.

The putative products of six Azotobacter vinelandii chromosomal open reading frames (ORFs) were suggested to be involved in dihydrogen (H2) metabolism [Chen and Mortenson (1992) Biochim Biophys Acta 1131, 199-202]. A promoterless lacZ-containing cassette was used to disrupt the ORFs. Qualitative analysis revealed that the lacZ genes were expressed only in those mutants where the directions of the inserted lacZ were identical to those of the ORFs, showing that the six ORFs were transcribed as predicted. Unlike wildtype (w.t.), none of the mutants could perform dioxygen (O2)-dependent H2-oxidation, even though Western immunoanalyses showed that the hydrogenase large subunit was present although in amounts less than in w.t. Only one of the mutants (a hypB mutant), grown in nickel-enriched media, showed meaningful restoration of the H2-oxidizing ability. From the above observations it is concluded that (a) the six-ORF region is transcriptionally active and involved in H2-oxidation, (b) the product of hypB is needed for nickel activation of hydrogenase, and (c) the six ORFs (genes) belong to two or more operons. Possible roles of the gene products for the assembly, modification, and processing of hydrogenase from its apoproteins and metal centers are discussed.

Health care policies affecting the treatment of patients with cancer and cancer research.

BACKGROUND: Reimbursement policies under the current health care financing system are chaotic, even for older individuals who might expect some measure of uniformity under federal statues that govern coverage under Medicare. Policy changes at the federal level have increased the complexity and uncertainty of reimbursement for providers and patients. METHODS: The Association of Community Cancer Centers, in conjunction with a number of state medical oncology societies, distributed surveys to medical oncologists in 20 states during 1993. Eight hundred, fifty-six medical oncologists in 282 practices responded with information on their practices and insurance problems. RESULTS: Data illustrate the broad diversity of coverage for cancer treatment and research and its impact on treatment decisions and patient access. The broad spectrum of results from various states indicates significant differences in the implementation of federal policies on key issues, such as access to chemotherapeutic agents for off-label indications and clinical trials. CONCLUSIONS: The data portray the significant influence of insurance company policies on clinical research and disease prevention and treatment. Under the current system, prevention measures and prevention trials are discouraged, and a growing number of insurers are not paying for clinical research. New state and federal legislation offers some hope that uniformity of access to providers, prevention, off-label drugs, and clinical trials may be on the horizon.

Resonance Raman studies of iron-only hydrogenases.

The nature of the iron-sulfur clusters in oxidized and reduced forms of Fe-only hydrogenases from Desulfovibrio vulgaris, Thermotoga maritima, and Clostridium pasteurianum has been investigated by resonance Raman spectroscopy. The results indicate the presence of ferredoxin-like [4Fe-4S]2+,+ and [2Fe-2S]2+,+ clusters in both T. maritima hydrogenase and C. pasteurianum hydrogenase I, but only [4Fe-4S]2+,+ clusters in D. vulgaris hydrogenase. This necessitates a reevaluation of the iron-sulfur cluster composition of C. pasteurianum hydrogenase I and indicates that the resonance Raman bands in the oxidized hydrogenase that were previously attributed to the hydrogen activating center [Macor, K. A., Czernuszewicz, R. S., Adams, M. W. W., & Spiro, T. G. (1987) J. Biol. Chem. 262, 9945-9947] arise from an indigenous [2Fe-2S]2+ cluster. No resonance Raman bands that could be uniquely attributed to the oxidized or reduced hydrogen activating center were observed. This suggests that the hydrogen activating center is a novel Fe center that is unrelated to any known type of Fe-S cluster.

The unusual metal clusters of nitrogenase: structural features revealed by x-ray anomalous diffraction studies of the MoFe protein from Clostridium pasteurianum.

Nitrogenase (EC 1.18.6.1) catalyzes the conversion of dinitrogen to ammonia, the central reaction of biological nitrogen fixation. X-ray anomalous diffraction data were analyzed to probe the structures of the metal clusters bound by nitrogenase MoFe protein. In addition to one FeMo cofactor, each half-molecule of MoFe protein binds one large FeS cluster of a type not previously observed in a protein. The FeS cluster contains roughly eight Fe atoms, comprises two subclusters, and is separated from the FeMo cofactor by an edge-to-edge distance of 14 A. The inorganic framework of the FeMo cofactor is not resolved into subclusters, but the Mo atom is located at its periphery. FeMo cofactors in different half-molecules are 70 A apart and cannot promote binuclear activation of dinitrogen by two Mo atoms.

Increasing nitrogenase catalytic efficiency for MgATP by changing serine 16 of its Fe protein to threonine: use of Mn2+ to show interaction of serine 16 with Mg2+.

MgATP-binding and hydrolysis are an integral part of the nitrogenase catalytic mechanism. We are exploring the function of MgATP hydrolysis in this reaction by analyzing the properties of the Fe protein (FeP) component of Azotobacter vinelandii nitrogenase altered by site-directed mutagenesis. We have previously (Seefeldt, L.C., Morgan, T.V., Dean, D.R., & Mortenson, L.E., 1992, J. Biol. Chem. 267, 6680-6688) identified a region near the N-terminus of FeP that is involved in interaction with MgATP. This region of FeP is homologous to the well-known nucleotide-binding motif GXXXXGKS/T. In the present work, we examined the function of the four hydroxyl-containing amino acids immediately C-terminal to the conserved lysine 15 that is involved in interaction with the gamma-phosphate of MgATP. We have established, by altering independently Thr 17, Thr 18, and Thr 19 to alanine, that a hydroxyl-containing residue is not needed at these positions for FeP to function. In contrast, an hydroxyl-containing amino acid at position 16 was found to be critical for FeP function. When the strictly conserved Ser 16 was altered to Ala, Cys, Asp, or Gly, the FeP did not support N2 fixation when expressed in place of the wild-type FeP in A. vinelandii. Altering Ser 16 to Thr (S16T), however, resulted in the expression of an FeP that was partially active. This S16T FeP was purified to homogeneity, and its biochemical examination allowed us to assign a catalytic function to this hydroxyl group in the nitrogenase mechanism. Of particular importance was the finding that the S16T FeP had a significantly higher affinity for MgATP than the wild-type FeP, with a measured Km of 20 microM compared to the wild-type FeP Km of 220 microM. This increased kinetic affinity for MgATP was reflected in a significantly stronger binding of the S16T FeP for MgATP. In contrast, the affinity for MgADP, which binds at the same site as MgATP, was unchanged. The catalytic efficiency (kcat/Km) of S16T FeP was found to be 5.3-fold higher than for the wild-type FeP, with the S16T FeP supporting up to 10 times greater nitrogenase activity at low MgATP concentrations. This indicates a role for the hydroxyl group at position 16 in interaction with MgATP but not MgADP. The site of interaction of this residue was further defined by examining the properties of wild-type and S16T FePs in utilizing MnATP compared with MgATP.(ABSTRACT TRUNCATED AT 400 WORDS)

Carboxyl-terminal processing may be essential for production of active NiFe hydrogenase in Azotobacter vinelandii.

The NiFe hydrogenase from Azotobacter vinelandii is a membrane-bound alpha beta heterodimer that can oxidize H2 to protons and electrons and thereby provide energy. Genes encoding the alpha and beta subunits, hoxG and hoxK respectively, followed by thirteen contiguous accessory genes potentially involved in H2 oxidation, have been previously sequenced. Mutations in some of these accessory genes give rise to inactive enzyme containing an alpha subunit with decreased electrophoretic mobility. Mass spectral analysis of the subunits demonstrated that the alpha subunit had a molecular weight 1,663 Da less than that predicted from hoxG. Since the N-terminal sequence of the purified alpha subunit matches the sequence predicted from hoxG we suggest this difference is due to removal of the C-terminus of the alpha subunit which may be an important step linked to metal insertion, localization, and formation of active hydrogenase.

Nucleotide sequences and genetic analysis of hydrogen oxidation (hox) genes in Azotobacter vinelandii.

Azotobacter vinelandii contains a heterodimeric, membrane-bound [NiFe]hydrogenase capable of catalyzing the reversible oxidation of H2. The beta and alpha subunits of the enzyme are encoded by the structural genes hoxK and hoxG, respectively, which appear to form part of an operon that contains at least one further potential gene (open reading frame 3 [ORF3]). In this study, determination of the nucleotide sequence of a region of 2,344 bp downstream of ORF3 revealed four additional closely spaced or overlapping ORFs. These ORFs, ORF4 through ORF7, potentially encode polypeptides with predicted masses of 22.8, 11.4, 16.3, and 31 kDa, respectively. Mutagenesis of the chromosome of A. vinelandii in the area sequenced was carried out by introduction of antibiotic resistance gene cassettes. Disruption of hoxK and hoxG by a kanamycin resistance gene abolished whole-cell hydrogenase activity coupled to O2 and led to loss of the hydrogenase alpha subunit. Insertional mutagenesis of ORF3 through ORF7 with a promoterless lacZ-Kmr cassette established that the region is transcriptionally active and involved in H2 oxidation. We propose to call ORF3 through ORF7 hoxZ, hoxM, hoxL, hoxO, and hoxQ, respectively. The predicted hox gene products resemble those encoded by genes from hydrogenase-related operons in other bacteria, including Escherichia coli and Alcaligenes eutrophus.

Identification of six open reading frames from a region of the Azotobacter vinelandii genome likely involved in dihydrogen metabolism.

We reported earlier the identification of two Azotobacter vinelandii open reading frames (ORFs), ORF1 and ORF2, downstream from the hydrogenase structural genes (Chen, J.C. and Mortenson, L.E. (1992) Biochim. Biophys. Acta 1131, 122-124). Sequencing of 6008 base pairs of DNA immediately downstream from ORF2 revealed six additional ORFs (ORF3 through ORF8). All six ORFs are transcribed from the same DNA strand as that of the ORF1 and ORF2. Deduced amino acid sequences of ORF3 through ORF5, and those of ORF4, ORF5, ORF7 and ORF8 have strong homology with genes required for dihydrogen (H2) metabolism in Rhodobacter capsulatus and in Escherichia coli, respectively. ORF4, ORF5, ORF6 and ORF8 would encode for polypeptides containing one or more 'Cys-X-X-Cys' motifs. The predicted products of ORF5 and ORF6 each contain a histidine-rich region, and the product of ORF5 also includes a 'Cys-Thr-Val-Cys-Gly-Cys' region near its amino-terminus. Implications of these findings with respect to metal binding, transport and incorporation, to hydrogenase assembly and to H2 metabolism are discussed.

Two open reading frames (ORFs) identified near the hydrogenase structural genes in Azotobacter vinelandii, the first ORF may encode for a polypeptide similar to rubredoxins.

Sequencing of 744 base pairs (bp) of a cloned section of DNA from Azotobacter vinelandii reveals two complete, closely-spaced open reading frames (ORF1 and ORF2). Both ORFs are transcribed from the same DNA strand as that of the structural genes for hydrogenase (hoxK and hoxG, Menon, A.L. et al. (1990) Gene 96, 67-74), and are located downstream from the latter genes. The distance between the end of hoxG and the beginning of ORF1 is approx. 3.0 kilobases (kb). Most of the deduced amino acid sequence of ORF1 shares high homology with rubredoxin sequences. Some of the deduced amino acid sequence of ORF2 shares homology with that of a reported partial ORF from Rhodobacter capsulatus, ORF located within a region of DNA required for dihydrogen oxidation in that organism. Implications of these findings with respect to dihydrogen metabolism are discussed.

Mapping the site(s) of MgATP and MgADP interaction with the nitrogenase of Azotobacter vinelandii. Lysine 15 of the iron protein plays a major role in MgATP interaction.

Nitrogenase binds and hydrolyzes 2MgATP yielding 2MgADP and 2Pi for each electron that is transferred from the iron protein to the MoFe protein. The iron protein alone binds but does not hydrolyze 2MgATP or 2MgADP and the binding of these nucleotides is competitive. Iron protein amino acid sequences all contain a putatitive mononucleotide-binding region similar to a region found in other mononucleotide-binding proteins. To examine the role of this region in MgATP interaction, we have substituted glutamine and proline for conserved lysine 15. The amino acid substitutions, K15Q and K15P, both yielded a non-N2-fixing phenotype when the genes coding for them were substituted into the Azotobacter vinelandii chromosome in place of the wild-type gene. The iron protein from the K15Q mutant was purified to homogeneity, whereas the protein from the K15P mutant could not be purified in its native form. Unlike wild-type iron protein, the purified K15Q iron protein showed no acetylene reduction, H2 evolution, or ATP hydrolysis activities when complemented with wild-type MoFe protein. The K15Q iron protein and the normal iron protein had a similar total iron content and both proteins showed the characteristic rhombic EPR signal resulting from the reduced state of the single 4Fe-4S cluster bridging the two subunits. Unlike the wild-type iron protein, addition of MgATP to the K15Q iron protein did not result in the perturbation necessary to change the EPR signal of its 4Fe-4S center from a rhombic to an axial line shape. Also unlike the wild-type iron protein, addition of MgATP to K15Q iron protein in the presence of the iron chelator, alpha,alpha'-dipyridyl, did not result in a time-dependent transfer of iron to the chelator. Thus, even though the K15Q iron protein contains a normal 4Fe-4S center, it does not respond to MgATP like the wild-type protein. Examination of the ability of the K15Q iron protein to bind MgADP showed no change from the wild-type iron protein, but its ability to bind MgATP decreased to 35% of the wild-type protein. Thus, in A. vinelandii iron protein, lysine 15 is not needed for interaction with MgADP but is involved in the binding of ATP, presumably through charge-charge interaction with the gamma-phosphate. Based on the above data, this lysine appears to be essential for the MgATP induced conformational change of wild-type iron protein that is required for activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Kinetic and spectroscopic analysis of the inactivating effects of nitric oxide on the individual components of Azotobacter vinelandii nitrogenase.

The effects of nitric oxide (NO) on the individual components of Azotobacter vinelandii nitrogenase have been examined by kinetic and spectroscopic methods. Incubation of the Fe protein (Av2) for 1 h with stoichiometries of 4- and 8-fold molar excesses of NO to Av2 dimer resulted in a complete loss of activity of Av2 in C2H2-reduction assays. The kinetics of inactivation indicated that the minimum stoichiometry of NO to Av2 required to fully inactivate Av2 lies between 1 and 2. The rate of inactivation of Av2 activity by NO was stimulated up to 2-fold by the presence of MgATP and MgADP but was unaffected by the presence of sodium dithionite. Unexpectedly, complete inactivation of Av2 by low ratios of NO to Av2 also resulted in a complete loss of its ability to bind MgATP and MgADP. UV-visible spectroscopy indicated that the effect of NO on Av2 involves oxidation of the [4Fe-4S] center. EPR spectroscopy revealed that the loss of activity during inactivation of Av2 by NO correlated with the loss of the S = 1/2 and S = 3/2 signals. Appearance of the classical and intense iron-nitrosyl signal (g = 20.3) was only observed when Av2 was incubated with large molar excesses of NO and the appearance of this signal did not correlate with the loss of Av2 activity. The effects of NO on the MoFe protein (Av1) were more complex than for Av2. A time-dependent inactivation of Av1 activity (C2H2 reduction) was observed which required considerably higher concentrations of NO than those required to inactivate Av2 (up to 10 kPa).(ABSTRACT TRUNCATED AT 250 WORDS)

Resonance Raman studies of the [4Fe-4S] to [2Fe-2S] cluster conversion in the iron protein of nitrogenase.

Resonance Raman spectroscopy has been used to investigate the Fe-S stretching modes of the [4Fe-4S]2+ cluster in the oxidized iron protein of Clostridium pasteurianum nitrogenase. The results are consistent with a cubane [4Fe-4S] cluster having effective Td symmetry with cysteinyl coordination for each iron. In accord with previous optical and EPR studies [(1984) Biochemistry 23, 2118-2122], treatment with the iron chelator alpha, alpha'-dipyridyl in the presence of MgATP is shown to effect cluster conversion to a [2Fe-2S]2+ cluster. Resonance Raman data also indicate that partial conversion to a [2Fe-2S]2+ cluster is induced by thionine-oxidation in the presence of MgATP in the absence of an iron chelator. This result suggests new explanations for the dramatic change in the CD spectrum that accompanies MgATP-binding to the oxidized Fe protein and the anomalous resonance Raman spectra of thionine-oxidized Clostridium pasteurianum bidirectional hydrogenase.

Cloning, sequencing and characterization of the [NiFe]hydrogenase-encoding structural genes (hoxK and hoxG) from Azotobacter vinelandii.

The Azotobacter vinelandii [NiFe]hydrogenase-encoding structural genes were isolated from an A. vinelandii genomic cosmid library. Nucleotide (nt) sequence analysis showed that the two genes, hoxK and hoxG, which encode the small and large subunits of the enzyme, respectively, form part of an operon that contains at least one other gene. The hoxK gene encodes a polypeptide of 358 amino acids (aa) (39,209 Da). The deduced aa sequence encodes a possible 45-aa N-terminus extension, not present in the purified A. vinelandii hydrogenase small subunit, which could be a cellular targeting sequence. The hoxG gene is downstream form, and overlaps hoxK by 4 nt and encodes a 602-aa polypeptide of 66,803 Da. The hoxK and hoxG gene products display homology to aa sequences of hydrogenase small and large subunits, respectively, from other organisms. The hoxG gene lies 16 nt upstream from a third open reading frame which could encode a 27,729-Da (240-aa) hydrophobic polypeptide containing 53% nonpolar and 11% aromatic aa. The significance of this possible third gene is not known at present.

Pulsed electron paramagnetic resonance studies of the interaction of Mg-ATP and D2O with the iron protein of nitrogenase.

Mg-ATP binds to the iron protein component of nitrogenase. The magnetic field dependence of the linear electric field effect (LEFE) in pulsed EPR is consistent with a single 4Fe-4S cluster. The LEFE is virtually unaltered when Mg-ATP is bound. Electron spin echo envelope modulation techniques were employed to evaluate the possibility of a magnetic interaction between 31P of Mg-ATP and the Fe-S center of the iron protein. None was detected. However, weak modulations possibly attributable to peptide 14N were seen, and these were slightly shifted by Mg-ATP addition. Further, protons in the vicinity of the Fe-S cluster of the protein readily exchange with D2O, and this process is unaffected by Mg-ATP.