OmoMYC blunts promoter invasion by oncogenic MYC to inhibit gene expression characteristic of MYC-dependent tumors.

MYC genes have both essential roles during normal development and exert oncogenic functions during tumorigenesis. Expression of a dominant-negative allele of MYC, termed OmoMYC, can induce rapid tumor regression in mouse models with little toxicity for normal tissues. How OmoMYC discriminates between physiological and oncogenic functions of MYC is unclear. We have solved the crystal structure of OmoMYC and show that it forms a stable homodimer and as such recognizes DNA in the same manner as the MYC/MAX heterodimer. OmoMYC attenuates both MYC-dependent activation and repression by competing with MYC/MAX for binding to chromatin, effectively lowering MYC/MAX occupancy at its cognate binding sites. OmoMYC causes the largest decreases in promoter occupancy and changes in expression on genes that are invaded by oncogenic MYC levels. A signature of OmoMYC-regulated genes defines subgroups with high MYC levels in multiple tumor entities and identifies novel targets for the eradication of MYC-driven tumors.

High accuracy FIONA-AFM hybrid imaging.

Multi-protein complexes are ubiquitous and play essential roles in many biological mechanisms. Single molecule imaging techniques such as electron microscopy (EM) and atomic force microscopy (AFM) are powerful methods for characterizing the structural properties of multi-protein and multi-protein-DNA complexes. However, a significant limitation to these techniques is the ability to distinguish different proteins from one another. Here, we combine high resolution fluorescence microscopy and AFM (FIONA-AFM) to allow the identification of different proteins in such complexes. Using quantum dots as fiducial markers in addition to fluorescently labeled proteins, we are able to align fluorescence and AFM information to ≥8nm accuracy. This accuracy is sufficient to identify individual fluorescently labeled proteins in most multi-protein complexes. We investigate the limitations of localization precision and accuracy in fluorescence and AFM images separately and their effects on the overall registration accuracy of FIONA-AFM hybrid images. This combination of the two orthogonal techniques (FIONA and AFM) opens a wide spectrum of possible applications to the study of protein interactions, because AFM can yield high resolution (5-10nm) information about the conformational properties of multi-protein complexes and the fluorescence can indicate spatial relationships of the proteins in the complexes.

Prophylaxis in factor IX deficiency product and patient variation.

To determine the dosing needed to maintain a prophylactic level of factor IX (FIX) >/=2%, 15 non-inhibitor severe (/=2%. Based on pharmacokinetic analysis the median amount of concentrate needed to maintain a prophylactic level >/=2% for 30 days when administered every third day is 677 IU kg(-1) pd-FIX (range 388-6005 IU kg(-1) pd-FIX) compared with 1168 IU kg(-1) r-FIX (range 268-13085 IU kg(-1) r-FIX). The median cost for 30 days of prophylaxis of an average 25-kg 8-year-old child at the current University of Iowa Price (0.87 US dollars Mononine/0.86 US dollars BeneFix as of December 2002) if given every third day would be 19,972 US dollars and 34,456 US dollars for r-FIX. However, because of wide inter-patient variability in recovery and half-life, pharmacokinetic evaluation of each patient is necessary to determine the appropriate dosing schedule and product best suited for prophylaxis.

Error-prone DNA polymerases: novel structures and the benefits of infidelity.

Studies on several recently discovered error-prone DNA polymerases reveal novel structures that may explain the low fidelity of this general class of enzymes, a number of which are involved in the replicative bypass (translesion synthesis) of base damage in DNA.

Crystal structure and deletion analysis show that the accessory subunit of mammalian DNA polymerase gamma, Pol gamma B, functions as a homodimer.

Polymerase gamma, which replicates and repairs mitochondrial DNA, requires the Pol gamma B subunit for processivity. We determined the crystal structure of mouse Pol gamma B, a core component of the mitochondrial replication machinery. Pol gamma B shows high similarity to glycyl-tRNA synthetase and dimerizes through an unusual intermolecular four-helix bundle. A human Pol gamma B mutant lacking the four-helix bundle failed to dimerize in solution or to stimulate the catalytic subunit Pol gamma A, but retained the ability to bind with Pol gamma A to a primer-template construct, indicating that the functional holoenzyme contains two Pol gamma B molecules. Other mutants retained stimulatory activity but lost the ability to bind folded ssDNA. These results suggest that the Pol gamma B dimer contains distinct sites for Pol gamma A binding, dimerization, and DNA binding.

Incidence of focal white matter lesions in a population of hemophiliac children and their normal siblings. Hemophilia Growth and Development Study.

OBJECTIVE: This analysis was undertaken to evaluate the etiology and sequelae of 2- to 5-mm focal white matter hyperintensities on T2-weighted MR images of some participants enrolled in the Hemophilia Growth and Development Study (HGDS). MATERIALS AND METHODS: The HGDS is a multicenter study of the growth and development, neurological, neuropsychological, and immune functioning of a cohort of children and adolescents, 62% of whom were infected with HIV through the use of clotting factor concentrates, and their non-hemophiliac, non-HIV infected male siblings. The current investigation was conducted with all three groups of HGDS participants: HIV-positive hemophiliacs (n = 207), HIV-negative hemophiliacs (n = 126), and their siblings (n = 47). Magnetic resonance imaging was performed at each center, with a variety of 0.3 to 1.5 T instruments. Standard examinations included 5-mm-thick T1-weighted sagittal and axial images, intermediate, and T2-weighted axial images. A study of abnormalities of the coagulation system known to be associated with thrombotic events was conducted among a subgroup of participants (n = 51) from eight centers. RESULTS: Lesions were not associated with hemophilia-related factors, immune function, hematologic, or neurologic factors. There were no associations between the presence of white matter lesions and defects of coagulation in any of the assays completed. CONCLUSION: The 2- to 5-mm focal white matter hyperintensities on T2-weighted MR images of the brain were incidental findings in our study population.

The Tetracycline Repressor-A Paradigm for a Biological Switch.

The excessive use of antibiotics has enabled bacteria to develop resistance through a variety of mechanisms. The most common bacteriostatic action of the broad-spectrum antibiotic tetracycline (Tc) is by the inactivation of the bacterial ribosome so that the protein biosynthesis is interrupted and the bacteria die. The most common mechanism of resistance in gram-negative bacteria against Tc is associated with the membrane-intrinsic protein TetA, which exports invaded Tc out of the bacterial cell before it can attack its target, the ribosome. The expression of TetA is tightly regulated by the homodimeric Tet repressor (TetR)(2), which binds specifically with two helix-turn-helix motifs of operator DNA (tetO; K(ass) approximately 10(11) M(-1)) located upstream from the tetA gene on a plasmid or transposon. When Tc diffuses into the cell it chelates Mg(2+) and the complex [MgTc](+) binds to (TetR)(2) to form the induced complex (TetR small middle dot[MgTc](+))(2). This process is associated with conformational changes, which sharply reduce the affinity of (TetR)(2) to tetO, so that expression of TetA can take place, thus conferring resistance to the bacteria cells against Tc. Crystallographic studies show sequence-specific protein-nucleic acid interactions in the (TetR)(2) small middle dottetO complex and how the binding of two [MgTc](+) to (TetR)(2) enforces conformational changes that are stabilized by cooperative binding of two chains of eight water molecules each so that the formed (TetR small middle dot[MgTc](+))(2) is no longer able to recognize and bind to tetO. Since the switching mechanisms of the TetR/[MgTc](+) system is so tight, it has proven very useful in the regulation of eukaryotic gene expression and may also be applicable in gene therapy.

The nucleotide excision repair protein UvrB, a helicase-like enzyme with a catch.

Nucleotide excision repair (NER) is a universal DNA repair mechanism found in all three kingdoms of life. Its ability to repair a broad range of DNA lesions sets NER apart from other repair mechanisms. NER systems recognize the damaged DNA strand and cleave it 3', then 5' to the lesion. After the oligonucleotide containing the lesion is removed, repair synthesis fills the resulting gap. UvrB is the central component of bacterial NER. It is directly involved in distinguishing damaged from undamaged DNA and guides the DNA from recognition to repair synthesis. Recently solved structures of UvrB from different organisms represent the first high-resolution view into bacterial NER. The structures provide detailed insight into the domain architecture of UvrB and, through comparison, suggest possible domain movements. The structure of UvrB consists of five domains. Domains 1a and 3 bind ATP at the inter-domain interface and share high structural similarity to helicases of superfamilies I and II. Not related to helicase structures, domains 2 and 4 are involved in interactions with either UvrA or UvrC, whereas domain 1b was implicated for DNA binding. The structures indicate that ATP binding and hydrolysis is associated with domain motions. UvrB's ATPase activity, however, is not coupled to the separation of long DNA duplexes as in helicases, but rather leads to the formation of the preincision complex with the damaged DNA substrate. The location of conserved residues and structural comparisons with helicase-DNA structures suggest how UvrB might bind to DNA. A model of the UvrB-DNA interaction in which a beta-hairpin of UvrB inserts between the DNA double strand has been proposed recently. This padlock model is developed further to suggest two distinct consequences of domain motion: in the UvrA(2)B-DNA complex, domain motions lead to translocation along the DNA, whereas in the tight UvrB-DNA pre-incision complex, they lead to distortion of the 3' incision site.

A closer look at the active site of gamma-class carbonic anhydrases: high-resolution crystallographic studies of the carbonic anhydrase from Methanosarcina thermophila.

The prototype of the gamma-class of carbonic anhydrase has been characterized from the methanogenic archaeon Methanosarcina thermophila. Previously reported kinetic studies of the gamma-class carbonic anhydrase are consistent with this enzyme having a reaction mechanism similar to that of the mammalian alpha-class carbonic anhydrase. However, the overall folds of these two enzymes are dissimilar, and apart from the zinc-coordinating histidines, the active site residues bear little resemblance to one another. The crystal structures of zinc-containing and cobalt-substituted gamma-class carbonic anhydrases from M. thermophila are reported here between 1.46 and 1.95 A resolution in the unbound form and cocrystallized with either SO(4)(2)(-) or HCO(3)(-). Relative to the tetrahedral coordination geometry seen at the active site in the alpha-class of carbonic anhydrases, the active site of the gamma-class enzyme contains additional metal-bound water ligands, so the overall coordination geometry is trigonal bipyramidal for the zinc-containing enzyme and octahedral for the cobalt-substituted enzyme. Ligands bound to the active site all make contacts with the side chain of Glu 62 in manners that suggest the side chain is likely protonated. In the uncomplexed zinc-containing enzyme, the side chains of Glu 62 and Glu 84 appear to share a proton; additionally, Glu 84 exhibits multiple conformations. This suggests that Glu 84 may act as a proton shuttle, which is an important aspect of the reaction mechanism of alpha-class carbonic anhydrases. A hydrophobic pocket on the surface of the enzyme may participate in the trapping of CO(2) at the active site. On the basis of the coordination geometry at the active site, ligand binding modes, the behavior of the side chains of Glu 62 and Glu 84, and analogies to the well-characterized alpha-class of carbonic anhydrases, a more-defined reaction mechanism is proposed for the gamma-class of carbonic anhydrases.

Human coagulation factor FVIIa (recombinant) in the management of limb-threatening bleeds unresponsive to alternative therapies: results from the NovoSeven emergency-use programme in patients with severe haemophilia or with acquired inhibitors.

This open-label, emergency-use study evaluated the efficacy and safety of activated human coagulation factor VIIa (recombinant) (rFVIIa) (NovoSeven; Novo Nordisk Pharmaceuticals, Inc., New Jersey, USA) in treating limb-threatening joint or muscle bleeds in 17 patients with haemophilia A or B and six patients with acquired inhibitors to factor VIII or factor IX. All patients had previously failed on one or more alternative therapies. rFVIIa administration was effective or partially effective in controlling joint or muscle bleeds in 34 out of 35 (97%) bleeding episodes; in 23 patients, 14 of 17 (82%) muscle bleeds and 16 of 18 (89%) joint bleeds were effectively controlled. These findings suggest that rFVIIa is an effective and well-tolerated therapeutic option in the management of joint or muscle haemorrhage in patients with haemophilia and in patients with acquired inhibitors.

Changes in hepatitis B serologic titers in HIV+ and HIV- children with haemophilia.

This longitudinal study examines differences in hepatitis B immune titres in children and adolescents with haemophilia to determine if they are dependent on how immunity was acquired (vaccination or natural infection), and whether they are related to the child's HIV status and/or are influenced by HIV disease progression. Serologic titres (HBcAb, HBsAb) and HBsAg were measured prospectively at baseline, and at years 1, 2 and 3 of follow-up in 126 HIV- and 207 HIV+ children and adolescents with haemophilia. Analyses were performed to assess the impact of HIV status on the measured titres, and for HIV+ subjects to examine the association with CD4+ lymphocyte counts and p24 antigen status. The results show that HIV+ children were more likely than HIV- children to lose vaccine-induced immunity as indicated by the loss of HBsAb. There was an increased risk of losing HBsAb with higher CD4+ counts and younger age. Re-immunization was not successful in seven of eight HIV+ children. Two subjects (one HIV+, one HIV-) entered the study HBsAg- but became HBsAg+ over the course of follow-up. Seven HIV+ subjects lost natural immunity as indicated by the loss of HBcAb. The loss of either HBsAb or HBcAb in HIV--subjects was negligible to absent. In conclusion, because of the loss of immunity in HIV+ children the viral safety of factor replacement concentrates for these children is an important consideration. HIV- children rarely lose immunity, therefore frequent measures of HBsAb are not necessary.

Crystal structure of UvrB, a DNA helicase adapted for nucleotide excision repair.

Nucleotide excision repair (NER) is a highly conserved DNA repair mechanism. NER systems recognize the damaged DNA strand, cleave it on both sides of the lesion, remove and newly synthesize the fragment. UvrB is a central component of the bacterial NER system participating in damage recognition, strand excision and repair synthesis. We have solved the crystal structure of UvrB in the apo and the ATP-bound forms. UvrB contains two domains related in structure to helicases, and two additional domains unique to repair proteins. The structure contains all elements of an intact helicase, and is evidence that UvrB utilizes ATP hydrolysis to move along the DNA to probe for damage. The location of conserved residues and structural comparisons allow us to predict the path of the DNA and suggest that the tight pre-incision complex of UvrB and the damaged DNA is formed by insertion of a flexible beta-hairpin between the two DNA strands.

Structural basis and mechanism of enoyl reductase inhibition by triclosan.

The enoyl-acyl carrier protein reductase (ENR) is involved in bacterial fatty acid biosynthesis and is the target of the antibacterial diazaborine compounds and the front-line antituberculosis drug isoniazid. Recent studies suggest that ENR is also the target for the broad-spectrum biocide triclosan. The 1.75 A crystal structure of EnvM, the ENR from Escherichia coli, in complex with triclosan and NADH reveals that triclosan binds specifically to EnvM. These data provide a molecular mechanism for the antibacterial activity of triclosan and substantiate the hypothesis that its activity results from inhibition of a specific cellular target rather than non-specific disruption of the bacterial cell membrane. This has important implications for the emergence of drug-resistant bacteria, since triclosan is an additive in many personal care products such as toothpastes, mouthwashes and soaps. Based on this structure, rational design of triclosan derivatives is possible which might be effective against recently identified triclosan-resistant bacterial strains.

Home treatment of mild to moderate bleeding episodes using recombinant factor VIIa (Novoseven) in haemophiliacs with inhibitors.

OBJECTIVE: To assess the safety and efficacy of a fixed dose of recombinant activated factor VII (rFVIIa; NovoSeven) in the home setting for mild to moderately severe joint, muscle; and mucocutaneous bleeding episodes in patients with haemophilia A or B with inhibitors. DESIGN: Multicentre, open-label, single arm, phase III study of one year duration. METHODS; Patients or their caregivers administered up to three doses of rFVIIa (90 microg/kg i.v.) at 3 h intervals within 8 h of the onset of a mild to moderate bleeding episode. Once the subject considered that rFVIIa had been "effective" with regard to haemostasis (after 1-3 injections), one further (maintenance) dose of rFVIIa was administered. RESULTS: Of 60 patients enrolled, 56 experienced at least one bleed, and 46 completed the one year study. 614 of 877 bleeds (70%) were evaluable according to protocol definitions. Haemostasis was rated as "effective" in 92% (566/614) of evaluable bleeds after a mean of 2.2 injections. For successfully treated episodes, the time from onset of bleeding until administration of the first injection was 1.1+/-2.0 h (mean+/-SD). Twenty-four hours after initial successful response, haemostasis was reported as having been maintained in 95% of cases. Efficacy was comparable for muscle, joint and target joint, and mucocutaneous bleeding episodes. In an intent-to-treat analysis of all 877 bleeding events, efficacy outcomes were equivalent to the evaluable bleeds, with an effective response in 88% of treated episodes. Treatment-related adverse events occurred in 32 (3% of all) bleeding episodes and consisted of re-bleeds/new bleeds in more than 50% (18/32) of these events. A single episode of superficial thrombophlebitis was the only thrombotic complication encountered, and there were no patient withdrawals due to adverse events. Development of FVII(a) antibodies could not be detected, and hypersensitivity reactions to rFVIIa were not reported. CONCLUSION: rFVIIa is effective and well tolerated when used in the home setting to treat mild to moderate bleeding episodes in patients with haemophilia A or B with inhibitors.

A structural comparison of molybdenum cofactor-containing enzymes.

This work gives an overview of the recent achievements which have contributed to the understanding of the structure and function of molybdenum and tungsten enzymes. Known structures of molybdo-pterin cofactor-containing enzymes will be described briefly and the structural differences between representatives of the same and different families will be analyzed. This comparison will show that the molybdo-pterin cofactor-containing enzymes represent a very heterogeneous group with differences in overall enzyme structure, cofactor composition and stoichiometry, as well as differences in the immediate molybdenum environment. Two recently discovered molybdo-pterin cofactor-containing enzymes will be described with regard to molecular and EPR spectroscopic properties, pyrogallol-phloroglucinol transhydroxylase from Pelobacter acidigallici and acetylene hydratase from Pelobacter acetylenicus. On the basis of its amino acid sequence, transhydroxylase can be classified as a member of the dimethylsulfoxide reductase family, whereas classification of the tungsten/molybdenum-containing acetylene hydratase has to await the determination of its amino acid sequence.

Shared management of children with cancer.

OBJECTIVE: To determine the risks and benefits of university-based pediatric oncologists and community-based primary care physicians sharing the management of children with cancer. DESIGN: Physicians participating in shared management of children with cancer were surveyed, and the outcomes of the children were measured. SETTING AND PARTICIPANTS: One hundred thirty-seven community-based primary care physicians participated in the management of the 226 children with cancer in Iowa and western Illinois during the past 15 years. The survival of the 226 children was compared with that of 240 randomly selected children treated using the identical treatment protocols but treated only by pediatric oncologists. INTERVENTION: A 7-point Likert scale questionnaire was completed by 97 (71%) of the participating primary care physicians. RESULTS AND OUTCOME MEASURES: There were no differences in the survival of children using shared management compared with those treated only by pediatric oncologists. Primary care physicians believed that shared management is of economic and psychosocial benefit to patients, improves the treatment choices available to patients, does not require excessive time, and does not result in loss of practice income. The system strengthens the primary care physicians' relationships with oncologists and results in additional referrals to the university-based pediatric oncologists. It is of educational value, is personally satisfying, and provides relief from the stress associated with caring for these families. Primary care physicians would like to see this system expanded to include other children with special health care needs. CONCLUSION: The shared-management approach to care is a viable attractive option of health care provision for children.