Apathy predicts rate of cognitive decline over 24 months in premanifest Huntington's disease.

BACKGROUND: Cognitive impairment is a core feature of Huntington's disease (HD), however, the onset and rate of cognitive decline is highly variable. Apathy is the most common neuropsychiatric symptom of HD, and is associated with cognitive impairment. The aim of this study was to investigate apathy as a predictor of subsequent cognitive decline over 2 years in premanifest and early HD, using a prospective, longitudinal design. METHODS: A total of 118 premanifest HD gene carriers, 111 early HD and 118 healthy control participants from the multi-centre TRACK-HD study were included. Apathy symptoms were assessed at baseline using the apathy severity rating from the Short Problem Behaviours Assessment. A composite of 12 outcome measures from nine cognitive tasks was used to assess cognitive function at baseline and after 24 months. RESULTS: In the premanifest group, after controlling for age, depression and motor signs, more apathy symptoms predicted faster cognitive decline over 2 years. In contrast, in the early HD group, more motor signs, but not apathy, predicted faster subsequent cognitive decline. In the control group, only older age predicted cognitive decline. CONCLUSIONS: Our findings indicate that in premanifest HD, apathy is a harbinger for cognitive decline. In contrast, after motor onset, in early diagnosed HD, motor symptom severity more strongly predicts the rate of cognitive decline.

Functionality of the three-site ferroxidase center of Escherichia coli bacterial ferritin (EcFtnA).

At least three ferritins are found in the bacterium Escherichia coli : the heme-containing bacterioferritin (EcBFR) and two nonheme bacterial ferritins (EcFtnA and EcFtnB). In addition to the conserved A and B sites of the diiron ferroxidase center, EcFtnA has a third iron-binding site (the C site) of unknown function that is nearby the diiron site. In the present work, the complex chemistry of iron oxidation and deposition in EcFtnA was further defined through a combination of oximetry, pH stat, stopped-flow and conventional kinetics, UV-vis, fluorescence, and EPR spectroscopic measurements on both the wild-type protein and site-directed variants of the A, B, and C sites. The data reveal that although H2O2 is a product of dioxygen reduction in EcFtnA and oxidation occurs with a stoichiometry of Fe(2+)/O2 ∼ 3:1 most of the H2O2 produced is consumed in subsequent reactions with a 2:1 Fe(2+)/H2O2 stoichiometry, thus suppressing hydroxyl-radical formation. Although the A and B sites are essential for rapid iron oxidation, the C site slows oxidation and suppresses iron turnover at the ferroxidase center. A tyrosyl radical, assigned to Tyr24 near the ferroxidase center, is formed during iron oxidation, and its possible significance to the function of the protein is discussed. Taken as a whole, the data indicate that there are multiple iron-oxidation pathways in EcFtnA with O2 and H2O2 as oxidants. Furthermore, our data do not support a universal mechanism for iron oxidation in all ferritins whereby the C site acts as transit site, as has been recently proposed.

Phase I clinical trial of the Src inhibitor dasatinib with dacarbazine in metastatic melanoma.

BACKGROUND: Src inhibitors sensitise melanoma cells to chemotherapy in preclinical models. The combination of dasatinib and dacarbazine was tested in a phase I trial in melanoma. METHODS: Patients had ECOG performance status 0-2 and normal organ function. Dacarbazine was administered on day 1 and dasatinib on day 2 through 19 of each 21-day cycle. Both were escalated from 50 mg b.i.d. of dasatinib and 800 mg m(-2) of dacarbazine. Available pre-treatment biopsies were sequenced for BRAF, NRAS, and C-Kit mutations. RESULTS: Dose-limiting toxicity was reached at dasatinib 70 mg b.i.d./dacarbazine 1000 mg m(-2), and was predominantly haematological. In 29 patients receiving dasatinib 70 mg b.i.d., the objective response rate (ORR) was 13.8%, the clinical benefit rate (ORR+SD) was 72.4%, the 6-month progression-free survival (PFS) was 20.7%, and the 12-month overall survival (OS) was 34.5%. Two out of three patients who were wild type for BRAF, NRAS, and c-KIT mutations had confirmed partial responses, and one had a minor response. CONCLUSION: The recommended phase II dose is dasatinib 70 mg b.i.d with dacarbazine 800 mg m(-2). PFS and OS data for dasatinib at 70 mg b.i.d. with dacarbazine compared favourably with historical controls. Preliminary data support evaluating tumour mutation status further as a biomarker of response.

Draft genome sequence of a uropathogenic Escherichia coli isolate (ST38 O1:H15) from Pakistan, an emerging multidrug-resistant sequence type with a high virulence profile.

Sequence type 38 is considered a uropathogenic Escherichia coli/enteroaggregative E. coli hybrid associated with multidrug resistance and urinary tract infections. The draft genome sequence of UEC59 from a woman in Pakistan revealed a 5 324 938 bp genome with 5386 coding sequences (CDS), 86 transfer RNA genes and multiple antibiotic resistance genes (bla TEM-1, CMY-2, sul1, sul2, dfrA17, tetA, mphA) and mobile elements (int1, two transposons, 30 insertion sequence elements, one integrative conjugative element, four plasmids, five prophages), along with many virulence genes.

Steady-state nonlinear pharmacokinetics of 5-fluorouracil during hepatic arterial and intravenous infusions in cancer patients.

Hepatic arterial catheters were placed for therapy in 8 patients with primary or metastatic liver cancer. Temporary hepatic venous catheters allowed direct sampling of blood for hepatic venous drug concentrations. Patients were administered from three to six infusions at rates of 10, 30, 90, 135, 180, 210, and 270 mg/kg/day (0.053 to 1.43 microM/kg/min), given over 2 h, of 5-fluorouracil (FUra). In Method 1, FUra was infused i.v., and FUra was measured in plasma from hepatic arterial and hepatic venous blood. In Method 2, FUra was given i.v. at one time and infused into hepatic arterial blood at another time, and FUra was measured in plasma from peripheral blood at the same site in both cases. Steady-state FUra plasma concentrations were measured by a sensitive and specific high-performance liquid chromatography method. Data were computer fitted to the equations appropriate for a physiological two-compartment flow model with Michaelis-Menten elimination from the peripheral compartment and blood flow rate, Q, between the central and peripheral compartment. Methods 1 and 2 gave mean Vmax and Km values which did not differ significantly; the overall mean Vmax was 2.02 microM/kg/min, and the overall mean Km was 10.9 microM. For Method 1 the mean Q1 value was 0.0803 liters/(kg X min) or 5.26 liters/min, which is the same as cardiac output, but for Method 2 the mean Q2 value was higher, namely 0.189 liters/(kg X min) or 13.0 liters/min. Steady-state systemic and intrinsic clearances and extraction ratios decreased progressively as the dose rate increased. Intra- and inter-subject variation of both Vmax and Km were of the same order of magnitude. As a result, dose rate escalation should be conservative for dose rates above 135 mg/kg/day. The results support hepatic arterial infusion as a means of improving the therapeutic index of FUra in the treatment of cancer of the liver.

Physical, chemical and immunological properties of the bacterioferritins of Escherichia coli, Pseudomonas aeruginosa and Azotobacter vinelandii.

The 70-amino-acid-residue N-terminal sequence of the bacterioferritin (BFR) of Azotobacter vinelandii was determined and shown to be highly similar to the N-terminal sequences of the Escherichia coli and Nitrobacter winogradskyi bacterioferritins. Electrophoretic and immunological analyses further indicate that the bacterioferritins of E. coli, A. vinelandii and Pseudomonas aeruginosa are closely related. A novel, two-subunit assembly state that predominates over the 24-subunit form of BFR at low pH was demonstrated. The results indicate that the bacterioferritins form a family of proteins that are distinct from the ferritins of plants and animals.

The high-resolution X-ray crystallographic structure of the ferritin (EcFtnA) of Escherichia coli; comparison with human H ferritin (HuHF) and the structures of the Fe(3+) and Zn(2+) derivatives.

The high-resolution structure of the non-haem ferritin from Escherichia coli (EcFtnA) is presented together with those of its Fe(3+) and Zn(2+) derivatives, this being the first high-resolution X-ray analysis of the iron centres in any ferritin. The binding of both metals is accompanied by small changes in the amino acid ligand positions. Mean Fe(A)(3+)-Fe(B)(3+) and Zn(A)(2+)-Zn(B)(2+) distances are 3.24 A and 3.43 A, respectively. In both derivatives, metal ions at sites A and B are bridged by a glutamate side-chain (Glu50) in a syn-syn conformation. The Fe(3+) derivative alone shows a third metal site (Fe( C)( 3+)) joined to Fe(B)(3+) by a long anti-anti bidentate bridge through Glu130 (mean Fe(B)(3+)-Fe(C)(3+) distance 5.79 A). The third metal site is unique to the non-haem bacterial ferritins. The dinuclear site lies at the inner end of a hydrophobic channel connecting it to the outside surface of the protein shell, which may provide access for dioxygen and possibly for metal ions shielded by water. Models representing the possible binding mode of dioxygen to the dinuclear Fe(3+) pair suggest that a gauche micro-1,2 mode may be preferred stereochemically. Like those of other ferritins, the 24 subunits of EcFtnA are folded as four-helix bundles that assemble into hollow shells and both metals bind at dinuclear centres in the middle of the bundles. The structural similarity of EcFtnA to the human H chain ferritin (HuHF) is remarkable (r.m.s. deviation of main-chain atoms 0.66 A) given the low amino acid sequence identity (22 %). Many of the conserved residues are clustered at the dinuclear centre but there is very little conservation of residues making inter-subunit interactions.

Iron storage in bacteria.

Iron is an essential nutrient for nearly all organisms but presents problems of toxicity, poor solubility and low availability. These problems are alleviated through the use of iron-storage proteins. Bacteria possess two types of iron-storage protein, the haem-containing bacterioferritins and the haem-free ferritins. These proteins are widespread in bacteria, with at least 39 examples known so far in eubacteria and archaebacteria. The bacterioferritins and ferritins are distantly related but retain similar structural and functional properties. Both are composed of 24 identical or similar subunits (approximately 19 kDa) that form a roughly spherical protein (approximately 450 kDa, approximately 120 A diameter) containing a large hollow centre (approximately 80 A diameter). The hollow centre acts as an iron-storage cavity with the capacity to accommodate at least 2000 iron atoms in the form of a ferric-hydroxyphosphate core. Each subunit contains a four-helix bundle which carries the active site or ferroxidase centre of the protein. The ferroxidase centres endow ferrous-iron-oxidizing activity and are able to form a di-iron species that is an intermediate in the iron uptake, oxidation and core formation process. Bacterioferritins contain up to 12 protoporphyrin IX haem groups located at the two-fold interfaces between pairs of two-fold related subunits. The role of the haem is unknown, although it may be involved in mediating iron-core reduction and iron release. Some bacterioferritins are composed of two subunit types, one conferring haem-binding ability (alpha) and the other (beta) bestowing ferroxidase activity. Bacterioferritin genes are often adjacent to genes encoding a small [2Fe-2S]-ferredoxin (bacterioferritin-associated ferredoxin or Bfd). Bfd may directly interact with bacterioferritin and could be involved in releasing iron from (or delivering iron to) bacterioferritin or other iron complexes. Some bacteria contain two bacterioferritin subunits, or two ferritin subunits, that in most cases co-assemble. Others possess both a bacterioferritin and a ferritin, while some appear to lack any type of iron-storage protein. The reason for these differences is not understood. Studies on ferritin mutants have shown that ferritin enhances growth during iron starvation and is also involved in iron accumulation in the stationary phase of growth. The ferritin of Campylobacter jejuni is involved in redox stress resistance, although this does not appear to be the case for Escherichia coli ferritin (FtnA). No phenotype has been determined for E. coli bacterioferritin mutants and the precise role of bacterioferritin in E. coli remains uncertain.

Bacterioferritins and ferritins are distantly related in evolution. Conservation of ferroxidase-centre residues.

Iron-storage proteins can be divided into two classes; the bacterioferritins and ferritins. In spite of many apparent structural and functional analogies, no significant amino acid sequence similarity has been detected previously. This report now reveals a distant evolutionary relationship between bacterioferritins and ferritins derived by 'Profile Analysis'. Optimum alignment of bacterioferritin and ferritin sequences suggests that key residues of the ferroxidase centres of ferritins are conserved in bacterioferritins.

Charge compensated binding of divalent metals to bacterioferritin: H+ release associated with cobalt(II) and zinc(II) binding at dinuclear metal sites.

Divalent metal ion binding to the bacterial iron-storage protein, bacterioferritin (BFR), which contains a dinuclear metal binding site within each of its 24 subunits, was investigated by potentiometric and spectrophotometric methods. Cobalt(II) and zinc(II) were found to bind at both high- and low-affinity sites. Cobalt(II) binding at the high-affinity site was observed at a level of two per subunit with the release of approximately 1.6 protons per metal ion, thus confirming the dinuclear metal centre as the high-affinity site. Zinc(II) binding at the dinuclear centre (high-affinity site) resulted in the release of approximately 2 protons per metal ion, but exhibited a binding stoichiometry which indicated that not all dinuclear centres were capable of binding two zinc(II) ions. Competition data showed that binding affinities for the dinuclear centre were in the order zinc(II) > cobalt(II), and also confirmed the unexpected stoichiometry of zinc(II) binding. This work emphasises the importance of charge neutrality at the dinuclear centre.

Direct observation of the iron binding sites in a ferritin.

X-Ray analysis of the ferritin of Escherichia coli (Ec-FTN) and of Ec-FTN crystals soaked in (NH4)2Fe(SO4)2 has revealed the presence of three iron-binding sites per subunit. Two of these form a di-iron site in the centre of the subunit as has been proposed for the 'ferroxidase centres' of human ferritin H chains. This di-iron site, lying within the 4-alpha-helix bundle, resemble those of ribonucleotide reductase, methane monoxygenase and haemerythrin. The third iron is bound by ligands unique to Ec-FTN on the inner surface of the protein shell. It is speculated that this state may represent the nucleation centre of a novel type of Fe(III) cluster, recently observed in Ec-FTN.

The haemoglobin-like protein (HMP) of Escherichia coli has ferrisiderophore reductase activity and its C-terminal domain shares homology with ferredoxin NADP+ reductases.

Three soluble ferrisiderophore reductases (FsrA, FsrB and FsrC) were detected in Escherichia coli. FsrB was purified and identified as the haemoglobin-like protein (HMP) by size and N-terminal sequence analyses. HMP was previously isolated as a dihydropteridine reductase and is now shown to have ferrisiderophore reductase activity. Database searches revealed that the C-terminal region of HMP (FsrB) is homologous to members of a family of flavoprotein oxidoreductases which includes ferredoxin NADP+ reductase (FNR). The combination of FNR-like and haemoglobin-like regions in HMP (FsrB) represents a novel pairing of functionally and structurally distinct domains. Structure-function properties of other FNR-like proteins, including LuxG and VanB, are also discussed.

An EPR investigation of non-haem iron sites in Escherichia coli bacterioferritin and their interaction with phosphate. A study using nitric oxide as a spin probe.

EPR studies of bacterioferritin (BFR), an iron-storage protein of Escherichia coli [1993, Biochem. J. 292, 47-56], have revealed the presence of non-haem iron (III) (NHI) sites within the protein coat which may be involved in iron uptake and release. When nitric oxide was used as an EPR spin probe of the Fe(II) state of the NHI sites, two distinct mononuclear NHI species were found. Under certain conditions, an iron dimer was also observed. The reaction of phosphate with NHI species has been investigated. Results point to a function for this anion in core nucleation.

Kinetic and structural characterization of an intermediate in the biomineralization of bacterioferritin.

The mechanism by which iron-storage proteins take up and oxidise iron(II) is not understood. We show by rapid-kinetic and EPR measurements that iron uptake, in vitro, by a bacterial iron-storage protein, bacterioferritin, involves at least three kinetically distinguishable phases: phase 1, the binding of Fe(II) ions, probably at a dimeric iron ferroxidase centre; phase 2, oxidation of the Fe(II) dimer and production of mononuclear Fe(III); and phase 3, iron core formation.

Treatment of cancers involving the liver and porta hepatis with external beam irradiation and intraarterial hepatic fluorodeoxyuridine.

A Phase I/II clinical trial was designed for patients with malignancies of the liver and porta hepatis. This protocol employed three concepts: a) boost treatment to gross tumor within the liver for selected patients, determined by the dose-volume histogram (DVH) of the normal liver that would be irradiated by boost treatment; b) concurrent use of intraarterial hepatic 5-fluorodeoxyuridine (FdUrd) as a radiosensitizer; and c) hyperfractionation (1.5 Gy fractions given bid greater than 4 hr apart). This report describes the results of treatment of the first 33 patients entered onto this study, with a minimum follow-up of 1 year. Twenty patients received only whole liver irradiation (33 Gy). Thirteen patients were treated with whole liver irradiation (30 Gy) plus a 15 Gy (6 patients) or 30 Gy (7 patients) boost (total 45 Gy and 60 Gy to the tumor, respectively). Forty-eight percent of the evaluable patients (14/29) had an objective response, based on CT scan. The median duration of response was 8 months. The chief toxicities were fatigue, nausea, gastritis, and diarrhea, which were less than or equal to grade 2 in severity. Two patients developed mild radiation hepatitis which was treated successfully with diuretics. These data suggest that the treatment of intrahepatic malignancies can be guided by the concept of DVH analysis of the normal liver to allow the safe administration of doses of radiation that are potentially tumoricidal and are well above those that would be predicted to be tolerable for the whole liver.

Topological analysis of DctQ, the small integral membrane protein of the C4-dicarboxylate TRAP transporter of Rhodobacter capsulatus.

Tripartite ATP-independent periplasmic ('TRAP') transporters are a novel group of bacterial and archaeal secondary solute uptake systems which possess a periplasmic binding protein, but which are unrelated to ATP-binding cassette (ABC) systems. In addition to the binding protein, TRAP transporters contain two integral membrane proteins or domains, one of which is 40-50 kDa with 12 predicted transmembrane (TM) helices, thought to be the solute import protein, while the other is 20-30 kDa and of unknown function. Using a series of plasmid-encoded beta-lactamase fusions, we have determined the topology of DctQ, the smaller integral membrane protein from the high-affinity C4-dicarboxylate transporter of Rhodobacter capsulatus, which to date is the most extensively characterised TRAP transporter. DctQ was predicted by several topology prediction programmes to have four TM helices with the N- and C-termini located in the cytoplasm. The levels of ampicillin resistance conferred by the fusions when expressed in Escherichia coli were found to correlate with this predicted topology. The data have provided a topological model which can be used to test hypotheses concerning the function of the different regions of DctQ and which can be applied to other members of the DctQ family.

Isolation of a ferritin from Bacteroides fragilis.

A ferritin was isolated from the obligate anaerobe Bacteroides fragilis. Estimated molecular masses were 400 kDa for the holomer and 16.7 kDa for the subunits. A 30-residue N-terminal amino acid sequence was determined and found to resemble the sequences of other ferritins (human H-chain ferritin, 43% identity; Escherichia coli gen-165 product, 37% identity) and to a lesser degree, bacterioferritins (E. coli bacterioferritin, 20% identity). The protein stained positively for iron, and incorporated 59Fe when B. fragilis was grown in the presence of [59Fe]citrate. However, the isolated protein contained only about three iron atoms per molecule, and contained no detectable haem. This represents the first isolation of a ferritin protein from bacteria. It may alleviate iron toxicity in the presence of oxygen.

Structure, function, and evolution of ferritins.

The ferritins of animals and plants and the bacterioferritins (BFRs) have a common iron-storage function in spite of differences in cytological location and biosynthetic regulation. The plant ferritins and BFRs are more similar to the H chains of mammals than to mammalian L chains, with respect to primary structure and conservation of ferroxidase center residues. Hence they probably arose from a common H-type ancestor. The recent discovery in E. coli of a second type of iron-storage protein (FTN) resembling ferritin H chains raises the question of what the relative roles of these two proteins are in this organism. Mammalian L ferritins lack ferroxidase centers and form a distinct group. Comparison of the three-dimensional structures of mammalian and invertebrate ferritins, as well as computer modeling of plant ferritins and of BFR, indicate a well conserved molecular framework. The characterisation of numerous ferritin homopolymer variants has allowed the identification of some of the residues involved in iron uptake and an investigation of some of the functional differences between mammalian H and L chains.

Caregiver burden and symptom distress in people with cancer receiving hospice care.

PURPOSE/OBJECTIVES: To examine the relationship between caregiver burden and symptom distress in patients with terminal cancer who are enrolled in hospice. DESIGN: Descriptive, quantitative. SETTING: A large, metropolitan, nonprofit-based organization in west central Florida. SAMPLE: Convenience sample of 30 patient-caregiver dyads enrolled in hospice. METHODS: Caregivers completed the Caregiver Reaction Scale to measure the level of caregiver burden; patients completed the Adopted Symptom Distress Scale. Results were correlated using a Pearson correlation. MAIN RESEARCH VARIABLES: Symptom distress and caregiver burden. FINDINGS: The patient sample exhibited low symptom distress, and the caregiver sample exhibited moderate caregiver burden. A statistically significant moderate correlation existed between symptom distress and caregiver burden. CONCLUSIONS: The significant moderate correlation confirms the idea that caregiver burden and patient symptom distress are related. Future studies are needed to obtain a more representative sample of caregivers of patients closer to death, even if those patients are nonresponsive. IMPLICATIONS FOR NURSING PRACTICE: This information can assist hospice nurses in assessing and formulating targeted care for symptom distress and caregiver burden in their patients,

The expression of ferritin, lactoferrin, transferrin receptor and solute carrier family 11A1 in the host response to BCG-vaccination and Mycobacterium tuberculosis challenge.

Iron is an essential cofactor for both mycobacterial growth during infection and for a successful protective immune response by the host. The immune response partly depends on the regulation of iron by the host, including the tight control of expression of the iron-storage protein, ferritin. BCG vaccination can protect against disease following Mycobacterium tuberculosis infection, but the mechanisms of protection remain unclear. To further explore these mechanisms, splenocytes from BCG-vaccinated guinea pigs were stimulated ex vivo with purified protein derivative from M. tuberculosis and a significant down-regulation of ferritin light- and heavy-chain was measured by reverse-transcription quantitative-PCR (P≤0.05 and ≤0.01, respectively). The mechanisms of this down-regulation were shown to involve TNFα and nitric oxide. A more in depth analysis of the mRNA expression profiles, including genes involved in iron metabolism, was performed using a guinea pig specific immunological microarray following ex vivo infection with M. tuberculosis of splenocytes from BCG-vaccinated and naïve guinea pigs. M. tuberculosis infection induced a pro-inflammatory response in splenocytes from both groups, resulting in down-regulation of ferritin (P≤0.05). In addition, lactoferrin (P≤0.002), transferrin receptor (P≤0.05) and solute carrier family 11A1 (P≤0.05), were only significantly down-regulated after infection of the splenocytes from BCG-vaccinated animals. The results show that expression of iron-metabolism genes is tightly regulated as part of the host response to M. tuberculosis infection and that BCG-vaccination enhances the ability of the host to mount an iron-restriction response which may in turn help to combat invasion by mycobacteria.