The right time to measure anti-Xa activity in critical illness: pharmacokinetics of therapeutic dose nadroparin.

Background: Peak anti-Xa activity of low-molecular-weight heparin nadroparin is measured 3 to 5 hours after subcutaneous injection. In critically ill patients, physiological changes and medical therapies may result in peak activities before or after this interval, possibly impacting dosing. Objectives: The primary objective was to determine the percentage of critically ill patients with adequately estimated peak activities drawn 3 to 5 hours after subcutaneous administration of a therapeutic dose of nadroparin. Adequate was defined as a peak activity of ≥80% of the actual peak anti-Xa activity. If ≥80% of patients had adequately estimated peak activities in the 3- to 5-hour interval, measurement in this interval was regarded as acceptable. The secondary objective was to determine the pharmacokinetic profile of nadroparin. Methods: In this single-center, prospective study, we evaluated anti-Xa activities in patients admitted to a general intensive care unit. After ≥4 equal doses of nadroparin, anti-Xa activity was measured according to a 12- to 24-hour sampling scheme. Results: In 25 patients, anti-Xa activities drawn between 3 and 5 hours after administration ranged 80% to 100% of the actual peak activity. Compared to the threshold level of an adequate estimation in at least 20 patients (≥80%), measuring anti-Xa activities in the 3- to 5-hour interval is an acceptable method (1-tailed binomial test; P < .02). We found a large interindividual variability for nadroparin exposure (mean ± SD area-under-the-curve0-12h, 10.3 ± 4.8 IU·h/mL) and delayed elimination (t1/2 range, 4.0-120.9 hours) despite adequate renal function. Conclusion: In critically ill patients, measuring anti-Xa activity in a 3- to 5-hour interval after subcutaneous injection of therapeutic nadroparin is an acceptable method to estimate the actual peak anti-Xa activity.

The ß6/ß7 region of the Hsp70 substrate-binding domain mediates heat-shock response and prion propagation.

Hsp70 is a highly conserved chaperone that in addition to providing essential cellular functions and aiding in cell survival following exposure to a variety of stresses is also a key modulator of prion propagation. Hsp70 is composed of a nucleotide-binding domain (NBD) and substrate-binding domain (SBD). The key functions of Hsp70 are tightly regulated through an allosteric communication network that coordinates ATPase activity with substrate-binding activity. How Hsp70 conformational changes relate to functional change that results in heat shock and prion-related phenotypes is poorly understood. Here, we utilised the yeast [PSI +] system, coupled with SBD-targeted mutagenesis, to investigate how allosteric changes within key structural regions of the Hsp70 SBD result in functional changes in the protein that translate to phenotypic defects in prion propagation and ability to grow at elevated temperatures. We find that variants mutated within the ß6 and ß7 region of the SBD are defective in prion propagation and heat-shock phenotypes, due to conformational changes within the SBD. Structural analysis of the mutants identifies a potential NBD:SBD interface and key residues that may play important roles in signal transduction between domains. As a consequence of disrupting the ß6/ß7 region and the SBD overall, Hsp70 exhibits a variety of functional changes including dysregulation of ATPase activity, reduction in ability to refold proteins and changes to interaction affinity with specific co-chaperones and protein substrates. Our findings relate specific structural changes in Hsp70 to specific changes in functional properties that underpin important phenotypic changes in vivo. A thorough understanding of the molecular mechanisms of Hsp70 regulation and how specific modifications result in phenotypic change is essential for the development of new drugs targeting Hsp70 for therapeutic purposes.

Clinical Course of Two Children with Unstable Hemoglobins: The Effect of Hydroxyurea Therapy.

Case reports on the effect of hydroxyurea (HU) therapy for unstable hemoglobins (Hbs) are sparse; only three adult cases have been reported. We report for the first time on the effect of HU therapy in children carrying unstable Hbs. The first case concerns a female child with a familial history of chronic hemolytic anemia. She was diagnosed with Hb Volga (HBB: c.83C>A) at the age of 7 months. At age 6, treatment options were reconsidered due to increasing fatigue and decreasing Hb concentration. The second case also concerns a female child with chronic hemolytic anemia and icterus since the age of 5. She was diagnosed with Hb Köln (HBB: c.295G>A) at the age of 9. At age 10, treatment options were reconsidered due to decreased general condition and poor school performance. Both children were started on HU therapy. The child with Hb Volga showed reduced clinical symptoms and increased average Hb concentrations. She has been on HU therapy for over 7 years at preparation of this manuscript. The child with Hb Köln showed decreasing Hb concentrations upon start of therapy; clinical symptoms did not improve. Therapy was discontinued after 3½ months. The Hb Volga case report suggests that HU therapy could improve clinical symptoms in some patients with unstable Hbs. Based on these and previously published cases, it was speculated that response can be predicted by the percentage of Hb F and reticulocyte counts.

Polymorphisms of the LEP, LEPR and HTR2C gene: obesity and BMI change in patients using antipsychotic medication in a naturalistic setting.

UNLABELLED: Weight gain is a frequently occurring serious somatic adverse effect of atypical antipsychotic agents. Genetic factors influence the risk of an individual to gain weight. OBJECTIVES: To determine whether LEPR Q223R, LEP -2548G/A and HTR2C -759C/T polymorphisms are associated with obesity and weight change in patients using atypical antipsychotic drugs. METHODS: A longitudinal study design was used in a naturalistic setting. The study population included 141 patients, all of whom were using an atypical antipsychotic drug. The body mass index was measured twice. Primary outcome measures were obesity at the moment of first measurement and body mass index change during treatment. Determinants were the LEPR Q223R (rs1137101), the LEP -2548G/A SNP (rs7799039) and the HTR2C -759C/T (rs3813929) polymorphisms. RESULTS: Of the 141 included patients, 35 (24.8%) were obese. In females, presence of the LEPR 223R allele was associated with an increased risk of obesity (47.6 vs 17.6%; p = 0.03). In males this association was not found. None of the SNPs were significantly associated with weight change during treatment. CONCLUSIONS: The LEPR Q223R polymorphism may be a risk factor for obesity in women with a psychotic disorder treated with atypical antipsychotic drugs. This is in line with earlier findings of our group.

Association between LEP and LEPR gene polymorphisms and dyslipidemia in patients using atypical antipsychotic medication.

BACKGROUND: Treatment with atypical antipsychotic agents is often complicated by dyslipidemia, which is a risk factor for cardiovascular disease. OBJECTIVES: To determine whether the LEPR Q223R, the LEP -2548G/A, and the HTR2C -759C/T polymorphisms are associated with dyslipidemia in patients using atypical antipsychotic drugs. METHODS: A cross-sectional study design was used. The study population included all patients who had been screened for dyslipidemia between January 2008 and March 2009 and had been using an atypical antipsychotic for at least 3 months at the moment of screening. Primary outcome measure was the mean total cholesterol (TC)/HDL ratio. Determinants were the LEPR Q223R (rs1137101), the LEP -2548G/A SNP (rs7799039), and the HTR2C -759C/T (rs3813929) polymorphisms. RESULTS: A total of 353 patients was included in the study, of which 184 (52.1%) were men and 169 (47.9%) were women. Overall, no significant differences were found between the different genotype groups. However, in patients with a first admission to the hospital less than a year ago, the LEP -2548G allele had a lower mean TC/HDL ratio compared with patients without the LEP -2548G allele (6.41 vs. 4.12, P-adj: 0.017) and patients with the LEPR 223R allele had a lower mean TC/HDL ratio compared with patients without the LEPR 223R allele (5.04 vs. 3.92, P-adj: 0.019). The association between the LEP -2548G/A allele and the TC/HDL ratio in recent patients was present only in men. CONCLUSION: Genetic variation in the LEP and LEPR gene may be associated with short-term dyslipidemia in patients using atypical antipsychotic agents.

Implementation of CYP2D6 genotyping in psychiatry.

BACKGROUND: Response to pharmacotherapy is subject to large inter-individual variation. Most medicine is metabolised by CYP450 enzymes, including CYP2D6. CYP2D6 activity is determined substantially by the genetic composition. Polymorphisms of CYP2D6 are associated with inadequate response on treatment with psychotropics. The large number of polymorphisms of CYP2D6 raises the question of the way associations between genotype and clinical outcome measurements should be analysed and implemented into a clinical setting. OBJECTIVE: Several methods used for grouping CYP2D6 genotypes are discussed, including the Predicted Phenotype method, the Semi-quantitative Gene Dose system and the Activity Score system. Possible ways to translate genetic information into dose recommendations and to implement genetic information into a clinical setting are discussed. CONCLUSIONS: A 100% accurate prediction of metabolic activity is not possible due to the effect of non-genetic factors on metabolic activity. When used for clinical purposes, none of the methods for grouping CYP2D6 genotypes has substantial advantages. The Semi-quantitative Gene Dose or Activity Score system might be preferred for research purposes. Large clinical studies on cost-efficiency will be needed to assure the cost-efficiency of routinely genotyping CYP2D6. Creating more awareness among prescribers of psychotropics about the genetic causes for aberrant drug response, and the possibility of genotyping, is desirable.

Alcohol oxidase (AOX1) from Pichia pastoris is a novel inhibitor of prion propagation and a potential ATPase.

Previous results suggest that methylotrophic yeasts may contain factors that modulate prion stability. Alcohol oxidase (AOX), a key enzyme in methanol metabolism, is an abundant protein that is specific to methylotrophic yeasts. We examined the effect of Pichia pastoris AOX1 on prion phenotypes in Saccharomyces cerevisiae. The S. cerevisiae prion states [PSI(+)] and [URE3] arise from aggregation of the proteins Sup35p and Ure2p respectively, and correlate with the ability of Sup35p and Ure2p to form amyloid-like fibrils in vitro. We found that expression of P. pastoris AOX1 in S. cerevisiae had no effect on propagation of the [PSI(+)] prion, but inhibited propagation of [URE3]. Addition of AOX1 early in the time-course of fibril formation inhibits Ure2p fibril formation in vitro. AOX1 has not previously been identified as an ATPase. However, we discovered that in addition to its flavin adenine dinucleotide-dependent AOX activity, AOX1 possesses ATPase activity. This study identifies AOX1 as a novel prion inhibitory factor and a potential ATPase.

Semi-quantitative CYP2D6 gene doses in relation to metabolic ratios of psychotropics.

PURPOSE: Genetic polymorphisms in cytochrome P450 (CYP) enzyme CYP2D6 have a substantial effect on the success of pharmacotherapy. Different models, including a predicted-phenotype model and a semi-quantitative gene dose (SGD) model, have been developed based on CYP genotype. The objective of this study was to investigate the surplus value of the SGD model in predicting the metabolic ratios (MRs) of the psychotropics venlafaxine, fluoxetine and risperidone. METHODS: Phenotype prediction and semi-quantitative gene doses were conducted after genotyping for CYP2D6 *3, *4, *5, *6, *9, *10, *41 and gene multiplication. RESULTS: The predicted-phenotype and SGD model showed increasing mean MRs with increasing predicted metabolic activity and decreasing SGD values, respectively, for all three psychotropics. The reliability of MR prediction was higher for the SGD model. CONCLUSIONS: Both models are suitable for venlafaxine, fluoxetine and risperidone. In this study, a surplus value of semi-quantitative gene dose model was present, but small, for all three psychotropics.

Regulation of phagocytosis in Dictyostelium by the inositol 5-phosphatase OCRL homolog Dd5P4.

Phosphoinositides are involved in endocytosis in both mammalian cells and the amoeba Dictyostelium discoideum. Dd5P4 is the Dictyostelium homolog of human OCRL (oculocerebrorenal syndrome of Lowe); both have a RhoGAP domain and a 5-phosphatase domain that acts on phosphatidylinositol 4,5-bisphosphate/phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3). Inactivation of Dd5P4 inhibits growth on liquid medium and on bacteria. Dd5p4-null cells are impaired in phagocytosis of yeast cells. In wild-type cells, PI(3,4,5)P3 is formed and converted to PI(3,4)P2 just before closure of the phagocytic cup. In dd5p4-null cells, a phagocytic cup is formed upon contact with the yeast cell, and PI(3,4,5)P3 is still produced, but the phagocytic cup does not close. We suggest that Dd5P4 regulates the conversion of PI(3,4,5)P3 to PI(3,4)P2 and that this conversion is essential for closure of the phagocytic cup. Phylogenetic analysis of OCRL-like 5-phosphatases with RhoGAP domains reveal that D. discoideum Dd5P4 is a surprisingly close homolog of human OCRL, the protein responsible for Lowe syndrome. We expressed human OCRL in dd5p4-null cells. Growth on bacteria and axenic medium is largely restored, whereas the rate of phagocytosis of yeast cells is partly restored, indicating that human OCRL can functionally replace Dictyostelium Dd5P4.

Hsp40 interacts directly with the native state of the yeast prion protein Ure2 and inhibits formation of amyloid-like fibrils.

Ure2 is the protein determinant of the [URE3] prion phenotype in Saccharomyces cerevisiae and consists of a flexible N-terminal prion-determining domain and a globular C-terminal glutathione transferase-like domain. Overexpression of the type I Hsp40 member Ydj1 in yeast cells has been found to result in the loss of [URE3]. However, the mechanism of prion curing by Ydj1 remains unclear. Here we tested the effect of overexpression of Hsp40 members Ydj1, Sis1, and Apj1 and also Hsp70 co-chaperones Cpr7, Cns1, Sti1, and Fes1 in vivo and found that only Ydj1 showed a strong curing effect on [URE3]. We also investigated the interaction of Ydj1 with Ure2 in vitro. We found that Ydj1 was able to suppress formation of amyloid-like fibrils of Ure2 by delaying the process of fibril formation, as monitored by thioflavin T binding and atomic force microscopy imaging. Controls using bovine serum albumin, Sis1, or the human Hsp40 homologues Hdj1 or Hdj2 showed no significant inhibitory effect. Ydj1 was only effective when added during the lag phase of fibril formation, suggesting that it interacts with Ure2 at an early stage in fibril formation and delays the nucleation process. Using surface plasmon resonance and size exclusion chromatography, we demonstrated a direct interaction between Ydj1 and both wild type and N-terminally truncated Ure2. In contrast, Hdj2, which did not suppress fibril formation, did not show this interaction. The results suggest that Ydj1 inhibits Ure2 fibril formation by binding to the native state of Ure2, thus delaying the onset of oligomerization.

Importance of the Hsp70 ATPase domain in yeast prion propagation.

The Saccharomyces cerevisiae non-Mendelian genetic element [PSI+] is the prion form of the translation termination factor Sup35p. The ability of [PSI+] to propagate efficiently has been shown previously to depend upon the action of protein chaperones. In this article we describe a genetic screen that identifies an array of mutants within the two major cytosolic Hsp70 chaperones of yeast, Ssa1p and Ssa2p, which impair the propagation of [PSI+]. All but one of the mutants was located within the ATPase domain of Hsp70, which highlights the important role of regulation of Hsp70-Ssa ATP hydrolysis in prion propagation. A subset of mutants is shown to alter Hsp70 function in a way that is distinct from that of previously characterized Hsp70 mutants that alter [PSI+] propagation and supports the importance of interdomain communication and Hsp70 interaction with nucleotide exchange factors in prion propagation. Analysis of the effects of Hsp70 mutants upon propagation of a second yeast prion [URE3] further classifies these mutants as having general or prion-specific inhibitory properties.

Distinct roles of PI(3,4,5)P3 during chemoattractant signaling in Dictyostelium: a quantitative in vivo analysis by inhibition of PI3-kinase.

The role of PI(3,4,5)P(3) in Dictyostelium signal transduction and chemotaxis was investigated using the PI3-kinase inhibitor LY294002 and pi3k-null cells. The increase of PI(3,4,5)P(3) levels after stimulation with the chemoattractant cAMP was blocked >95% by 60 microM LY294002 with half-maximal effect at 5 microM. This correlated well with the inhibition of the membrane translocation of the PH-domain protein, PHcracGFP. LY294002 did not reduce cAMP-mediated cGMP production, but significantly reduced the cAMP response up to 75% in wild type and completely in pi3k-null cells. LY294002-treated cells were round, not elongated as control cells. Interestingly, cAMP induced a time and dose-dependent recovery of cell elongation. These elongated LY294002-treated wild-type and pi3k-null cells exhibited chemotactic orientation toward cAMP that is statistically identical to chemotactic orientation of control cells. In control cells, PHcrac-GFP and F-actin colocalize upon cAMP stimulation. However, inhibition of PI3-kinases does not affect the first phase of the actin polymerization at a wide range of chemoattractant concentrations. Our data show that severe inhibition of cAMP-mediated PI(3,4,5)P(3) accumulation leads to inhibition of cAMP relay, cell elongation and cell aggregation, but has no detectable effect on chemotactic orientation, provided that cAMP had sufficient time to induce cell elongation.

Sensitization of Dictyostelium chemotaxis by phosphoinositide-3-kinase-mediated self-organizing signalling patches.

The leading edge of Dictyostelium cells in chemoattractant gradients can be visualized using green fluorescent protein (GFP) tagged to the pleckstrin-homology (PH) domain of cytosolic regulator of adenylyl cyclase (CRAC), which presumable binds phosphatidylinositol-(3,4,5)triphosphate [PtdIns(3,4,5)P(3)]. Uniform cyclic AMP (cAMP) concentrations induce persistent translocation of PH(Crac)-GFP from the cytosol to multiple patches, which are similar to the single patch of PH(Crac)-GFP at the leading edge in a cAMP gradient. We show that cAMP determines the probability of patch formation (half-maximal effect at 0.5 nM cAMP) but not the size, lifetime or intensity of patches, indicating that patches are self-organizing structures. A pseudopod is extended from the area of the cell with a PH(Crac)-GFP patch at about 10 seconds after patch formation. Cells treated with the F-actin inhibitor latrunculin A are round without pseudopodia; uniform cAMP still induces localized patches of PH(Crac)-GFP. Inhibition of phosphoinositide-3-kinase (PI3K) activity with LY294002 inhibits PH(Crac)-GFP patches and inhibits chemotaxis towards nanomolar cAMP but has no effect at higher cAMP concentrations. Thus, very low cAMP concentrations induce self-organizing PH(Crac)-GFP patches that serve as a spatial cue for pseudopod formation, which enhances the sensitivity and amplitude of chemotactic movement.

Chemotaxis: signalling modules join hands at front and tail.

Chemotaxis is the result of a refined interplay among various intracellular molecules that process spatial and temporal information. Here we present a modular scheme of the complex interactions between the front and the back of cells that allows them to navigate. First, at the front of the cell, activated Rho-type GTPases induce actin polymerization and pseudopod formation. Second, phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) is produced in a patch at the leading edge, where it binds pleckstrin-homology-domain-containing proteins, which enhance actin polymerization and translocation of the pseudopod. Third, in Dictyostelium amoebae, a cyclic-GMP-signalling cascade has been identified that regulates myosin filament formation in the posterior of the cell, thereby inhibiting the formation of lateral pseudopodia that could misdirect the cell.

A diverse family of inositol 5-phosphatases playing a role in growth and development in Dictyostelium discoideum.

Inositol phosphate-containing molecules play an important role in a broad range of cellular processes. Inositol 5-phosphatases participate in the regulation of these signaling molecules. We have identified four inositol 5-phosphatases in Dictyostelium discoideum, Dd5P1-4, showing a high diversity in domain composition. Dd5P1 possesses only a inositol 5-phosphatase catalytic domain. An unique domain composition is present in Dd5P2 containing a RCC1-like domain. RCC1 has a seven-bladed propeller structure and interacts with G-proteins. Dd5P3 and Dd5P4 have a domain composition similar to human Synaptojanin with a SacI domain and OCRL with a RhoGAP domain, respectively. We have expressed the catalytic domains and show that these inositol 5-phosphatases have different substrate preferences. Single and double gene inactivation suggest a functional redundancy for Dd5P1, Dd5P2, and Dd5P3. Inactivation of the gene coding for Dd5P4 leads to defects in growth and development. These defects are restored by the expression of the complete protein but not by the 5-phosphatase catalytic domain.