Correlation of Fragile Histidine Triad (Fhit) Protein Structural Features with Effector Interactions and Biological Functions

We have previously shown that Fhit tumor suppressor protein interacts with Hsp60 chaperone machinery and ferredoxin reductase (Fdxr) protein. Fhit-effector interactions are associated with a Fhit-dependent increase in Fdxr stability, followed by generation of reactive oxygen species and apoptosis induction under conditions of oxidative stress. To define Fhit structural features that affect interactions, downstream signaling, and biological outcomes, we used cancer cells expressing Fhit mutants with amino acid substitutions that alter enzymatic activity, enzyme substrate binding, or phosphorylation at tyrosine 114. Gastric cancer cell clones stably expressing mutants that do not bind substrate or cannot be phosphorylated showed decreased binding to Hsp60 and Fdxr and reduced mitochondrial localization. Expression of Fhit or mutants that bind interactor proteins results in oxidative damage and accumulation of cells in G₂/M or sub-G₁ fractions after peroxide treatment; noninteracting mutants are defective in these biological effects. Gastric cancer clones expressing noncomplexing Fhit mutants show reduction of Fhit tumor suppressor activity, confirming that substrate binding, interaction with heat shock proteins, mitochondrial localization, and interaction with Fdxr are important for Fhit tumor suppressor function.Fhit protein is a powerful tumor suppressor that is frequently lost or reduced in cancer cells because of rearrangement of the exquisitely DNA damage-sensitive fragile FHIT gene. Restoration of Fhit expression suppresses tumorigenicity of cancer cells of various types, and the ability to induce apoptosis in cancer cells in vitro is reduced by specific Fhit mutations (1, 2).Through studies of signal pathways affected by Fhit expression, by searches for Fhit protein effectors, and by in vitro analyses of Fhit activity, we and others have defined Fhit enzymatic activity in vitro (3), apoptotic activity in cells and tumors (4–6), and most recently identification of a Fhit protein complex that affects Fhit stability, mitochondrial localization, and interaction with ferredoxin reductase (Fdxr)⁵ (7). The complex includes Hsp60 and Hsp10 that mediate Fhit stability and may affect import into mitochondria, where Fhit interacts with Fdxr, which is responsible for transferring electrons from NADPH to cytochrome P450 via ferredoxin. Virally mediated Fhit restoration in Fhit-deficient cancer cells increases production of intracellular reactive oxygen species (ROS), followed by increased apoptosis of cancer cells under oxidative stress conditions; conversely, Fhit-negative cells escape apoptosis, likely carrying oxidative DNA damage that contributes to accumulation of mutations.The Fhit protein sequence, showing high homology to the histidine triad (HIT) family of proteins, suggested that the protein product would hydrolyze diadenosine tetraphosphate or diadenosine triphosphate (Ap₃A) (8), and in vitro studies showed that Ap₃A was cleaved into ADP and AMP by Fhit. The catalytic histidine triad within Fhit was essential for catalytic activity (3), and a Fhit mutant that substituted Asn for His at the central histidine (H96N mutant) was catalytically inactive, although it bound substrate well (3). Early tumor suppression studies showed that cancer cells stably transfected with wild type (WT) or H96N mutant Fhit were suppressed for tumor growth in nude mice. This suggested the hypothesis that the Fhit-substrate complex sends the tumor suppression signal (9, 10). To test this hypothesis, a series of FHIT alleles was designed to reduce substrate-binding and/or hydrolytic rates and was characterized by quantitative cell-death assays on cancer cells virally infected with each allele. The allele series covered defects as great as 100,000-fold in k_cat and increases as large as 30-fold in K_m. Mutants with 2–7-fold increases in K_m had significantly reduced apoptotic indices and the mutant with a 30-fold increase in K_m retained little apoptotic function. Thus, the proapoptotic function of Fhit, which is likely associated with tumor suppressor function, is limited by substrate binding and is unrelated to substrate hydrolysis (11).Fhit, a homodimeric protein of 147 amino acids, is a target of tyrosine phosphorylation by the Src family protein kinases, which can phosphorylate Tyr-114 of Fhit in vitro and in vivo (12). After co-expression of Fhit with the Elk tyrosine kinase in Escherichia coli to generate phosphorylated forms of Fhit, unphosphorylated, mono-, and diphosphorylated Fhit were purified, and enzyme kinetics studies showed that monophosphorylated Fhit exhibited monophasic kinetics with K_m and k_cat values ∼2- and ∼7-fold lower, respectively, than for unphosphorylated Fhit. Diphosphorylated Fhit exhibited biphasic kinetics; one site had K_m and k_cat values ∼2- and ∼140-fold lower, respectively, than for unphosphorylated Fhit; the second site had a K_m ∼60-fold higher and a k_cat ∼6-fold lower than for unphosphorylated Fhit (13). Thus, it was possible that the alterations in K_m and k_cat values for phosphorylated forms of Fhit might favor formation and lifetime of the Fhit-Ap₃A complex and enhance tumor suppressor activity (see

A food safety control low mass-range proteomics platform for the detection of illicit treatments in veal calves by MALDI-TOF-MS serum profiling

Performance enhancing agents (PEAs) are illegally used in cattle and other meat producing species to increase food conversion and lean meat production. Due to the very short breeding cycle, veal calves represent the meat producing bovine category mostly subjected to illicit treatments. These chemical agents are difficult to detect by conventional analytical approaches due to the employment of synergistic formulations at very low dosage and given the use of uncharacterized novel compounds. Such a scenario has fostered a strong interest in the discovery of functional molecular biomarkers for the detection of growth promoting agents in meat producing species. A multivariate MALDI-TOF-MS proteomics platform has been developed using bovine serum samples. Analytical performances have been thoroughly evaluated in order to enable reproducible profiles from 10 μL sera samples. We propose univariate and multivariate discrimination models capable to identify calves undergoing illicit treatments. In particular, we found a strong discrimination power associated with a polypeptide fragment from β2-glycoprotein-I. We provide a fundamental proof of concept in the potential application of MALDI-TOF-MS proteomics profiling in the food safety control.     

Computing parametric beta diversity with unequal plot weights: a solution based on resampling methods

Jost (Ecology, 88:2427–2439, 2007) recently showed that the Shannon diversity is the only standard diversity measure that can be partitioned into meaningful independent alpha and beta components when plot weights are unequal. This conclusion is very disappointing if one wants to calculate the beta diversity of unequal weighted plots using a parametric measure with varying sensitivities to the occurrence of rare and abundant species. To overcome this impasse, at least partially, in this paper, I propose a parametric measure of beta diversity that is based on the combination of Shannon’s entropy with Hurlbert’s ‘expected species diversity’. Unlike most parametric measures of diversity, the proposed index has a clear probabilistic interpretation, allowing at the same time a multiplicative partition of diversity into independent alpha and beta components for unequally weighted plots.     

Homeodomain Interacting Protein Kinase 2 Activation Compromises Endothelial Cell Response to Laminar Flow: Protective Role of p21waf1,cip1,sdi1

Background

In the cardiovascular system, laminar shear stress (SS) is one of the most important source of endothelial protecting signals. Physical and chemical agents, however, including ionising radiations and anticancer drugs, may injure endothelial cells determining an increase in oxidative stress and genotoxic damage. Whether the SS protective function remains intact in the presence of strong oxidants or DNA damage is currently unclear.

Methods and Results

To investigate this aspect a series of experiments were performed in which HUVEC were exposed to sub-lethal doses of the radio-mimetic compound Bleomycin (Bleo; 10 µg/ml) which generated free radicals (ROS) without significantly compromising cell survival. Remarkably, the application of a SS of 12 dyne/cm² did not protect endothelial cells but markedly accelerated apoptosis compared to controls kept in static culture and in the presence of Bleo. Experiments with the inducible nitric oxide synthase (iNOS) inhibitor {"type":"entrez-nucleotide","attrs":{"text":"GW274150","term_id":"282552565","term_text":"GW274150"}}GW274150 significantly reduced the SS-dependent apoptosis indicating that the production of NO was relevant for this effect. At molecular level, the ataxia-telangectasia-mutated (ATM) kinase, the homeodomain-interacting protein kinase-2 (HIPK2) and p53 were found activated along a pro-apoptotic signalling pathway while p21^{waf1,cip1,sdi1} was prevented from its protective action. RNA interference experiments revealed that HIPK2 and p53 were both important for this process, however, only the forced expression p21^{waf1,cip1,sdi1} fully restored the SS-dependent pro-survival function.

Conclusions

This study provides the first evidence that, in the presence of genotoxic damage, laminar flow contributes to endothelial toxicity and death and identifies molecular targets potentially relevant in endothelial dysfunction and cardiovascular disease pathogenesis.     

Nitric Oxide Administration Using an Oxygen Hood: A Pilot Trial

Background

We have shown earlier that inhaled nitric oxide (iNO) administered by oxygen hood reduces pulmonary hypertension in an animal model (J Perinatol 2002; 22:50-6). Our objective in this study was to determine feasibility of iNO by oxygen hood in neonates with elevated alveolar-arterial oxygen gradients (A-aDO₂).

Methods/Principal Findings

Masked randomized controlled pilot trial. Inclusion criteria were: gestation≥34 weeks, age<7 days, with post-ductal arterial line, and A-aDO₂ 400–600. Infants were randomized to study gas (iNO 20 ppm or equivalent O₂ flow) for 1 hr which was then weaned over the next 4 hours. Primary outcome was PaO₂ one hour post-randomization. Four infants each were randomized to iNO or O₂ (controls). Two of the four infants given iNO had an increase in PaO₂ of >100 torr, while oxygenation was unchanged in the controls. Methemoglobinemia and other adverse effects were not noted in any infant. Environmental levels of NO and NO₂ were minimal (<1 ppm) at >0.3 m from the hood.

Conclusions

Administration of iNO by oxygen hood is feasible. Larger randomized controlled trials are required to measure the efficacy and determine an appropriate target population for this technique.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00041548","term_id":"NCT00041548"}}NCT00041548     

Synergy between Repellents and Organophosphates on Bed Nets: Efficacy and Behavioural Response of Natural Free-Flying An. gambiae Mosquitoes

Background

Chemicals are used on bed nets in order to prevent infected bites and to kill aggressive malaria vectors. Because pyrethroid resistance has become widespread in the main malaria vectors, research for alternative active ingredients becomes urgent. Mixing a repellent and a non-pyrethroid insecticide seemed to be a promising tool as mixtures in the laboratory showed the same features as pyrethroids.

Methodology/Principal Findings

We present here the results of two trials run against free-flying Anopheles gambiae populations comparing the effects of two insect repellents (either DEET or KBR 3023, also known as icaridin) and an organophosphate insecticide at low-doses (pirimiphos-methyl, PM) used alone and in combination on bed nets. We showed that mixtures of PM and the repellents induced higher exophily, blood feeding inhibition and mortality among wild susceptible and resistant malaria vectors than compounds used alone. Nevertheless the synergistic interactions are only involved in the high mortality induced by the two mixtures.

Conclusion

These field trials argue in favour of the strategy of mixing repellent and organophosphate on bed nets to better control resistant malaria vectors.     

RAPD profiles distinguish <Emphasis Type="Italic">Paracentrotus lividus</Emphasis> populations living in a stressing environment (Amvrakikos Gulf,Greece)

Random amplified polymorphic DNA (RAPD) analysis was performed to assess genetic markers of Paracentrotus lividus populations living in stressing environment in the Amvrakikos Gulf (Western Greece, Ionian Sea) where two populations distinguishable in body size, smaller than the open sea ones, were detected. The UPGMA dendrogram, constructed from pairwise Φ_st values among population nuclear DNA markers, revealed that the small and medium-sized populations living inside the Amvrakikos presented a lower polymorphism, and form a cluster that shows the genetic distance with normal-sized populations (Ionian and Tyrrhenian Seas) living in open sea. AMOVA analysis indicated a genetic distance among the sea urchin populations from the Tyrrhenian sea and Ionian sea. The article is published in the original.     

Modeling the 3-D Structure of a Recombinant Laccase from Trametes trogii Active at a pH Close to Neutrality

A cDNA encoding a novel laccase from the white-rot fungus Trametes trogii was cloned and expressed in Pichia pastoris. The recombinant protein (Lcc2) exhibited kinetic parameters for both phenolic and non phenolic substrates that were different from the previously described Lcc1, the main laccase isoform expressed by T. trogii; in addition, the pH/activity profiles for phenolic substrates of Lcc2 were shifted upward by 1–1.5 pH units towards neutrality as compared to Lcc1. Comparative modeling of the two laccases (69.2% identity) showed that the overall fold of Lcc2 is very similar to Lcc1 and other laccases. The substrate cavity of Lcc2 contains the Asp residue which is thought to mediate the laccase activity at acidic pHs, whereas two hydrophobic residues (Phe, Ile) on the cavity orifice of Lcc2 replace the two polar residues (Thr, Ser) of Lcc1. These structural differences may be responsible for the unique kinetic performances of Lcc2.     

Detection of allergen specific immunoglobulins by microarrays coupled to microfluidics

Allergen microarrays are under development for a component‐resolved diagnosis of Type I (IgE‐mediated) allergies. Here we report an improved microarray coupled to microfluidics for the detection of allergen specific immunoglobulin E (IgE). The signal intensity for IgE detection in serum has been improved by using glass slides coated with a novel poly[DMA‐co‐NAS] brush copolymer which is able to immobilize allergens in their native conformation and by carrying out the incubation step in dynamic conditions. The assay, fully automated, was performed in a microcell, using a software‐controlled fluidic processor, to bring assay reagents on the surface of the array. Microfluidics turns the binding between serum immunoglobulins and immobilized allergens from a diffusion‐limited to a kinetic‐limited process by ensuring an efficient mixing of serum samples on the surface of the microarray. As a result of this, the binding of high affinity IgE antibodies is enhanced whereas that of low affinity IgG antibodies, which are present at higher concentration, is impaired paving the way to more accurate and sensitive results.     

Role of Tryptophan 95 in substrate specificity and structural stability of Sulfolobus solfataricus alcohol dehydrogenase

A mutant of the thermostable NAD⁺-dependent (S)-stereospecific alcohol dehydrogenase from Sulfolobus solfataricus (SsADH) which has a single substitution, Trp95Leu, located at the substrate binding pocket, was fully characterized to ascertain the role of Trp95 in discriminating between chiral secondary alcohols suggested by the wild-type SsADH crystallographic structure. The Trp95Leu mutant displays no apparent activity with short-chain primary and secondary alcohols and poor activity with aromatic substrates and coenzyme. Moreover, the Trp → Leu substitution affects the structural stability of the archaeal ADH, decreasing its thermal stability without relevant changes in secondary structure. The double mutant Trp95Leu/Asn249Tyr was also purified to assist in crystallographic analysis. This mutant exhibits higher activity but decreased affinity toward aliphatic alcohols, aldehydes as well as NAD⁺ and NADH compared to the wild-type enzyme. The crystal structure of the Trp95Leu/Asn249Tyr mutant apo form, determined at 2.0 Å resolution, reveals a large local rearrangement of the substrate site with dramatic consequences. The Leu95 side-chain conformation points away from the catalytic metal center and the widening of the substrate site is partially counteracted by a concomitant change of Trp117 side chain conformation. Structural changes at the active site are consistent with the reduced activity on substrates and decreased coenzyme binding.