A Quantitative Approach to the Prioritization of Zoonotic Diseases in North America: A Health Professionals’ Perspective

Background

Currently, zoonoses account for 58% to 61% of all communicable diseases causing illness in humans globally and up to 75% of emerging human pathogens. Although the impact of zoonoses on animal health and public health in North America is significant, there has been no published research involving health professionals on the prioritization of zoonoses in this region.

Methodology/Principal Findings

We used conjoint analysis (CA), a well-established quantitative method in market research, to identify the relative importance of 21 key characteristics of zoonotic diseases for their prioritization in Canada and the US. Relative importance weights from the CA were used to develop a point-scoring system to derive a recommended list of zoonoses for prioritization in Canada and the US. Study participants with a background in epidemiology, public health, medical sciences, veterinary sciences and infectious disease research were recruited to complete the online survey (707 from Canada and 764 from the US). Hierarchical Bayes models were fitted to the survey data to derive CA-weighted scores for disease criteria. Scores were applied to 62 zoonotic diseases to rank diseases in order of priority.

Conclusions/Significance

We present the first zoonoses prioritization exercise involving health professionals in North America. Our previous study indicated individuals with no prior knowledge in infectious diseases were capable of producing meaningful results with acceptable model fits (79.4%). This study suggests health professionals with some knowledge in infectious diseases were capable of producing meaningful results with better-fitted models than the general public (83.7% and 84.2%). Despite more similarities in demographics and model fit between the combined public and combined professional groups, there was more uniformity across priority lists between the Canadian public and Canadian professionals and between the US public and US professionals. Our study suggests that CA can be used as a potential tool for the prioritization of zoonoses.     

Dissecting the Mechanisms of Tissue Transglutaminase-induced Cross-linking of α-Synuclein: IMPLICATIONS FOR THE PATHOGENESIS OF PARKINSON DISEASE*S?

Tissue transglutaminase (tTG) has been implicated in the pathogenesis of Parkinson disease (PD). However, exactly how tTG modulates the structural and functional properties of α-synuclein (α-syn) and contributes to the pathogenesis of PD remains unknown. Using site-directed mutagenesis combined with detailed biophysical and mass spectrometry analyses, we sought to identify the exact residues involved in tTG-catalyzed cross-linking of wild-type α-syn and α-syn mutants associated with PD. To better understand the structural consequences of each cross-linking reaction, we determined the effect of tTG-catalyzed cross-linking on the oligomerization, fibrillization, and membrane binding of α-syn in vitro. Our findings show that tTG-catalyzed cross-linking of monomeric α-syn involves multiple cross-links (specifically 2-3). We subjected tTG-catalyzed cross-linked monomeric α-syn composed of either wild-type or Gln → Asn mutants to sequential proteolysis by multiple enzymes and peptide mapping by mass spectrometry. Using this approach, we identified the glutamine and lysine residues involved in tTG-catalyzed intramolecular cross-linking of α-syn. These studies demonstrate for the first time that Gln⁷⁹ and Gln¹⁰⁹ serve as the primary tTG reactive sites. Mutating both residues to asparagine abolishes tTG-catalyzed cross-linking of α-syn and tTG-induced inhibition of α-syn fibrillization in vitro. To further elucidate the sequence and structural basis underlying these effects, we identified the lysine residues that form isopeptide bonds with Gln⁷⁹ and Gln¹⁰⁹. This study provides mechanistic insight into the sequence and structural basis of the inhibitory effects of tTG on α-syn fibrillogenesis in vivo, and it sheds light on the potential role of tTG cross-linking on modulating the physiological and pathogenic properties of α-syn.Parkinson disease (PD)² is a progressive movement disorder that is caused by the loss of dopaminergic neurons in the substantia nigra, the part of the brain responsible for controlling movement. Clinically, PD is manifested in symptoms that include tremors, rigidity, and difficulty in initiating movement (bradykinesia). Pathologically, PD is characterized by the presence of intraneuronal, cytoplasmic inclusions known as Lewy bodies (LB), which are composed primarily of the protein “α-synuclein” (α-syn) (1) and are seen in the post-mortem brains of PD patients with the sporadic or familial forms of the disease (2). α-Syn is a presynaptic protein of 140 residues with a “natively” unfolded structure (3). Three missense point mutations in α-syn (A30P, E46K, and A53T) are associated with the early-onset, dominant, inherited form of PD (4, 5). Moreover, duplication or triplication of the α-syn gene has been linked to the familial form of PD, suggesting that an increase in α-syn expression is sufficient to cause PD. Together, these findings suggest that α-syn plays a central role in the pathogenesis of PD.The molecular and cellular determinants that govern α-syn oligomerization and fibrillogenesis in vivo remain poorly understood. In vitro aggregation studies have shown that the mutations associated with PD (A30P, E46K, and A53T) accelerate α-syn oligomerization, but only E46K and A53T α-syn show higher propensity to fibrillize than wild-type (WT) α-syn (6-8). This suggests that oligomerization, rather than fibrillization, is linked to early-onset familial PD (9). Our understanding of the molecular composition and biochemical state of α-syn in LBs has provided important clues about protein-protein interactions and post-translational modifications that may play a role in modulating oligomerization, fibrillogenesis, and LB formation of the protein. In addition to ubiquitination (10), phosphorylation (11, 12), nitration (13, 14), and C-terminal truncation (15, 16), analysis of post-mortem brain tissues from PD and Lewy bodies in dementia patients has confirmed the colocalization of tissue transglutaminase (tTG)-catalyzed cross-linked α-syn monomers and higher molecular aggregates in LBs within dopaminergic neurons (17, 18). Tissue transglutaminase catalyzes a calcium-dependent transamidating reaction involving glutamine and lysine residues, which results in the formation of a covalent cross-link via ε-(γ-glutamyl) lysine bonds (Fig. 2F). To date, seven different isoforms of tTGs have been reported, of which only tTG2 seems to be expressed in the human brain (19), whereas tTG1 and tTG3 are more abundantly found in stratified squamous epithelia (20). Subsequent immuno-histochemical, colocalization, and immunoprecipitation studies have shown that the levels of tTG and cross-linked α-syn species are increased in the substantia nigra of PD brains (17). These findings, combined with the known role of tTG in cross-linking and stabilizing bimolecular assemblies, led to the hypothesis that tTG plays an important role in the initiation and propagation of α-syn fibril formation and that it contributes to fibril stability in LBs. This hypothesis was initially supported by in vitro studies demonstrating that tTG catalyzes the polymerization of the α-syn-derived non-amyloid component (NAC) peptide via intermolecular covalent cross-linking of residues Gln⁷⁹ and Lys⁸⁰ (21) and by other studies suggesting that tTG promotes the fibrillization of amyloidogenic proteins implicated in the pathogenesis of other neurodegenerative diseases such as Alzheimer disease, supranuclear palsy, Huntington disease, and other polyglutamine diseases (22-24). However, recent in vitro studies with full-length α-syn have shown that tTG catalyzes intramolecular cross-linking of monomeric α-syn and inhibits, rather than promotes, its fibrillization in vitro (25, 26). The structural basis of this inhibitory effect and the exact residues involved in tTG-mediated cross-linking of α-syn, as well as structural and functional consequences of these modifications, remain poorly understood.Open in a separate windowFIGURE 2.tTG-catalyzed cross-linking of α-syn involves one to three intramolecular cross-links. A-C, MALDI-TOF/TOF analysis of native (—) and cross-linked (- - -) α-syn, showing that most tTG-catalyzed cross-linking products of WT or disease-associated mutant forms of α-syn are intramolecularly linked (predominant peak with two cross-links), and up to three intramolecular cross-links can occur (left shoulder). The abbreviations M and m/cl are used to designate native and cross-linked α-synuclein, respectively. D and E, kinetic analysis of α-syn (A30P) cross-linking monitored by MALDI-TOF and SDS-PAGE. F, schematic depiction of the tTG-catalyzed chemical reaction (isodipeptide formation) between glutamine and lysine residues.In this study, we have identified the primary glutamine and lysine residues involved in tTG-catalyzed, intramolecularly cross-linked monomeric α-syn and investigated how cross-linking these residues affects the oligomerization, fibrillization, and membrane binding of α-syn in vitro. Using single-site mutagenesis and mass spectrometry applied to exhaustive proteolytic digests of native and cross-linked monomeric α-syn, we identified Gln¹⁰⁹ and Gln⁷⁹ as the major tTG substrates. We demonstrate that the altered electrophoretic mobility of the intramolecularly cross-linked α-syn in SDS-PAGE occurs as a result of tTG-catalyzed cross-linking of Gln¹⁰⁹ to lysine residues in the N terminus of α-syn, which leads to the formation of more compact monomers. Consistent with previous studies, we show that intramolecularly cross-linked α-syn forms off-pathway oligomers that are distinct from those formed by the wild-type protein and that do not convert to fibrils within the time scale of our experiments (3-5 days). We also show that membrane-bound α-syn is a substrate of tTG and that intramolecular cross-linking does not interfere with the ability of monomeric α-syn to adopt an α-helical conformation upon binding to synthetic membranes. These studies provide novel mechanistic insight into the sequence and structural basis of events that allow tTG to inhibit α-syn fibrillogenesis, and they shed light on the potential role of tTG-catalyzed cross-linking in modulating the physiological and pathogenic properties of α-syn.     

Focus: The Science of Stress: Potential Societal and Cultural Implications of Transgenerational Epigenetic Methylation of Trauma and PTSD: Pathology or Resilience?

Psychological trauma is unique in that it is an environmental event that could induce biological changes and post-traumatic stress disorder (PTSD), depression, or other mood disorders in some patients. On the other hand, there may be no psychopathology (in most cases), or even sometimes post-traumatic growth and resilience. According to the DSM-5, trauma is a prerequisite for PTSD and traumatic stress disorder, but not for depressive episodes or mood disorders, or other psychiatric conditions. This paper brings attention to the preliminary literature on transgenerational inheritance due to trauma exposure and its societal and cultural implications. There is accumulating evidence that exposure to trauma can be passed transgenerationally through epigenetic inheritance leading to changes in gene expression and possible disorders or resilience. The effects of resilience from transgenerational inheritance have not been studied, but should be, for a full understanding not only of the disease risk across generations, but also of its social and cultural implications. The epigenetic pathologic effects across generations also need further studies, as the current research is preliminary; larger replications are needed for definitive and more complete understanding. I present here a glimpse of where we are, a vision of where we should go in terms of future research direction for disease risk transmission, and recommend studies of resilience and post-traumatic growth across generations, as well as other studies related to the societal implications at the population level.     

Intracellular β-Carbonic Anhydrase of the Unicellular Green Alga Coccomyxa : Cloning of the cDNA and Characterization of the Functional Enzyme Overexpressed in Escherichia coli

Carbonic anhydrase (CA) (EC 4.2.1.1) enzymes catalyze the reversible hydration of CO₂, a reaction that is important in many physiological processes. We have cloned and sequenced a full-length cDNA encoding an intracellular β-CA from the unicellular green alga Coccomyxa. Nucleotide sequence data show that the isolated cDNA contains an open reading frame encoding a polypeptide of 227 amino acids. The predicted polypeptide is similar to β-type CAs from Escherichia coli and higher plants, with an identity of 26% to 30%. The Coccomyxa cDNA was overexpressed in E. coli, and the enzyme was purified and biochemically characterized. The mature protein is a homotetramer with an estimated molecular mass of 100 kD. The CO₂-hydration activity of the Coccomyxa enzyme is comparable with that of the pea homolog. However, the activity of Coccomyxa CA is largely insensitive to oxidative conditions, in contrast to similar enzymes from most higher plants. Fractionation studies further showed that Coccomyxa CA is extrachloroplastic.     

Selective Inhibition of Carotenoid Cleavage Dioxygenases: PHENOTYPIC EFFECTS ON SHOOT BRANCHING*S?

Members of the carotenoid cleavage dioxygenase family catalyze the oxidative cleavage of carotenoids at various chain positions, leading to the formation of a wide range of apocarotenoid signaling molecules. To explore the functions of this diverse enzyme family, we have used a chemical genetic approach to design selective inhibitors for different classes of carotenoid cleavage dioxygenase. A set of 18 arylalkyl-hydroxamic acids was synthesized in which the distance between an iron-chelating hydroxamic acid and an aromatic ring was varied; these compounds were screened as inhibitors of four different enzyme classes, either in vitro or in vivo. Potent inhibitors were found that selectively inhibited enzymes that cleave carotenoids at the 9,10 position; 50% inhibition was achieved at submicromolar concentrations. Application of certain inhibitors at 100 μm to Arabidopsis node explants or whole plants led to increased shoot branching, consistent with inhibition of 9,10-cleavage.Carotenoids are synthesized in plants and micro-organisms as photoprotective molecules and are key components in animal diets, an example being β-carotene (pro-vitamin A). The oxidative cleavage of carotenoids occurs in plants, animals, and micro-organisms and leads to the release of a range of apocarotenoids that function as signaling molecules with a diverse range of functions (1). The first gene identified as encoding a carotenoid cleavage dioxygenase (CCD)² was the maize Vp14 gene that is required for the formation of abscisic acid (ABA), an important hormone that mediates responses to drought stress and aspects of plant development such as seed and bud dormancy (2). The VP14 enzyme cleaves at the 11,12 position (Fig. 1) of the epoxycarotenoids 9′-cis-neoxanthin and/or 9-cis-violaxanthin and is now classified as a 9-cis-epoxycarotenoid dioxygenase (NCED) (3), a subclass of the larger CCD family.Open in a separate windowFIGURE 1.Reactions catalyzed by the carotenoid cleavage dioxygenases. a, 11,12-oxidative cleavage of 9′-cis-neoxanthin by NCED; b, oxidative cleavage reactions on β-carotene and zeaxanthin.Since the discovery of Vp14, many other CCDs have been shown to be involved in the production of a variety of apocarotenoids (Fig. 1). In insects, the visual pigment retinal is formed by oxidative cleavage of β-carotene by β-carotene-15,15′-dioxygenase (4). Retinal is produced by an orthologous enzyme in vertebrates, where it is also converted to retinoic acid, a regulator of differentiation during embryogenesis (5). A distinct mammalian CCD is believed to cleave carotenoids asymmetrically at the 9,10 position (6) and, although its function is unclear, recent evidence suggests a role in the metabolism of dietary lycopene (7). The plant volatiles β-ionone and geranylacetone are produced from an enzyme that cleaves at the 9,10 position (8) and the pigment α-crocin found in the spice saffron results from an 7,8-cleavage enzyme (9).Other CCDs have been identified where biological function is unknown, for example, in cyanobacteria where a variety of cleavage specificities have been described (10-12). In other cases, there are apocarotenoids with known functions, but the identity or involvement of CCDs have not yet been described: grasshopper ketone is a defensive secretion of the flightless grasshopper Romalea microptera (13), mycorradicin is produced by plant roots during symbiosis with arbuscular mycorrhyza (14), and strigolactones (15) are plant metabolites that act as germination signals to parasitic weeds such as Striga and Orobanche (16).Recently it was discovered that strigolactones also function as a branching hormone in plants (17, 18). The existence of such a branching hormone has been known for some time, but its identity proved elusive. However, it was known that the hormone was derived from the action of at least two CCDs, max3 and max4 (more axillary growth) (19), because deletion of either of these genes in Arabidopsis thaliana, leads to a bushy phenotype (20, 21). In Escherichia coli assays, AtCCD7 (max3) cleaves β-carotene at the 9,10 position and the apocarotenoid product (10-apo-β-carotene) is reported to be further cleaved at 13,14 by AtCCD8 (max4) to produce 13-apo-β-carotene (22). Also recent evidence suggests that AtCCD8 is highly specific, cleaving only 10-apo-β-carotene (23). How the production of 13-apo-β-carotene leads to the synthesis of the complex strigolactone is unknown. The possibility remains that the enzymes may have different specificities and cleavage activities in planta. In addition, a cytochrome P450 enzyme (24) is believed to be involved in strigolactone synthesis and acts in the pathway downstream of the CCD genes. Strigolactone is thought to effect branching by regulating auxin transport (25). Because of the involvement of CCDs in strigolactone synthesis, the possibility arises that plant architecture and interaction with parasitic weeds and mycorrhyza could be controlled by the manipulation of CCD activity.Although considerable success has been obtained using genetic approaches to probe function and substrate specificity of CCDs in their native biological contexts, particularly in plant species with simple genetic systems or that are amenable to transgenesis, there are many systems where genetic approaches are difficult or impossible. Also, when recombinant CCDs are studied either in vitro or in heterologous in vivo assays, such as in E. coli strains engineered to accumulate carotenoids (26), they are often active against a broad range of substrates (5, 21, 27), and in many cases the true in vivo substrate of a particular CCD remains unknown. Therefore additional experimental tools are needed to investigate both apocarotenoid and CCD functions in their native cellular environments.In the reverse chemical genetics approach, small molecules are identified that are active against known target proteins; they are then applied to a biological system to investigate protein function in vivo (28, 29). This approach is complementary to conventional genetics since the small molecules can be applied easily to a broad range of species, their application can be controlled in dose, time, and space to provide detailed studies of biological functions, and individual proteins or whole protein classes may be targeted by varying the specificity of the small molecules. Notably, functions of the plant hormones gibberellin, brassinosteroid, and abscisic acid have been successfully probed using this approach by adapting triazoles to inhibit specific cytochrome P450 monooxygenases involved in the metabolism of these hormones (30).In the case of the CCD family, the tertiary amines abamine (31) and the more active abamineSG (32) were reported as specific inhibitors of NCED, and abamine was used to show new functions of abscisic acid in legume nodulation (33). However, no selective inhibitors for other types of CCD are known. Here we have designed a novel class of CCD inhibitor based on hydroxamic acids, where variable chain length was used to direct inhibition of CCD enzymes that cleave carotenoids at specific positions. We demonstrate the use of such novel inhibitors to control shoot branching in a model plant.     

Promiscuous Usage of Nucleotides by the DNA Helicase of Bacteriophage T7: DETERMINANTS OF NUCLEOTIDE SPECIFICITY*S?

The multifunctional protein encoded by gene 4 of bacteriophage T7 (gp4) provides both helicase and primase activity at the replication fork. T7 DNA helicase preferentially utilizes dTTP to unwind duplex DNA in vitro but also hydrolyzes other nucleotides, some of which do not support helicase activity. Very little is known regarding the architecture of the nucleotide binding site in determining nucleotide specificity. Crystal structures of the T7 helicase domain with bound dATP or dTTP identified Arg-363 and Arg-504 as potential determinants of the specificity for dATP and dTTP. Arg-363 is in close proximity to the sugar of the bound dATP, whereas Arg-504 makes a hydrogen bridge with the base of bound dTTP. T7 helicase has a serine at position 319, whereas bacterial helicases that use rATP have a threonine in the comparable position. Therefore, in the present study we have examined the role of these residues (Arg-363, Arg-504, and Ser-319) in determining nucleotide specificity. Our results show that Arg-363 is responsible for dATP, dCTP, and dGTP hydrolysis, whereas Arg-504 and Ser-319 confer dTTP specificity. Helicase-R504A hydrolyzes dCTP far better than wild-type helicase, and the hydrolysis of dCTP fuels unwinding of DNA. Substitution of threonine for serine 319 reduces the rate of hydrolysis of dTTP without affecting the rate of dATP hydrolysis. We propose that different nucleotides bind to the nucleotide binding site of T7 helicase by an induced fit mechanism. We also present evidence that T7 helicase uses the energy derived from the hydrolysis of dATP in addition to dTTP for mediating DNA unwinding.Helicases are molecular machines that translocate unidirectionally along single-stranded nucleic acids using the energy derived from nucleotide hydrolysis (1–3). The gene 4 protein encoded by bacteriophage T7 consists of a helicase domain and a primase domain, located in the C-terminal and N-terminal halves of the protein, respectively (4). The T7 helicase functions as a hexamer and has been used as a model to study ring-shaped replicative helicases. In the presence of dTTP, T7 helicase binds to single-stranded DNA (ssDNA)³ as a hexamer and translocates 5′ to 3′ along the DNA strand using the energy of hydrolysis of dTTP (5–7). T7 helicase hydrolyzes a variety of ribo and deoxyribonucleotides; however, dTTP hydrolysis is optimally coupled to DNA unwinding (5).Most hexameric helicases use rATP to fuel translocation and unwind DNA (3). T7 helicase does hydrolyze rATP but with a 20-fold higher K_m as compared with dTTP (5, 8). It has been suggested that T7 helicase actually uses rATP in vivo where the concentration of rATP is 20-fold that of dTTP in the Escherichia coli cell (8). However, hydrolysis of rATP, even at optimal concentrations, is poorly coupled to translocation and unwinding of DNA (9). Other ribonucleotides (rCTP, rGTP, and rUTP) are either not hydrolyzed or the poor hydrolysis observed is not coupled to DNA unwinding (8). Furthermore, Patel et al. (10) found that the form of T7 helicase found in vivo, an equimolar mixture of the full-length gp4 and a truncated form lacking the zinc binding domain of the primase, prefers dTTP and dATP. Therefore, in the present study we have restricted our examination of nucleotides to the deoxyribonucleotides.The nucleotide binding site of the replicative DNA helicases, such as T7 gene 4 protein, bind nucleotides at the subunit interface (Fig. 1) located between two RecA-like subdomains that bind ATP (11, 12). The location of the nucleotide binding site at the subunit interface provides multiple interactions of residues with the bound NTP. A number of cis- and trans-acting amino acids stabilize the bound nucleotide in the nucleotide binding site and also provide for communication between subunits (13–15). Earlier reports revealed that the arginine finger (Arg-522) in T7 helicase is positioned to interact with the γ-phosphate of the bound nucleotide in the adjacent subunit (12, 16). However, His-465 (phosphate sensor), Glu-343 (catalytic base), and Asp-424 (Walker motif B) interacts with the γ-phosphate of the bound nucleotide in the same subunit (12, 17, 18). The arginine finger and the phosphate sensor have been proposed to couple NTP hydrolysis to DNA unwinding. Substitution of Glu-343, the catalytic base, eliminates dTTP hydrolysis (19), and substitution of Asp-424 with Asn leads to a severe reduction in dTTP hydrolysis (20). The conserved Lys-318 in Walker motif A interacts with the β-phosphate of the bound nucleotide and plays an important role in dTTP hydrolysis (21).Open in a separate windowFIGURE 1.Crystal structure of T7 helicase. A, crystal structure of the hexameric helicase C-terminal domain of gp4 (17). The structure reveals a ring-shaped molecule with a central core through which ssDNA passes. The inset shows the interface between two subunits of the helicase with adenosine 5′-{β,γ-imidol}-triphosphate in the nucleotide binding site. B, the nucleotide binding site of a monomer of the gp4 with the crucial amino acid residues reported earlier and in the present study is shown in sticks. The crystal structures of the T7 gene 4 helicase domain (12) with bound dTTP (C) and dATP (D). The structures shown are the nucleotide binding site of T7 helicase as viewed in Pymol by analyzing the PDB files 1cr1 and 1cr2 (12). Arg-504 and Tyr-535 sandwiches the base of the bound dNTP. Additionally, Arg-504 forms a hydrogen bridge with dTTP. Arg-363 interacts specifically with the 3-OH group of bound dATP. AMPPNP, adenosine 5′-(β,γ-imino)triphosphate.Considering the wealth of information on the above residues that are involved in the hydrolysis of dTTP and the coupling of hydrolysis to unwinding, it is intriguing that little information is available on nucleotide specificity. Several crystal structures of T7 helicase in complex with a nucleotide triphosphate are available. However, most of structures were crystallized with a non-hydrolyzable analogue of dTTP or the nucleotide was diffused into the crystal. The crystal structure of the T7 helicase domain bound with dTTP or dATP was reported by Sawaya et al. (12). These structures assisted us in identifying two basic residues (Arg-363 and Arg-504) in close proximity to the sugar and base of the bound nucleotide whose orientation suggested that these residues could be involved in nucleotide selection. Arg-504 together with Tyr-535 sandwich the base of the bound nucleotide at the subunit interface of the hexameric helicase (Fig. 1). Arg-504 and Tyr-535 are structurally well conserved in various helicases (12). However, Arg-504 could make a hydrogen bridge with the OH group of thymidine, thus suggesting a role in dTTP specificity. On the other hand, Arg-363 is in close proximity (∼3.4 Å) to the sugar 3′-OH of bound dATP, whereas in the dTTP-bound structure this residue is displaced by 7.12 Å (Fig. 1) from the equivalent position. Consequently Arg-363 could play a role in dATP binding. The crystal structures do not provide any information on different interaction of residues with the phosphates of dATP and dTTP. However, alignment of the residues in the P-loops of different hexameric helicases reveals that the serine adjacent to the invariant lysine at position 319 (Ser-319) is conserved in bacteriophages, whereas bacterial helicases have a conserved threonine in the equivalent position (supplemental Fig. 1). Bacterial helicases use rATP in the DNA unwinding reactions. whereas T7 helicase preferentially uses dTTP, and bacteriophage T4 gene 41 uses rGTP or rATP (22).Although considerable information is available on the role of residues in nucleotide binding and dTTP hydrolysis, very little is known on the determinants of nucleotide specificity. In the present study we made an attempt to address the role of a few selected residues (Arg-363, Arg-504, and Ser-319) in determining nucleotide specificity, especially dTTP and dATP, both of which are hydrolyzed and mediate DNA unwinding. We show that under physiological conditions T7 helicase uses the energy derived from the hydrolysis of dATP in addition to dTTP for mediating DNA unwinding.     

Regulation of Rac1 by Simvastatin in Endothelial Cells: DIFFERENTIAL ROLES OF AMP-ACTIVATED PROTEIN KINASE AND CALMODULIN-DEPENDENT KINASE KINASE-��*

These studies explore the connections between simvastatin, Rac1, and AMP-activated protein kinase (AMPK) pathways in cultured vascular endothelial cells and in arterial preparations isolated from statin-treated mice. In addition to their prominent effects on lipoprotein metabolism, statins can regulate the small GTPase Rac1, and may also affect the phosphorylation of the ubiquitous AMPK. We explored pathways of statin-modulated Rac1 and AMPK activation both in arterial preparations from statin-treated mice as well as in cultured endothelial cells. We treated adult mice with simvastatin daily for 2 weeks and then harvested and analyzed arterial preparations. Simvastatin treatment of mice led to a significant increase in AMPK and LKB1 phosphorylation and to a decrease in protein kinase A activity relative to control animals, associated with a marked increase in Rac1 activation. Exposure of bovine aortic endothelial cells to simvastatin for 24 h strikingly increased GTP-bound Rac1 and led to increased phosphorylation of AMPK as well as the AMPK kinase LKB1. These responses to simvastatin were blocked by mevalonate or geranylgeranyl pyrophosphate but not by farnesyl pyrophosphate. Small interfering RNA (siRNA)-mediated knockdown of AMPK abrogated simvastatin-induced Rac1 activation and LKB1 phosphorylation. Importantly, siRNA-mediated knockdown of the key AMPK kinase, calcium/calmodulin-dependent protein kinase kinase β, completely blocked simvastatin-induced endothelial cell migration and also abrogated statin-promoted phosphorylation of AMPK and LKB1, as did pharmacological inhibition with the specific calcium/calmodulin-dependent protein kinase β inhibitor STO-609. Moreover, siRNA-mediated knockdown of Rac1 completely blocked simvastatin-induced LKB1 phosphorylation, but without affecting simvastatin-induced AMPK phosphorylation. These findings establish a key role for simvastatin in activation of a novel Rac1-dependent signaling pathway in the vascular wall.HMG-CoA² reductase inhibitors, commonly known as statins, are widely prescribed for the prevention and treatment of hypercholesterolemia and cardiovascular diseases (1, 2). The salutary clinical effects of these drugs derive in part from their effects on the levels of serum lipoproteins, yet other statin responses appear to be mediated by alterations in vascular function involving the endothelial isoform of nitric-oxide synthase (3) and related signaling pathways. Inhibition of HMG-CoA reductase suppresses the cellular levels of its enzymatic product mevalonate, thereby attenuating formation both of cholesterol as well as the synthesis of distinct isoprenoid compounds such as farnesyl pyrophosphate (Fpp) and geranylgeranyl pyrophosphate (GGpp). Many key signaling proteins are covalently modified by these isoprenoids, which are the products of a metabolic pathway that diverges from the pathway that leads to cholesterol synthesis downstream of HMG-CoA reductase. These isoprenoid compounds can provide lipophilic anchors that facilitate membrane targeting and modulate protein-protein interactions of many key signaling proteins. One such iso-prenylated signaling protein is the GTP-binding cytoskeletonassociated protein Rac1, a member of the Rho GTPase small G protein family that undergoes geranylgeranylation at its C terminus. Statins also affect post-translational modification of another small GTPase, RhoA, that, like Rac1, is a geranylgeranylated protein that is an important determinant of vascular signaling (4–8). Rac1 has particularly important roles in vascular endothelial cells, where this cytoskeleton regulatory protein modulates activity of the endothelial isoform of nitric-oxide synthase (eNOS), a key determinant of vascular homeostasis (9). Rac1 activation in endothelial cells is influenced by the AMP-activated protein kinase (AMPK) (6), which itself is phosphorylated by the protein kinase LKB1 and by the calcium-calmodulin-dependent protein kinase β (CaMKKβ) (see review (10)). In recent years, numerous reports have described effects of statins on variety of these signaling proteins in different experimental systems (11–14).Statins have been shown to promote the phosphorylation of AMPK (13), a heterotrimeric enzyme involved in the modulation of cellular energy pathways that has also been implicated in eNOS regulation (3, 15–17). AMPK was originally discovered and characterized as a cellular “energy sensor” that can be activated by increases in the intracellular AMP:ATP ratio (18). However, in recent years, it has become clear that AMPK is also regulated through AMP-independent pathways involving enzyme phosphorylation on threonine 172 of the enzyme''s α subunit, leading to marked enzyme activation (19). Protein kinases that phosphorylate AMPK include the tumor suppressor LKB1 and the calcium/calmodulin-dependent kinase CaMKKβ. LKB1 itself is a phosphoprotein. The pathways that regulate LKB1 are incompletely understood, and a variety of upstream protein kinases have been implicated in LKB1 regulation (see review (20)). CaMKKβ is principally regulated by calcium binding, but this kinase may also be phosphorylated by the cAMP-dependent protein kinase PKA (21, 22). Another substrate for PKA in vascular cells is the actin-binding phosphoprotein VASP (23, 24); the phosphorylation state of VASP at its PKA site can serve as a surrogate marker for the activity of cAMP-dependent signaling pathways in the vascular wall (25). CaMKKβ has been shown to be involved in AMPK regulation in endothelial cells in response to receptor tyrosine kinase activation and via G protein-coupled receptor pathways (6). Activated AMPK directly phosphorylates eNOS, and this kinase thereby appears be an important determinant of NO-dependent signaling in endothelial cells. However, much remains to be learned about the molecular mechanisms whereby statins enhance AMPK activation.In cultured cells, statins have been shown to inhibit the geranylgeranylation of Rac1, associated with an increase in Rac1 GTP binding and activation (26). The activation of Rac1 is a key step in eNOS activation: siRNA-mediated Rac1 “knockdown” in endothelial cells markedly suppresses receptor signaling to eNOS (5, 7). siRNA-mediated AMPK knockdown suppresses Rac1 activation, again leading to the attenuation of receptor-dependent activation of eNOS (6). The relationships among these various statin-modulated signaling pathways are incompletely characterized. The present studies identify CaMKKβ and LKB1 as critical determinants of simvastatin-dependent activation of AMPK- and Rac1-modulated signaling and reveal that Rac1 in turn regulates LKB1 phosphorylation.     

Mechanism of Activation and Functional Role of Protein Kinase C�� in Human Platelets

The novel class of protein kinase C (nPKC) isoform η is expressed in platelets, but not much is known about its activation and function. In this study, we investigated the mechanism of activation and functional implications of nPKCη using pharmacological and gene knock-out approaches. nPKCη was phosphorylated (at Thr-512) in a time- and concentration-dependent manner by 2MeSADP. Pretreatment of platelets with MRS-2179, a P2Y₁ receptor antagonist, or YM-254890, a G_q blocker, abolished 2MeSADP-induced phosphorylation of nPKCη. Similarly, ADP failed to activate nPKCη in platelets isolated from P2Y₁ and G_q knock-out mice. However, pretreatment of platelets with P2Y₁₂ receptor antagonist, AR-C69331MX did not interfere with ADP-induced nPKCη phosphorylation. In addition, when platelets were activated with 2MeSADP under stirring conditions, although nPKCη was phosphorylated within 30 s by ADP receptors, it was also dephosphorylated by activated integrin α_IIbβ₃ mediated outside-in signaling. Moreover, in the presence of SC-57101, a α_IIbβ₃ receptor antagonist, nPKCη dephosphorylation was inhibited. Furthermore, in murine platelets lacking PP1cγ, a catalytic subunit of serine/threonine phosphatase, α_IIbβ₃ failed to dephosphorylate nPKCη. Thus, we conclude that ADP activates nPKCη via P2Y₁ receptor and is subsequently dephosphorylated by PP1γ phosphatase activated by α_IIbβ₃ integrin. In addition, pretreatment of platelets with η-RACK antagonistic peptides, a specific inhibitor of nPKCη, inhibited ADP-induced thromboxane generation. However, these peptides had no affect on ADP-induced aggregation when thromboxane generation was blocked. In summary, nPKCη positively regulates agonist-induced thromboxane generation with no effects on platelet aggregation.Platelets are the key cellular components in maintaining hemostasis (1). Vascular injury exposes subendothelial collagen that activates platelets to change shape, secrete contents of granules, generate thromboxane, and finally aggregate via activated α_IIbβ₃ integrin, to prevent further bleeding (2, 3). ADP is a physiological agonist of platelets secreted from dense granules and is involved in feedback activation of platelets and hemostatic plug stabilization (4). It activates two distinct G-protein-coupled receptors (GPCRs) on platelets, P2Y₁ and P2Y₁₂, which couple to G_q and G_i, respectively (5–8). G_q activates phospholipase Cβ (PLCβ), which leads to diacyl glycerol (DAG)² generation and calcium mobilization (9, 10). On the other hand, G_i is involved in inhibition of cAMP levels and PI 3-kinase activation (4, 6). Synergistic activation of G_q and G_i proteins leads to the activation of the fibrinogen receptor integrin α_IIbβ₃. Fibrinogen bound to activated integrin α_IIbβ₃ further initiates feed back signaling (outside-in signaling) in platelets that contributes to the formation of a stable platelet plug (11).Protein kinase Cs (PKCs) are serine/threonine kinases known to regulate various platelet functional responses such as dense granule secretion and integrin α_IIbβ₃ activation (12, 13). Based on their structure and cofactor requirements, PKCs are divided in to three classes: classical (cofactors: DAG, Ca²⁺), novel (cofactors: DAG) and atypical (cofactors: PIP₃) PKC isoforms (14). All the members of the novel class of PKC isoforms (nPKC), viz. nPKC isoforms δ, θ, η, and ε, are expressed in platelets (15), and they require DAG for activation. Among all the nPKCs, PKCδ (15, 16) and PKCθ (17–19) are fairly studied in platelets. Whereas nPKCδ is reported to regulate protease-activated receptor (PAR)-mediated dense granule secretion (15, 20), nPKCθ is activated by outside-in signaling and contributes to platelet spreading on fibrinogen (18). On the other hand, the mechanism of activation and functional role of nPKCη is not addressed as yet.PKCs are cytoplasmic enzymes. The enzyme activity of PKCs is modulated via three mechanisms (14, 21): 1) cofactor binding: upon cell stimulus, cytoplasmic PKCs mobilize to membrane, bind cofactors such as DAG, Ca²⁺, or PIP3, release autoinhibition, and attain an active conformation exposing catalytic domain of the enzyme. 2) phosphorylations: 3-phosphoinositide-dependent kinase 1 (PDK1) on the membrane phosphorylates conserved threonine residues on activation loop of catalytic domain; this is followed by autophosphorylations of serine/threonine residues on turn motif and hydrophobic region. These series of phosphorylations maintain an active conformation of the enzyme. 3) RACK binding: PKCs in active conformation bind receptors for activated C kinases (RACKs) and are lead to various subcellular locations to access the substrates (22, 23). Although various leading laboratories have elucidated the activation of PKCs, the mechanism of down-regulation of PKCs is not completely understood.The premise of dynamic cell signaling, which involves protein phosphorylations by kinases and dephosphorylations by phosphatases has gained immense attention over recent years. PP1, PP2A, PP2B, PHLPP are a few of the serine/threonine phosphatases reported to date. Among them PP1 and PP2 phosphatases are known to regulate various platelet functional responses (24, 25). Furthermore, PP1c, is the catalytic unit of PP1 known to constitutively associate with α_IIb and is activated upon integrin engagement with fibrinogen and subsequent outside-in signaling (26). Among various PP1 isoforms, recently PP1γ is shown to positively regulate platelet functional responses (27). Thus, in this study we investigated if the above-mentioned phosphatases are involved in down-regulation of nPKCη. Furthermore, reports from other cell systems suggest that nPKCη regulates ERK/JNK pathways (28). In platelets ERK is known to regulate agonist induced thromboxane generation (29, 30). Thus, we also investigated if nPKCη regulates ERK phosphorylation and thereby agonist-induced platelet functional responses.In this study, we evaluated the activation of nPKCη downstream of ADP receptors and its inactivation by an integrin-associated phosphatase PP1γ. We also studied if nPKCη regulates functional responses in platelets and found that this isoform regulates ADP-induced thromboxane generation, but not fibrinogen receptor activation in platelets.     

Health psychology: a new form of psychotherapy?

Psychotherapy in its traditional form is being challenged due to managed care pressures. Psychotherapy using the model of health psychology can adapt well in a managed care environment. Differences between traditional psychotherapy and the psychotherapeutic approach of health psychology are discussed in this article, with a focus on an overview of health psychology and its applications to primary care, and the concept of single-session therapy. A case example of a sample treatment emphasizing the model of brief health psychology treatment is illustrated.     

Differential regulation of cell death programs in males and females by Poly (ADP-Ribose) Polymerase-1 and 17 β estradiol

Cell death can be divided into the anti-inflammatory process of apoptosis and the pro-inflammatory process of necrosis. Necrosis, as apoptosis, is a regulated form of cell death, and Poly-(ADP-Ribose) Polymerase-1 (PARP-1) and Receptor-Interacting Protein (RIP) 1/3 are major mediators. We previously showed that absence or inhibition of PARP-1 protects mice from nephritis, however only the male mice. We therefore hypothesized that there is an inherent difference in the cell death program between the sexes. We show here that in an immune-mediated nephritis model, female mice show increased apoptosis compared to male mice. Treatment of the male mice with estrogens induced apoptosis to levels similar to that in female mice and inhibited necrosis. Although PARP-1 was activated in both male and female mice, PARP-1 inhibition reduced necrosis only in the male mice. We also show that deletion of RIP-3 did not have a sex bias. We demonstrate here that male and female mice are prone to different types of cell death. Our data also suggest that estrogens and PARP-1 are two of the mediators of the sex-bias in cell death. We therefore propose that targeting cell death based on sex will lead to tailored and better treatments for each gender.     

Systematics of Old World Odontacolus Kieffer s.l. (Hymenoptera,Platygastridae s.l.): parasitoids?of?spider?eggs

The genera Odontacolus Kieffer and Cyphacolus Priesner are among the most distinctive platygastroid wasps because of their laterally compressed metasomal horn; however, their generic status has remained unclear. We present a morphological phylogenetic analysis comprising all 38 Old World and four Neotropical Odontacolus species and 13 Cyphacolus species, which demonstrates that the latter is monophyletic but nested within a somewhat poorly resolved Odontacolus. Based on these results Cyphacolus syn. n. is placed as a junior synonym of Odontacolus which is here redefined. The taxonomy of Old World Odontacolus s.str. is revised; the previously known species Odontacolus longiceps Kieffer (Seychelles), Odontacolus markadicus Veenakumari (India), Odontacolus spinosus (Dodd) (Australia) and Odontacolus hackeri (Dodd) (Australia) are re-described, and 32 new species are described: Odontacolus africanus Valerio & Austin sp. n. (Congo, Guinea, Kenya, Madagascar, Mozambique, South Africa, Uganda, Zimbabwe), Odontacolus aldrovandii Valerio & Austin sp. n. (Nepal), Odontacolus anningae Valerio & Austin sp. n. (Cameroon), Odontacolus australiensis Valerio & Austin sp. n. (Australia), Odontacolus baeri Valerio & Austin sp. n. (Australia), Odontacolus berryae Valerio & Austin sp. n. (Australia, New Zealand, Norfolk Island), Odontacolus bosei Valerio & Austin sp. n. (India, Malaysia, Sri Lanka), Odontacolus cardaleae Valerio & Austin sp. n. (Australia), Odontacolus darwini Valerio & Austin sp. n. (Thailand), Odontacolus dayi Valerio & Austin sp. n. (Indonesia), Odontacolus gallowayi Valerio & Austin sp. n. (Australia), Odontacolus gentingensis Valerio & Austin sp. n. (Malaysia), Odontacolus guineensis Valerio & Austin sp. n. (Guinea), Odontacolus harveyi Valerio & Austin sp. n. (Australia), Odontacolus heratyi Valerio & Austin sp. n. (Fiji), Odontacolus heydoni Valerio & Austin sp. n. (Malaysia, Thailand), Odontacolus irwini Valerio & Austin sp. n. (Fiji), Odontacolus jacksonae Valerio & Austin sp. n. (Cameroon, Guinea, Madagascar), Odontacolus kiau Valerio & Austin sp. n. (Papua New Guinea), Odontacolus lamarcki Valerio & Austin sp. n. (Thailand), Odontacolus madagascarensis Valerio & Austin sp. n. (Madagascar), Odontacolus mayri Valerio & Austin sp. n. (Indonesia, Thailand), Odontacolus mot Valerio & Austin sp. n. (India), Odontacolus noyesi Valerio & Austin sp. n. (India, Indonesia), Odontacolus pintoi Valerio & Austin sp. n. (Australia, New Zealand, Norfolk Island), Odontacolus schlingeri Valerio & Austin sp. n. (Fiji), Odontacolus sharkeyi Valerio & Austin sp. n. (Thailand), Odontacolus veroae Valerio & Austin sp. n. (Fiji), Odontacolus wallacei Valerio & Austin sp. n. (Australia, Indonesia, Malawi, Papua New Guinea), Odontacolus whitfieldi Valerio & Austin sp. n. (China, India, Indonesia, Sulawesi, Malaysia, Thailand, Vietnam), Odontacolus zborowskii Valerio & Austin sp. n. (Australia), and Odontacolus zimi Valerio & Austin sp. n. (Madagascar). In addition, all species of Cyphacolus are here transferred to Odontacolus: Odontacolus asheri (Valerio, Masner & Austin) comb. n. (Sri Lanka), Odontacolus axfordi (Valerio, Masner & Austin) comb. n. (Australia), Odontacolus bhowaliensis (Mani & Mukerjee) comb. n. (India), Odontacolus bouceki (Austin & Iqbal) comb. n. (Australia), Odontacolus copelandi (Valerio, Masner & Austin) comb. n. (Kenya, Nigeria, Zimbabwe, Thailand), Odontacolus diazae (Valerio, Masner & Austin) comb. n. (Kenya), Odontacolus harteni (Valerio, Masner & Austin) comb. n. (Yemen, Ivory Coast, Paskistan), Odontacolus jenningsi (Valerio, Masner & Austin) comb. n. (Australia), Odontacolus leblanci (Valerio, Masner & Austin) comb. n. (Guinea), Odontacolus lucianae (Valerio, Masner & Austin) comb. n. (Ivory Coast, Madagascar, South Africa, Swaziland, Zimbabwe), Odontacolus normani (Valerio, Masner & Austin) comb. n. (India, United Arab Emirates), Odontacolus sallyae (Valerio, Masner & Austin) comb. n. (Australia), Odontacolus tessae (Valerio, Masner & Austin) comb. n. (Australia), Odontacolus tullyae (Valerio, Masner & Austin) comb. n. (Australia), Odontacolus veniprivus (Priesner) comb. n. (Egypt), and Odontacolus watshami (Valerio, Masner & Austin) comb. n. (Africa, Madagascar). Two species of Odontacolus are transferred to the genus Idris Förster: Idris longispinosus (Girault) comb. n. and Idris amoenus (Kononova) comb. n., and Odontacolus doddi Austin syn. n. is placed as a junior synonym of Odontacolus spinosus (Dodd). Odontacolus markadicus, previously only known from India, is here recorded from Brunei, Malaysia, Sri Lanka, Thailand and Vietnam. The relationships, distribution and biology of Odontacolus are discussed, and a key is provided to identify all species.     

Characterization of Recombinant Rhamnogalacturonan α-l-Rhamnopyranosyl-(1,4)-α-d-Galactopyranosyluronide Lyase from Aspergillus aculeatus : An Enzyme That Fragments Rhamnogalacturonan I Regions of Pectin

The four major oligomeric reaction products from saponified modified hairy regions (MHR-S) from apple, produced by recombinant rhamnogalacturonan (RG) α-l-rhamnopyranosyl-(1,4)-α-d-galactopyranosyluronide lyase (rRG-lyase) from Aspergillus aculeatus, were isolated and characterized by ¹H-nuclear magnetic resonance spectroscopy. They contain an alternating RG backbone with a degree of polymerization of 4, 6, 8, and 10 and with an α-Δ-(4,5)-unsaturated d-galactopyranosyluronic acid at the nonreducing end and an l-rhamnopyranose at the reducing end. l-Rhamnopyranose units are substituted at C-4 with β-galactose. The maximum reaction rate of rRG-lyase toward MHR-S at pH 6.0 and 31°C was 28 units mg⁻¹. rRG-lyase and RG-hydrolase cleave the same alternating RG I subunit in MHR. Both of these enzymes fragment MHR by a multiple attack mechanism. The catalytic efficiency of rRG-lyase for MHR increases with decreasing degree of acetylation. Removal of arabinose side chains improves the action of rRG-lyase toward MHR-S. In contrast, removal of galactose side chains decreased the catalytic efficiency of rRG-lyase. Native RG-lyase was purified from A. aculeatus, characterized, and found to be similar to the rRG-lyase expressed in Aspergillus oryzae.     

Genome-wide analysis of Saccharomyces cerevisiae identifies cellular processes affecting intracellular aggregation of Alzheimer's amyloid-β42: importance of lipid homeostasis

Amyloid-β (Aβ)–containing plaques are a major neuropathological feature of Alzheimer''s disease (AD). The two major isoforms of Aβ peptide associated with AD are Aβ40 and Aβ42, of which the latter is highly prone to aggregation. Increased presence and aggregation of intracellular Aβ42 peptides is an early event in AD progression. Improved understanding of cellular processes affecting Aβ42 aggregation may have implications for development of therapeutic strategies. Aβ42 fused to green fluorescent protein (Aβ42-GFP) was expressed in ∼4600 mutants of a Saccharomyces cerevisiae genome-wide deletion library to identify proteins and cellular processes affecting intracellular Aβ42 aggregation by assessing the fluorescence of Aβ42-GFP. This screening identified 110 mutants exhibiting intense Aβ42-GFP–associated fluorescence. Four major cellular processes were overrepresented in the data set, including phospholipid homeostasis. Disruption of phosphatidylcholine, phosphatidylserine, and/or phosphatidylethanolamine metabolism had a major effect on intracellular Aβ42 aggregation and localization. Confocal microscopy indicated that Aβ42-GFP localization in the phospholipid mutants was juxtaposed to the nucleus, most likely associated with the endoplasmic reticulum (ER)/ER membrane. These data provide a genome-wide indication of cellular processes that affect intracellular Aβ42-GFP aggregation and may have important implications for understanding cellular mechanisms affecting intracellular Aβ42 aggregation and AD disease progression.