Distribution pattern of phthirapterans infesting certain common Indian birds

The prevalence and frequency distribution patterns of 10 phthirapteran species infesting house sparrows, Indian parakeets, common mynas, and white breasted kingfishers were recorded in the district of Rampur, India, during 2004-05. The sample mean abundances, mean intensities, range of infestations, variance to mean ratios, values of the exponent of the negative binomial distribution, and the indices of discrepancy were also computed. Frequency distribution patterns of all phthirapteran species were skewed, but the observed frequencies did not correspond to the negative binomial distribution. Thus, adult-nymph ratios varied in different species from 1:0.53 to 1:1.25. Sex ratios of different phthirapteran species ranged from 1:1.10 to 1:1.65 and were female biased.     

p-Hydroxyphenylpyruvate dioxygenase is a herbicidal target site for beta-triketones from Leptospermum scoparium

p-Hydroxyphenylpyruvate dioxygenase (HPPD) is a key enzyme in tyrosine catabolism and is the molecular target site of β-triketone pharmacophores used to treat hypertyrosinemia in humans. In plants, HPPD is involved in the biosynthesis of prenyl quinones and tocopherols, and is the target site of β-triketone herbicides. The β-triketone-rich essential oil of manuka (Leptospermum scoparium), and its components leptospermone, grandiflorone and flavesone were tested for their activity in whole-plant bioassays and for their potency against HPPD. The achlorophyllous phenotype of developing plants exposed to manuka oil or its purified β-triketone components was similar to that of plants exposed to the synthetic HPPD inhibitor sulcotrione. The triketone-rich fraction and leptospermone were approximatively 10 times more active than that of the crude manuka oil, with I₅₀ values of 1.45, 0.96 and 11.5 μg mL⁻¹, respectively. The effect of these samples on carotenoid levels was similar. Unlike their synthetic counterpart, steady-state O₂ consumption experiments revealed that the natural triketones were competitive reversible inhibitors of HPPD. Dose-response curves against the enzyme activity of HPPD provided apparent I₅₀ values 15.0, 4.02, 3.14, 0.22 μg mL⁻¹ for manuka oil, triketone-rich fraction, leptospermone and grandiflorone, respectively. Flavesone was not active. Structure-activity relationships indicate that the size and lipophilicity of the side-chain affected the potency of the compounds. Computational analysis of the catalytic domain of HPPD indicates that a lipophilic domain proximate from the Fe²⁺ favors the binding of ligands with lipophilic moieties.     

Population expansion of the poultry fluff louse, Goniocotes gallinae (De Geer, 1778) (Ischnocera, Phthiraptera)

A study was undertaken to determine the rate of population expansion of an ischnoceran Phthiraptera, Goniocotes gallinae (infesting the domestic fowl, Gallus gallus domesticus), on the basis of in vitro and in vivo studies. Data obtained from in vitro rearing were utilized to construct the life history of the louse. The values of the gross reproductive rate (12.49 female eggs/female), net reproductive rate (8.31 female eggs/female), mean length of generation (36.91 days), precise generation time (35.65 days), finite rate of increase (1.06 female/days), and intrinsic rate of natural increase (0.059) of G. gallinae were determined. However, doubling-time values recorded during in vivo studies (14 days) was comparatively higher than those during in vitro rearing (11.73 days).     

USP3 counteracts RNF168 via deubiquitinating H2A and γH2AX at lysine 13 and 15

Histone ubiquitination plays a vital role in DNA damage response (DDR), which is important for maintaining genomic integrity in eukaryotic cells. In DDR, ubiquitination of histone H2A and γH2AX by the concerted action of ubiquitin (Ub) ligases, RNF168 and RNF8, generates a cascade of ubiquitination signaling. However, little is known about deubiquitinating enzymes (DUBs) that may catalyze the removal of Ub from these histones. This study demonstrated that USP3, an apparent DUB for mono-ubiquitinated H2A, is indeed the enzyme for deubiquitinating Ub conjugates of γH2AX and H2A from lysine sites, where the ubiquitination is initiated by RNF168. Here, we showed that ectopic expression of USP3 led to the deubiquitination of both H2A and γH2AX in response to UV-induced DNA damage. Moreover, ectopic USP3 expression abrogated FK2 antibody-reactive Ub-conjugate foci, which co-localize with damage-induced γH2AX foci. In addition, USP3 overexpression impaired the accumulation of downstream repair factors BRCA1 and 53BP1 at the damage sites in response to both UV and γ-irradiation. We further identified that the USP3 removes Ub at lysine 13 and 15 of H2A and γH2AX, as well as lysine 118 and 119 of H2AX in response to DNA damage. Taken together, the results suggested that USP3 is a negative regulator of ubiquitination signaling, counteracting RNF168- and RNF8-mediated ubiquitination.     

Plasmodium falciparum Infection Induces Expression of a Mosquito Salivary Protein (Agaphelin) That Targets Neutrophil Function and Inhibits Thrombosis without Impairing Hemostasis

Background

Invasion of mosquito salivary glands (SGs) by Plasmodium falciparum sporozoites is an essential step in the malaria life cycle. How infection modulates gene expression, and affects hematophagy remains unclear.

Principal Findings

Using Affimetrix chip microarray, we found that at least 43 genes are differentially expressed in the glands of Plasmodium falciparum-infected Anopheles gambiae mosquitoes. Among the upregulated genes, one codes for Agaphelin, a 58-amino acid protein containing a single Kazal domain with a Leu in the P1 position. Agaphelin displays high homology to orthologs present in Aedes sp and Culex sp salivary glands, indicating an evolutionarily expanded family. Kinetics and surface plasmon resonance experiments determined that chemically synthesized Agaphelin behaves as a slow and tight inhibitor of neutrophil elastase (K_D∼10 nM), but does not affect other enzymes, nor promotes vasodilation, or exhibit antimicrobial activity. TAXIscan chamber assay revealed that Agaphelin inhibits neutrophil chemotaxis toward fMLP, affecting several parameter associated with cell migration. In addition, Agaphelin reduces paw edema formation and accumulation of tissue myeloperoxidase triggered by injection of carrageenan in mice. Agaphelin also blocks elastase/cathepsin-mediated platelet aggregation, abrogates elastase-mediated cleavage of tissue factor pathway inhibitor, and attenuates neutrophil-induced coagulation. Notably, Agaphelin inhibits neutrophil extracellular traps (NETs) formation and prevents FeCl₃-induced arterial thrombosis, without impairing hemostasis.

Conclusions

Blockade of neutrophil elastase emerges as a novel antihemostatic mechanism in hematophagy; it also supports the notion that neutrophils and the innate immune response are targets for antithrombotic therapy. In addition, Agaphelin is the first antihemostatic whose expression is induced by Plasmodium sp infection. These results suggest that an important interplay takes place in parasite-vector-host interactions.     

A Highlights from MBoC Selection: Systematic identification of pathological lamin A interactors

Laminopathies are a collection of phenotypically diverse diseases that include muscular dystrophies, cardiomyopathies, lipodystrophies, and premature aging syndromes. Laminopathies are caused by >300 distinct mutations in the LMNA gene, which encodes the nuclear intermediate filament proteins lamin A and C, two major architectural elements of the mammalian cell nucleus. The genotype–phenotype relationship and the basis for the pronounced tissue specificity of laminopathies are poorly understood. Here we seek to identify on a global scale lamin A–binding partners whose interaction is affected by disease-relevant LMNA mutations. In a screen of a human genome–wide ORFeome library, we identified and validated 337 lamin A–binding proteins. Testing them against 89 known lamin A disease mutations identified 50 disease-associated interactors. Association of progerin, the lamin A isoform responsible for the premature aging disorder Hutchinson–Gilford progeria syndrome, with its partners was largely mediated by farnesylation. Mapping of the interaction sites on lamin A identified the immunoglobulin G (IgG)–like domain as an interaction hotspot and demonstrated that lamin A variants, which destabilize the Ig-like domain, affect protein–protein interactions more globally than mutations of surface residues. Analysis of a set of LMNA mutations in a single residue, which result in three phenotypically distinct diseases, identified disease-specific interactors. The results represent a systematic map of disease-relevant lamin A interactors and suggest loss of tissue-specific lamin A interactions as a mechanism for the tissue-specific appearance of laminopathic phenotypes.     

Identification and Validation of Novel Spinophilin-associated Proteins in Rodent Striatum Using an Enhanced ex Vivo Shotgun Proteomics Approach

Spinophilin regulates excitatory postsynaptic function and morphology during development by virtue of its interactions with filamentous actin, protein phosphatase 1, and a plethora of additional signaling proteins. To provide insight into the roles of spinophilin in mature brain, we characterized the spinophilin interactome in subcellular fractions solubilized from adult rodent striatum by using a shotgun proteomics approach to identify proteins in spinophilin immune complexes. Initial analyses of samples generated using a mouse spinophilin antibody detected 23 proteins that were not present in an IgG control sample; however, 12 of these proteins were detected in complexes isolated from spinophilin knock-out tissue. A second screen using two different spinophilin antibodies and either knock-out or IgG controls identified a total of 125 proteins. The probability of each protein being specifically associated with spinophilin in each sample was calculated, and proteins were ranked according to a χ² analysis of the probabilities from analyses of multiple samples. Spinophilin and the known associated proteins neurabin and multiple isoforms of protein phosphatase 1 were specifically detected. Multiple, novel, spinophilin-associated proteins (myosin Va, calcium/calmodulin-dependent protein kinase II, neurofilament light polypeptide, postsynaptic density 95, α-actinin, and densin) were then shown to interact with GST fusion proteins containing fragments of spinophilin. Additional biochemical and transfected cell imaging studies showed that α-actinin and densin directly interact with residues 151–300 and 446–817, respectively, of spinophilin. Taken together, we have developed a multi-antibody, shotgun proteomics approach to characterize protein interactomes in native tissues, delineating the importance of knock-out tissue controls and providing novel insights into the nature and function of the spinophilin interactome in mature striatum.Genomic sequencing has revealed the full repertoire of ∼20,000 proteins that can be expressed in most mammals. Innate biochemical or enzymatic activities of many proteins are critical to their function, but these activities are often modified by interactions with other proteins. Moreover, many proteins have no known catalytic activity and are thought to serve structural roles in assembling protein complexes, greatly increasing the efficiency and fidelity of intracellular processes. Thus, systematic definition of protein interactomes promises tremendous insight into biochemical mechanisms underlying the functions of many proteins.A prime example of the importance of protein-protein interactions for modifying biological function is the postsynaptic density (PSD),¹ an actin-rich organelle localized to neuronal dendritic spines that contains receptors, kinases, phosphatases, and scaffolding proteins (1, 2). Dynamic changes in enzymatic activities and protein-protein interactions underlie changes in the size and shape of both PSDs and dendritic spines as well as the modulation of PSD-targeted neurotransmitter receptors that are critical for synaptic plasticity, learning, and memory. Furthermore, dendritic spine morphology and number are altered in many neurological disorders, including Parkinson disease (PD), Angelman syndrome, and fragile X syndrome (3–7).Spinophilin (neurabin II) is an F-actin- and protein phosphatase 1 (PP1)-binding protein with no known catalytic function (8–10). It is highly expressed in brain and is localized to dendritic spines and PSDs where it plays a key role targeting PP1 to regulate synaptic plasticity, learning, and memory (11–14). Spinophilin associates with its homolog neurabin, which is also a PP1- and F-actin-binding protein that regulates synaptic plasticity and dendrite morphology (14–16). The interaction between spinophilin and the γ1 isoform of PP1 is enhanced in an animal model of PD (17), perhaps contributing to the altered phosphorylation of synaptic proteins, such as CaMKII and glutamate receptor subunits observed following dopamine (DA) depletion (18–20). DA depletion also decreases the number of dendritic spines on striatal medium spiny neurons (4, 5). Spine density is regulated by dynamic changes in the F-actin cytoskeleton, and spinophilin regulates dendritic spine density during development (21). Indeed, candidate protein or generic protein-protein interaction screens have identified many additional spinophilin-associated proteins (SpAPs) that modulate F-actin dynamics and/or cell morphology (22–27; for a review, see Ref. 28), consistent with the idea that spinophilin is an archetypical scaffolding protein. However, these interactions have mostly been characterized in vitro and/or following protein overexpression in cultured cells, and the inter-relationship of these interactions in vivo is largely unknown. Although the spinophilin interactome appears to dictate the biological roles of spinophilin, the composition of these complexes in the mature brain is poorly understood.Co-immunoprecipitation is commonly used to confirm the biological relevance of specific bivalent protein-protein interactions in native tissues that were initially identified using generic molecular approaches, such as yeast two-hybrid screening. Prior studies combined this approach with mass spectrometry-based proteomics methods to more broadly characterize the composition of mammalian signaling complexes and the PSD interactome, such as the signalosome associated with synaptic N-methyl-d-aspartate receptors (29) and complexes associated with other PSD-enriched proteins (30). In addition, proteomics methodologies were used to identify over 1100 protein components of the PSD (30). Indeed, the potential for shotgun proteomics studies to provide novel insights into protein function in the brain is increasingly recognized (31). Moreover, computational approaches are being developed to identify potential protein-protein interactions (32). However, validation of specific interactions among the very large data sets of candidates typically identified using these approaches can be daunting. In addition, most proteomics analyses have relied on a single antibody to the target protein of interest with, at best, an unrelated non-immune IgG as a negative control, necessitating the use of very high quality antibodies.We developed a systematic shotgun proteomics approach to define protein interactomes in a native tissue context. We used this approach to characterize the composition of spinophilin complexes isolated from rodent striatum and confirmed the association of multiple, novel SpAPs. Furthermore, we extensively characterized the interaction of two additional SpAPs, α-actinin and densin, using biochemical and imaging techniques. Our studies directly illustrate the importance of appropriate subcellular fractionation conditions, using multiple antibodies to the protein of interest, and the underappreciated, critical role of analyzing parallel samples prepared from knock-out (KO) animals. Thus, our findings demonstrate a methodological framework with key controls that can be broadly applied to characterizing protein interactomes, in addition to providing novel insights into the role of spinophilin in controlling synaptic signaling.     

Phaeohyphomycosis Caused by <Emphasis Type="Italic">Rhytidhysteron rufulum</Emphasis> and Review of Literature

Phaeohyphomycosis is caused by a heterogeneous group of mycelial dematiaceous (phaeoid) fungi, which produce melanin pigment. This condition is often confused with chromoblastomycosis. Rhytidhysteron is a dematiaceous fungus, which has been recently found to be causing human infections. Till date only three cases of infection with Rhytidhysteron rufulum have been reported in the literature. All three cases have been from North India. Hereby, we present another two cases where Rhytidhysteron was isolated. Both the patients belonged to Chandigarh (India) and presented with subcutaneous lesions. The isolates were confirmed by ITS sequencing. Both the patients were immunocompetent and gave no history of trauma or any other predisposing factor. Phaeohyphomycosis are often missed due to lack of knowledge regarding the fungi causing the infections and there is need for clinical, pathological and microbiological correlation for effective diagnosis and treatment in these cases.