Crystallization of Hevamine, an Enzyme with Lysozyme/Chitinase Activity from Hevea brasiliensis Latex

Hevamine, an enzyme with both lysozyme and chitinase activity, was isolated and purified from Hevea brasiliensis (rubber tree) latex. The enzyme (molecular weight 29,000) is homologous to certain “pathogenesis-related” proteins from plants, but not to hen egg-white or phage T4 lysozyme. To investigate the atomic details of the substrate specificity and the cause for hevamine’s low pH optimum (pH 4.0), we have crystallized two hevamine isozymes as a first step towards a high-resolution X-ray structure determination. Suitable crystals were obtained at room temperature from hanging drop experiments by vapor diffusion against 1.7 M to 3.4 M-NaCl (pH 5.0 to 9.0) for the major isozyme, and by vapor diffusion against 2.5 M to 4.3 M-NaCl (pH 5.0 to 8.0) for the minor one. Both isozymes give the same crystal morphology and space group. Their space group is P212121 with cell dimensions a = 82.3 Å, b = 58.1 Å and c = 52.5 Å (1 Å = 0.1 nm). The crystals diffract to at least 2.0 Å resolution

Human Adenovirus come indicatore di contaminazione biologica in matrici idriche

In Italia la normativa vigente non prevede virus come indicatori di contaminazione biologica. Nello specifico per le acque reflue, in uscita dall’impianto di recupero, gli unici indicatori presi in considerazione sono E. Coli e Salmonella (Allegato D.M. 185/03). Mentre per le acque di balneazione sono E. Coli ed Enterococchi (2006/7/CE), così come per le acque destinate al consumo umano (D.lgs. n. 31/2001). Per aerosol e superfici non vi sono normative a riguardo. Numerosi studi hanno però fatto emergere che la presenza di questi indicatori non è esaustiva per predire la contaminazione biologica delle matrici idriche in esame. Se da un lato sono in grado di rivelare una contaminazione e quindi un rischio per la salute, dall’altro non sono parametri correlabili con la presenza di virus e protozoi. Infatti, laddove E. Coli ed Enterococchi sono assenti, possono essere presenti virus umani come Human Adenovirus, Rotavirus, Epatite A (HAV), Enterovirus, Norovirus GII. Tra questi Human Adenovirus risulta essere ritrovato con maggior frequenza nelle matrici. In studi effettuati su acqua di mare tale virus risulta presente nel 21% dei campioni in cui invece erano totalmente assenti E. Coli ed Enterococchi (patogeni indice suggeriti dalla normativa). Tale dato si è riscontrato anche nelle acque di fiume dove il 72% dei campioni, negativo per E. Coli ed Enterococchi, è per il 63% positivo per Human Adenovirus. Nelle acque trattate il 46% dei campioni che è negativo per E. Coli ed Enterococchi è invece positivo invece per il 57% a Human Adenovirus. Questo lavoro si è concentrato appunto su Human Adenovirus, un virus della famiglia di Adenoviridae, genere Mastadenovirus, che conta 51 diversi sierotipi in grado di infettare l’uomo, divisi in 6 sottogeneri ( indicati con le lettere da A a F) e 51 sierotipi ( indicati con i numeri). E’ un virus a doppio filamento di DNA, formato da un capside icosaedrico privo di envelope e con un diametro di circa 35-40 nm. La trasmissione del virus avviene per via oro-fecale e respiratoria, mediante secrezioni respiratorie e oculari. Si localizza a livello di del tratto respiratorio, intestinale e della congiuntiva provocando molte patologie come: gastroenteriti, congiuntiviti, patologie respiratorie e infezioni del tratto urinario. E’ eliminato all’esterno mediante feci, urina, secrezioni oro-faringee e liquido congiuntivale. Lo scopo di questo lavoro è proporre Human Adenovirus come patogeno indice di contaminazione biologica in differenti matrici idriche da affiancare agli indicatori tradizionali. Tale scelta deriva dal fatto che Human Adenovirus è ubiquitario, ha elevata resistenza ambientale e ai trattamenti di disinfezione chimico-fisici. Inoltre ha elevata specificità per l’ospite umano il quale rilascia nell’ambiente un’alta concentrazione di particelle virali anche dopo molto tempo dall’infezione. Sono stati analizzati campioni di liquame in entrata e uscita dal depuratore, acqua sperimentalmente trattata, acqua di mare e acqua di fiume. I campioni successivamente sono stati esaminati con procedure standardizzate in house per passare poi all’analisi molecolare mediante la qPCR (Real Time PCR) con protocollo specifico per Human Adenovirus. Nella maggior parte dei campioni analizzati si è riscontrata la presenza del genoma di Human Adenovirus a concentrazioni variabili (liquame in entrata(1,7 x 1010 ± 7,9) e uscita dal depuratore(4,7 x 108 ± 11), acqua sperimentalmente trattata(1,6 x 102 ± 31), acqua di mare(9,3 x 103 ± 2,4) e acqua di fiume(8,5 x 102 ± 27), confermando la sua diffusione ubiquitaria nell’ambiente. I risultati ottenuti con questo lavoro concordano con quelli di precedenti monitoraggi confermano che Human Adenovirus potrebbe essere un ottimo candidato come indicatore di contaminazione biologica da affiancare a quelli tradizionali, per diverse matrici idriche, grazie anche alla facilità con cui, tramite qPCR, si può rilevare e quantificare la sua presenza

Ancient DNA Elucidates the Controversy about the Flightless Island Hens (Gallinula sp.) of Tristan da Cunha

A persistent controversy surrounds the flightless island hen of Tristan da Cunha, Gallinula nesiotis. Some believe that it became extinct by the end of the 19th century. Others suppose that it still inhabits Tristan. There is no consensus about Gallinula comeri, the name introduced for the flightless moorhen from the nearby island of Gough. On the basis of DNA sequencing of both recently collected and historical material, we conclude that G. nesiotis and G. comeri are different taxa, that G. nesiotis indeed became extinct, and that G. comeri now inhabits both islands. This study confirms that among gallinules seemingly radical adaptations (such as the loss of flight) can readily evolve in parallel on different islands, while conspicuous changes in other morphological characters fail to occur

Functional Role of Glutamine 28 and Arginine 39 in Double Stranded RNA Cleavage by Human Pancreatic Ribonuclease

Human pancreatic ribonuclease (HPR), a member of RNase A superfamily, has a high activity on double stranded (ds) RNA. By virtue of this activity HPR appears to be involved in the host-defense against pathogenic viruses. To delineate the mechanism of dsRNA cleavage by HPR, we have investigated the role of glutamine 28 and arginine 39 of HPR in its activity on dsRNA. A non-basic residue glycine 38, earlier shown to be important for dsRNA cleavage by HPR was also included in the study in the context of glutamine 28 and arginine 39. Nine variants of HPR respectively containing Q28A, Q28L, R39A, G38D, Q28A/R39A, Q28L/R39A, Q28A/G38D, R39A/G38D and Q28A/G38D/R39A mutations were generated and functionally characterized. The far-UV CD-spectral analysis revealed all variants, except R39A, to have structures similar to that of HPR. The catalytic activity of all HPR variants on single stranded RNA substrate was similar to that of HPR, whereas on dsRNA, the catalytic efficiency of all single residue variants, except for the Q28L, was significantly reduced. The dsRNA cleavage activity of R39A/G38D and Q28A/G38D/R39A variants was most drastically reduced to 4% of that of HPR. The variants having reduced dsRNA cleavage activity also had reduction in their dsDNA melting activity and thermal stability. Our results indicate that in HPR both glutamine 28 and arginine 39 are important for the cleavage of dsRNA. Although these residues are not directly involved in catalysis, both arginine 39 and glutamine 28 appear to be facilitating a productive substrate-enzyme interaction during the dsRNA cleavage by HPR

Protist-Type Lysozymes of the Nematode Caenorhabditis elegans Contribute to Resistance against Pathogenic Bacillus thuringiensis

Pathogens represent a universal threat to other living organisms. Most organisms express antimicrobial proteins and peptides, such as lysozymes, as a protection against these challenges. The nematode Caenorhabditis elegans harbours 15 phylogenetically diverse lysozyme genes, belonging to two distinct types, the protist- or Entamoeba-type (lys genes) and the invertebrate-type (ilys genes) lysozymes. In the present study we characterized the role of several protist-type lysozyme genes in defence against a nematocidal strain of the Gram-positive bacterium Bacillus thuringiensis. Based on microarray and subsequent qRT-PCR gene expression analysis, we identified protist-type lysozyme genes as one of the differentially transcribed gene classes after infection. A functional genetic analysis was performed for three of these genes, each belonging to a distinct evolutionary lineage within the protist-type lysozymes (lys-2, lys-5, and lys-7). Their knock-out led to decreased pathogen resistance in all three cases, while an increase in resistance was observed when two out of three tested genes were overexpressed in transgenic lines (lys-5, lys-7, but not lys-2). We conclude that the lysozyme genes lys-5, lys-7, and possibly lys-2 contribute to resistance against B. thuringiensis, thus highlighting the particular role of lysozymes in the nematode's defence against pathogens

A Honey Bee Hexamerin, HEX 70a, Is Likely to Play an Intranuclear Role in Developing and Mature Ovarioles and Testioles

Insect hexamerins have long been known as storage proteins that are massively synthesized by the larval fat body and secreted into hemolymph. Following the larval-to-pupal molt, hexamerins are sequestered by the fat body via receptor-mediated endocytosis, broken up, and used as amino acid resources for metamorphosis. In the honey bee, the transcript and protein subunit of a hexamerin, HEX 70a, were also detected in ovaries and testes. Aiming to identify the subcellular localization of HEX 70a in the female and male gonads, we used a specific antibody in whole mount preparations of ovaries and testes for analysis by confocal laser-scanning microscopy. Intranuclear HEX 70a foci were evidenced in germ and somatic cells of ovarioles and testioles of pharate-adult workers and drones, suggesting a regulatory or structural role. Following injection of the thymidine analog EdU we observed co-labeling with HEX 70a in ovariole cell nuclei, inferring possible HEX 70a involvement in cell proliferation. Further support to this hypothesis came from an injection of anti-HEX 70a into newly ecdysed queen pupae where it had a negative effect on ovariole thickening. HEX 70a foci were also detected in ovarioles of egg laying queens, particularly in the nuclei of the highly polyploid nurse cells and in proliferating follicle cells. Additional roles for this storage protein are indicated by the detection of nuclear HEX 70a foci in post-meiotic spermatids and spermatozoa. Taken together, these results imply undescribed roles for HEX 70a in the developing gonads of the honey bee and raise the possibility that other hexamerins may also have tissue specific functions

THE PRIMARY STRUCTURE OF LANGUR (PRESBYTIS-ENTELLUS) PANCREATIC RIBONUCLEASE - ADAPTIVE FEATURES IN DIGESTIVE ENZYMES IN MAMMALS

The primary structure of pancreatic ribonuclease from langur (Presbytis entellus) has been determined. This sequence differs from that of human pancreatic ribonuclease at 14 (11%) of the amino acid positions. Eight of these 14 differences involve changes of charge, with the langur enzyme having five fewer positive charges than the human enzyme. The difference in charge between human and langur ribonuclease may be an adaptation to the different requirements for a nondigestive and a digestive role, respectively. A number of similarities in expression, gene duplications, and properties between mammalian ribonucleases and lysozymes have been observed, indicating similar adaptations in both enzyme systems