Analysis of the quaternary structure of the putative HCMV portal protein PUL104

In this report we analyze the UL104 open reading frame of human cytomegalovirus (HCMV) genome that encodes the putative portal protein. An affinity-purified monospecific antiserum directed against a GST-UL104 fusion protein identified proteins of approximate M(r) 73000 and 145000 in HCMV-infected cells and purified virions. Furthermore, using an in vitro assay the ability of pUL104 to bind double-stranded DNA was shown. Analysis under native conditions of pUL104 revealed that the monomeric and dimeric forms of the protein also form high molecular weight complexes upon sucrose gradient centrifugation. The protein has been purified from recombinant baculovirus UL104 infected cells. The quaternary structure of rpUL104 was investigated by gel permeation chromatography and electron microscopy. The purified rpUL104 was found to assemble into high molecular weight complexes, a prerequisite of portal proteins which form channels for DNA import into capsids.     

Insights into the structure of human cytomegalovirus large terminase subunit pUL56

Terminases are a class of proteins which catalyze the generation of unit-length genomes during DNA packaging. These essential proteins are conserved throughout the herpesviruses and many double-stranded DNA bacteriophages. We have determined the structure of the large terminase subunit pUL56 of human cytomegalovirus, a highly pathogenic virus, to 2.6 nm resolution. Image analysis of purified pUL56 suggests that the molecule exists as a dimer formed by the association of two ring-like structures positioned on top of each other and connected by a pronounced density on one side. The 3D reconstruction of pUL56 provides first structural insights into the active protein.     

Specific short hairpin RNA-mediated inhibition of viral DNA packaging of human cytomegalovirus

To clearly demonstrate the role of the human cytomegalovirus (HCMV) portal protein pUL104 RNA interference was applied. Expressing cell lines were constructed by transduction of shRNAs via the infection with retroviral vectors. After infection of these cells with HCMV AD169 the expression of pUL104 was reduced up to 80% for shRNA S1 and 54% for shRNA S2 at late times of infection compared to controls. In addition, the inhibitory effect was corresponding with a decrease in viral mRNA and plaque formations. Electron microscopic analysis demonstrated that infection of cells expressing pUL104-specific shRNAs resulted in the inhibition of formation of replicative particles.     

Genetic and environmental heterogeneity of residual variance of weight traits in Nellore beef cattle

Background

Many studies have provided evidence of the existence of genetic heterogeneity of environmental variance, suggesting that it could be exploited to improve robustness and uniformity of livestock by selection. However, little is known about the perspectives of such a selection strategy in beef cattle.

Methods

A two-step approach was applied to study the genetic heterogeneity of residual variance of weight gain from birth to weaning and long-yearling weight in a Nellore beef cattle population. First, an animal model was fitted to the data and second, the influence of additive and environmental effects on the residual variance of these traits was investigated with different models, in which the log squared estimated residuals for each phenotypic record were analyzed using the restricted maximum likelihood method. Monte Carlo simulation was performed to assess the reliability of variance component estimates from the second step and the accuracy of estimated breeding values for residual variation.

Results

The results suggest that both genetic and environmental factors have an effect on the residual variance of weight gain from birth to weaning and long-yearling in Nellore beef cattle and that uniformity of these traits could be improved by selecting for lower residual variance, when considering a large amount of information to predict genetic merit for this criterion. Simulations suggested that using the two-step approach would lead to biased estimates of variance components, such that more adequate methods are needed to study the genetic heterogeneity of residual variance in beef cattle.     

Phylogenetic relationships of aroids and duckweeds (Araceae) inferred from coding and noncoding plastid DNA

Familial, subfamilial, and tribal monophyly and relationships of aroids and duckweeds were assessed by parsimony and Bayesian phylogenetic analyses of five regions of coding (rbcL, matK) and noncoding plastid DNA (partial trnK intron, trnL intron, trnL-trnF spacer) for exemplars of nearly all aroid and duckweed genera. Our analyses confirm the position of Lemna and its allies (formerly Lemnaceae) within Araceae as the well-supported sister group of all aroids except Gymnostachydoideae and Orontioideae. The last two subfamilies form the sister clade of the rest of the family. Monophyly of subfamilies Orontioideae, Pothoideae, Monsteroideae, and Lasioideae is supported, but Aroideae are paraphyletic if Calla is maintained in its own subfamily (Calloideae). Our results suggest expansion of the recently proposed subfamily Zamioculcadoideae (Zamioculcas, Gonatopus) to include Stylochaeton and identify problems in the current delimitation of tribes Anadendreae, Heteropsideae, and Monstereae (Monsteroideae), Caladieae/Zomicarpeae, and Colocasieae (Aroideae). Canalization of traits of the spathe and spadix considered typical of Araceae evolved after the split of Gymnostachydoideae, Orontioideae, and Lemnoideae. An association with aquatic habitats is a plesiomorphic attribute in Araceae, occurring in the helophytic Orontioideae and free-floating Lemnoideae, but evolving independently in various derived aroid lineages including free-floating Pistia (Aroideae).     

Identification of Glycoprotein gpTRL10 as a Structural Component of Human Cytomegalovirus

Human cytomegalovirus (HCMV) has a coding capacity for glycoproteins which far exceeds that of other herpesviruses. Few of these proteins have been characterized. We have investigated the gene product(s) of reading frame 10, which is present in both the internal and terminal repeat regions of HCMV strain AD169 and only once in clinical isolates. The putative protein product is a 171-amino-acid glycoprotein with a theoretical mass of 20.5 kDa. We characterized the protein encoded by this reading frame in the laboratory strain AD169 and a recent isolate, TB40E. The results from both strains were comparable. Northern blot analyses showed that the gene was transcribed with early/late kinetics. Two proteins of 22 and 23.5-kDa were detected in virus-infected cells and in cells transiently expressing recombinant TRL10. Both forms contained only high-mannose-linked carbohydrate modifications. In addition, virus-infected cells expressed small amounts of the protein modified with complex N-linked sugars. Image analysis localized transiently expressed TRL10 to the endoplasmic reticulum. Immunoblot analyses as well as immunoelectron microscopy of purified virions demonstrated that TRL10 represents a structural component of the virus particle. Immunoblot analysis in the absence of reducing agents indicated that TRL10, like the other HCMV envelope glycoproteins, is present in a disulfide-linked complex. Sequence analysis of the TRL10 coding region in nine low-passage clinical isolates revealed strain-specific variation. In summary, the protein product of the TRL10 open reading frame represents a novel structural glycoprotein of HCMV and was termed gpTRL10.     

Lack of Significant Elevation of Myeloid-Derived Suppressor Cells in Peripheral Blood of Chronically Hepatitis C Virus-Infected Individuals

Myeloid-derived suppressor cells (MDSC) are immature myeloid cells with immunosuppressive function. Compared to the level in healthy controls (HC), no elevation of MDSC in chronic hepatitis C (cHEP-C) patients was found, and there was no difference in MDSC based on genotype or viral load (P > 0.25). Moreover, MDSC of cHEP-C patients inhibited CD8 T cell function as efficiently as MDSC of HC did. Since we detected neither quantitative nor qualitative differences in MDSC of cHEP-C patients relative to those of HC, we postulate that MDSC in peripheral blood are most likely not significant regarding immune dysfunction in cHEP-C.     

Single cell recordings reveal hydroxydanaidal as the volatile compound attracting insects to pyrrolizidine alkaloids

Employment of electrophysiology in combination with simple chemical techniques elucidated the volatile which permit the localization of sources of pyrrolizidine alkaloids (PAs) by insects exploiting these secondary plant metabolites.Single cell recordings in Rhodogastria moths (Arctiidae) revealed a physiologically clearly separable type of antennal sensilla basiconica (SB II) which responds to headspace air of certain PAs (e.g. monocrotaline), but not to a great variety of odorants. However, stimulus sources of 1 g were required to elicit responses and the maximum frequency (obtained with stimulus sources of 5 mg) was only 50 imps/s. This suggested the occurrence of small amounts of airborne PA degradation products.Extraction and hydrolysis experiments in combination with thin-layer chromatography and using the sensory responses of antennal receptor cells as biological detectors eventually demonstrated that the dihydropyrrolizine hydroxydanaidal emanates in small amounts from sources of those PAs which contain retronecine and heliotridine, respectively, as necine moiety. This substance was also implicated as the volatile mediating attraction of the insects to PA-containing plants, as well as to artificial PA-baits. With respect to the high sensitivity and specificity of SB II-receptor cells to hydroxydanaidal, in particular to its R(-)-enantiomer, they are analogous to the well studied receptor cells for sex-attractant pheromones in Lepidoptera. Similar results were obtained with Danaus (Danainae) and Euchromia (Ctenuchiidae). Initial behavioural tests have proven the attractive power of hydroxydanaidal for PA-insects and thus corroborate our interpretation of the electrophysiological findings.

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Zusammenfassung Elektrophysiologische und einfache chemische Verfahren erlaubten die Eingrenzung und Identifikation des reizwirksamen Prinzips, welches Insekten, die Pyrrolizidin-Alkaloide (PA) nutzen, die Orientierung zu PA-Quellen ermöglicht.Einzelzellableitungen von antennalen Sensilla basiconica bei Rhodogastria (Lepidoptera: Arctiidae) zeigten einen morphologisch nicht unterscheidbaren physiologischen Typ, dessen Rezeptorzellen sich durch eine sehr geringe Spontanaktivität auszeichneten und ausschließlich auf PA-Duft reagierten (SB II). Allerdings wurden (z. B. mit Monocrotalin) Reizquellen-Beladungen von 1 g benötigt, um überschwellige Antworten auszulösen, und die maximal erreichte Aktivität (bei Reizung mit 5 mg) betrug lediglich 50 Imp./s; dies deutete auf das Auftreten von kleinen Mengen eines volatilen Abbauprodukts von PA hin.SB II wurden als biologische Detektoren verwendet, um — kombiniert mit Dünnschichtchromatographie — die Reizwirksamkeit von Produkten von Hydrolyse- und Extraktions-Experimenten mit PA zu testen. Auf diese Weise konnte schließlich gezeigt werden, daß von PA mit Retronecin bzw. Heliotridin als Necin geringe Mengen des Dihydropyrrolizins Hydroxydanaidal (Fig. 1G, H) ausgehen. Synthetisches Hydroxydanaidal stellte sich als der bestwirksame Reiz für SB II-Rezeptorzellen heraus, die bzgl. Spezifität und Sensitivität (insbesondere für R(-)-Hydroxydanaidal) den gut untersuchten Rezeptoren für weibliche Sexualpheromone bei Nachtfaltern vergleichbar sind. Entsprechende Ergebnisse wurden auch mit Danaus (Danainae) und Euchromia (Ctenuchiidae) gewonnen. Erste Verhaltensversuche beweisen die Lockwirkung von Hydroxydanaidal für PA-Insekten und bestätigen die Interpretation der elektrophysiologischen Befunde.