Characterization of novel components of the baculovirus per os infectivity factor complex

Baculovirus occlusion-derived virus (ODV) infects insect midgut cells under alkaline conditions, a process mediated by highly conserved per os infectivity factors (PIFs), P74 (PIF0), PIF1, PIF2, PIF3, PIF4, and PIF5 (ODV-E56). Previously, a multimolecular complex composed of PIF1, PIF2, PIF3, and P74 was identified which was proposed to play an essential role during ODV entry. Recently, more proteins have been identified that play important roles in ODV oral infectivity, including PIF4, PIF5, and SF58, which might work in concert with previously known PIFs to facilitate ODV infection. In order to understand the ODV entry mechanism, the identification of all components of the PIF complex is crucial. Hence, the aim of this study was to identify additional components of the PIF complex. Coimmunoprecipitation (CoIP) combined with proteomic analysis was used to identify the components of the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) PIF complex. PIF4 and P95 (AC83) were identified as components of the PIF complex while PIF5 was not, and this was confirmed with blue native PAGE and a second CoIP. Deletion of the pif4 gene impaired complex formation, but deletion of pif5 did not. Differentially denaturing SDS-PAGE further revealed that PIF4 forms a stable complex with PIF1, PIF2, and PIF3. P95 and P74 are more loosely associated with this complex. Three other proteins, AC5, AC68, and AC108 (homologue of SF58), were also found by the proteomic analysis to be associated with the PIF complex. Finally the functional significance of the PIF protein interactions is discussed.     

Parasitoids, predators and PCR: the use of diagnostic molecular markers in biological control of Arthropods

Abstract:  The polymerase chain reaction (PCR) revolutionized the field of diagnostics, and today it has routine applications in medical, veterinary, forensic and botanical sciences. The fields of biological control and insect pest management have generally been slow to adopt PCR-based diagnostics in comparison with other fields of science. However, there has been increasing interest in the use of molecular diagnostic tools in arthropod biological control. In applied entomology, molecular techniques have generally been used for insect identification and systematics; however, PCR-based techniques are increasingly becoming recognized as valuable tools in ecological studies. Here, we review research that has used PCR-based techniques for parasitoid and predator/prey identification and detection, and place these studies in the context of their contributions to biological control of arthropods. The status and future directions of diagnostic molecular markers in applied entomology and insect pest management are also discussed.     

Protein and lipid content of the rotifer Brachionus plicatilis during variable growth and feeding condition

Rotifers (Brachionus plicatilis) grown atdifferent growth rate ( = 0.05–0.39 d^–1)were analyzed for protein, lipid, fatty acids, aminoacids and free amino acids, and values are expressedin terms of individuals and dry weight. Increase ingrowth rate is equivalent with increased food rationof the individual rotifer, which responded by higheregg ratio. The protein content per individualrotifer increased by 60–80% when the growth rate increased, whereas the protein content per dryweight showed a slight, although insignificant,increase (p > 0.05). The lipid content perindividual was constant, whereas lipid per dryweight decreased when the growth rate increased. Theratio DHA/EPA decreased when the growth ratesincreased. The amino acids profile in percent oftotal amino acids showed low variation betweencultures maintained at different growth rates,whereas the values expressed in terms of amino acidper individual showed higher variation. The range ofvariation for free amino acids was more pronouncedthan for total amino acids.Short-term food enrichment of poorly fed rotifers( = 0.05 d^–1) with balanced protein richdiet resulted in increased protein and lipid contentper rotifer. The protein content per dry weightshowed only minor changes whereas lipid per dryweight increased. Contrary, short term enrichmentwith a lipid rich diet resulted in increased lipidcontent per individual rotifer and per dry weight,whereas the protein content per individual remainedconstant and the protein content per dry weightshowed a slight decrease.Our experiments show that the amount of protein, wasquite variable in rotifers, and that feeding andgrowth condition were decisive factors affecting it.The range of variation was large enough to be animportant factor during first feeding of marinelarvae, and should therefore be considered infeeding larvae.     

Investigation of the potential for microbial contamination of deep granitic aquifers during drilling using 16S rRNA gene sequencing and culturing methods

Total number of bacteria, viable counts of aerobic and anaerobic heterotrophic bacteria and 16S rRNA gene diversity were investigated during drilling of three boreholes in the walls of the Äspö hard rock laboratory tunnel, at depths ranging from 380 to 446 m below sea level. Water samples were taken from the drill water source, the drilling equipment and from the drilled boreholes. The drill water was kept under nitrogen atmosphere and all equipment was steam cleaned before the start of a new drilling. Total and viable counts of bacteria in the drilled boreholes were several orders of magnitude lower than in the samples from the drilling equipment, except for sulphate reducing bacteria. A total of 158 16S rRNA genes that were cloned from the drill water source, the drilling equipment and the drilled boreholes were partially sequenced. The drilled boreholes generally had a 16S rRNA diversity that differed from what was found in samples from the drilling equipment. Several of the sequences obtained could be identified on genus level as one of the genera Acinetobacter, Methylophilus, Pseudomonas and Shewanella. In conclusion, the tubing used for drill water supply constituted a source of bacterial contamination to the rest of the drilling equipment and the boreholes. The results show, using molecular and culturing methods, that although large numbers of contaminating bacteria were introduced to the boreholes during drilling, they did not establish in the borehole groundwater at detectable levels.     

Stimulation or inhibition of K+(86Rb+)influx in wheat roots depending on different ABA treatments

The regulatory role of abscisic acid (ABA) and kinetin on influx of K+(86RB+) IN tools of 7day old intact winter wheat which plant (Fritieun aestivum I ass starke 1 and 11) Was studied the inhibitory effect of 40,80 μM ABA in the uptake solution on K+(86RB+)influx was transiently stipulated pretreatment of the plants with ABA kinetin content enacted inhibitors effect caused by ABA. At low water potential in the uptake solution (05MPa)K+(86RB+) influx was slights higher in the presence of ABA than in is absence High humidity 123kpa ca 100% relative humidity (RID)around the shoots counteracted the inhibitory effect on k+(86RB+) influx caused by A,B,A IN the uptake solution the present data contain the hypothesis that when plants are subjected to conditions such as low water potential and low temperature. ABA stimulates K influx to facilitate water uptake.     

Margareta Ryberg (1946–2012): a personal tribute

We pay tribute to the life and work of Margareta Ryberg (1946-2012). She was an expert on the different forms of protochlorophyll(ide), their protein partners, and their transformations in angiosperms; on the structural aspects, and the nature of prolamellar bodies, as well as on the localization of light-dependent NADPH:protochlorophyllide oxido-reductase. She was a great teacher, who also loved gardening and handicraft. But above all, she was a beloved wife, mother, grandmother, and friend who will be deeply missed.     

Autographa californica Multiple Nucleopolyhedrovirus Core Gene ac96 Encodes a Per Os Infectivity Factor (pif-4)

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ac96 is a core gene, but its role in virus replication is still unknown. To determine its role in the baculovirus life cycle, we used the AcMNPV bacmid system to generate an ac96-null virus (vAc^96null). Our analyses showed that the absence of ac96 does not affect budded virus (BV) production or viral DNA replication in infected Sf9 cells. Western blotting and confocal immunofluorescence analysis showed that AC96 is expressed in both the cytoplasm and the nucleus throughout infection. In addition, AC96 was detected in the envelope fractions of both BV and occlusion-derived virus. Injection of vAc^96null BV into the hemocoel killed Trichoplusia ni larvae as efficiently as repaired and control viruses; however, vAc^96null was unable to infect the midgut tissue of Trichoplusia ni larvae when inoculated per os. Therefore, the results of this study show that ac96 encodes a new per os infectivity factor (PIF-4).The Baculoviridae comprise a large and diverse group of viruses that are pathogens of insects, mainly from the Lepidoptera, Hymenoptera, and Diptera. During the typical biphasic infection cycle, two structurally and functionally distinct enveloped virion phenotypes are produced: occlusion-derived virus (ODV) and budded virus (BV) (35). The primary infection cycle in animals begins in the midgut cell after occlusion bodies (OBs) are ingested. Upon ingestion, the OBs dissolve in the alkaline environment of the midgut, and the ODVs are released into the lumen of midgut (15, 16, 20). Virions pass through a disrupted peritrophic membrane, a process often facilitated by enhancins, a group of virus-encoded metalloproteases (38). Subsequently, ODVs bind to and fuse directly with the microvilli of midgut columnar epithelial cells. A protein receptor is proposed to mediate the process, since binding is proteinase sensitive and saturable (15, 16, 20). After the nucleocapsids are transported to the nuclei of the midgut cells, viral DNA is released, followed by gene expression, DNA replication, and assembly of progeny nucleocapsids. In the late phase of infection, newly formed nucleocapsids are transported to the cell membrane, bud from the cell, and acquire a new envelope from the basal membrane. The BVs spread via the hemolymph (16) and the tracheal system (8) into the other tissues of the insect, causing the secondary infection.Baculoviruses encode per os infectivity factors (PIFs) on the envelope surface of ODV to initiate the efficient primary infection in midgut. So far, four highly conserved core genes, p74 (pif-0), pif-1, pif-2, and pif-3, have been identified. The deletion of the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) p74 gene results in the complete elimination of the per os infectivity of OBs, while virions purified from mutant OBs were infectious when injected into the hemocoels of Trichoplusia ni or Heliothis virescens larvae (13, 17, 22). P74 is proposed to function as an ODV attachment protein that binds to a specific 30-kDa receptor protein on the primary target cells within the midgut (17, 39). PIF-1 was originally identified in Spodoptera littoralis NPV, where the deletion of pif-1 (spli7) resulted in viruses that were unable to infect S. littoralis larvae per os (21). PIF-2 was first identified in Spodoptera exigua MNPV, and the disruption of pif-2 resulted in the complete loss of per os infectivity for the host (11, 31). PIF-1 and PIF-2 have also been shown to participate in the binding of ODV to target cells in the midgut (28). PIF-3 (ac115) is also an essential factor for oral infection of AcMNPV. Although PIF-3 is not required for ODV attachment and fusion, it may mediate a critical downstream event, such as the translocation of ODV along microvilli during primary infection (28).AcMNPV, the archetype Alphabaculovirus of the Baculoviridae, has a double-stranded DNA genome of approximately 134 kbp that contains 154 predicted open reading frames (ORFs) (1). Comparative analysis of the 49 completely sequenced baculovirus genomes reveals 31 core genes that are conserved in all baculovirus genomes and are therefore likely to serve important roles in baculovirus life cycles (14, 26, 32, 37). Most core genes are related either to DNA replication, gene expression, packaging and assembly, or per os infection (37). Four core genes, ac68, p33 (ac92), ac96, and ac109, still have no known function or sequence similarities to proteins of known functions.In this study, an ac96-null mutant was constructed utilizing an AcMNPV bacmid, and the results showed that in tissue culture, ac96 was nonessential and was not required for viral DNA replication, ODV production, or BV production. However, in vivo assays demonstrated that the ac96-null virus was unable to infect midgut tissue when T. ni larvae were inoculated per os. The core gene ac96 therefore encodes a new per os infectivity factor, PIF-4.     

The expression of syndecan-1, syndecan-4 and decorin in healthy human breast tissue during the menstrual cycle

Background  

In order to unravel the interactions between the epithelium and the extra cellular matrix (ECM) in breast tissue progressing to cancer, it is necessary to understand the relevant interactions in healthy tissue under normal physiologic settings. Proteoglycans in the ECM play an important role in the signaling between the different tissue compartments. The proteoglycan decorin is abundant in the breast stroma. Decreased expression in breast cancer tissue is a sign of a poor tumor prognosis. The heparane sulphate proteoglycans syndecan-1 and syndecan-4 promote the integration of cellular adhesion and proliferation. The aim of this study was to investigate the gene expression and location of decorin, syndecan-1 and syndecan-4 in the healthy breast during the menstrual cycle.     

Formation of disulfide bonds and homodimers of the major cat allergen Fel d 1 equivalent to the natural allergen by expression in Escherichia coli

Dander from the domestic cat (Felis domesticus) is one of the most common causes of IgE-mediated allergy. Attempts to produce tetrameric folded major allergen Fel d 1 by recombinant methods with structural features similar to the natural allergen have been only partially successful. In this study, a recombinant folded Fel d 1 with molecular and biological properties similar to the natural counterpart was produced. A synthetic gene coding for direct fusion of the Fel d 1 chain 2 N-terminally to chain 1 was constructed by overlapping oligonucleotides in PCR. Escherichia coli expression resulted in a non-covalently associated homodimer with an apparent molecular mass of 30 kDa defined by size exclusion chromatography. Furthermore, each 19,177-Da subunit displayed a disulfide pattern identical to that found in the natural Fel d 1, i.e. Cys3(1) Cys73(2), Cys44(1)-Cys48(2), Cys70(1)-Cys7(2), as determined by electrospray mass spectrometry after tryptic digestion. Circular dichroism analysis showed identical folds of natural and recombinant Fel d 1. Furthermore, recombinant Fel d l reacted specifically with serum IgE, inducing expression of CD203c on basophils and lymphoproliferative responses in cat-allergic patients. The results show that the overall fold and immunological properties of the recombinant Fel d 1 are very similar to those of natural Fel d 1. Moreover, the recombinant Fel d 1 construct provides a tool for defining the three-dimensional structure of Fel d 1 and represents a reagent for diagnosis and allergen-specific immunotherapy of cat allergy.