Efficient Nuclear Transport of Structurally Disturbed Cargo: Mutations in a Cargo Protein Switch Its Cognate Karyopherin

The Karyopherin (Kap) family of nuclear transport receptors enables trafficking of proteins to and from the nucleus in a precise, regulated manner. Individual members function in overlapping pathways, while simultaneously being very specific for their main cargoes. The details of this apparent contradiction and rules governing pathway preference remain to be further elucidated. S. cerevisiae Lhp1 is an abundant protein that functions as an RNA chaperone in a variety of biologically important processes. It localizes almost exclusively to the nucleus and is imported by Kap108. We show that mutation of 3 of the 275 residues in Lhp1 alters its import pathway to a Kap121-dependent process. This mutant does not retain wild-type function and is bound by several chaperones. We propose that Kap121 also acts as a chaperone, one that can act as a genetic buffer by transporting mutated proteins to the nucleus

The role of cow urine in the oviposition site preference of culicine and Anopheles mosquitoes

<p>Abstract</p> <p>Background</p> <p>Chemical and behavioural ecology of mosquitoes plays an important role in the development of chemical cue based vector control. To date, studies available have focused on evaluating mosquito attractants and repellents of synthetic and human origins. This study, however, was aimed at seasonal evaluation of the efficiency of cow urine in producing oviposition cues to <it>Anopheles gambiae </it>s.l. and <it>Culex quinquefasciatus </it>in both laboratory and field conditions.</p> <p>Methods</p> <p>Oviposition response evaluation in laboratory conditions was carried out in mosquito rearing cages. The oviposition substrates were located in parallel or in diagonal positions inside the cage. Urine evaluation against gravid females of <it>An. arabiensis </it>and <it>Cx. quinquefasciatus </it>was carried out at Day 1, Day 3 and Day 7. Five millilitres (mls) of cow urine was added to oviposition substrate while de-chlorinated water was used as a control. In field experiments, 500 mls of cow urine was added in artificial habitats with 2500 mls of de-chlorinated water and 2 kgs of soil. The experiment was monitored for thirty consecutive days, eggs were collected daily from the habitats at 7.00 hrs. Data analysis was performed using parametric and non-parametric tests for treatments and controls while attraction of the oviposition substrate in each species was presented using Oviposition Activity Index (OAI).</p> <p>Results</p> <p>The OAI was positive with ageing of cattle urine in culicine species in both laboratory and field experiments. The OAI for anopheline species was positive with fresh urine. The OAI during the rainy season was positive for all species tested while in the dry season the OAI for culicine spp and <it>Anopheles gambiae </it>s.l., changed with time from positive to negative values.</p> <p>Based on linear model analysis, seasons and treatments had a significant effect on the number of eggs laid in habitats, even though the number of days had no effect.</p> <p>Conclusion</p> <p>Oviposition substrates treated with cow urine in both laboratory and field conditions have shown that cow urine left to age from 1-7 days has an influence on oviposition behavioural response in mosquitoes. The analysis of microbial colonies for decaying urine should be investigated along with its associated by-products.</p

Comparative study of fungal cell disruption—scope and limitations of the methods

Simple and effective protocols of cell wall disruption were elaborated for tested fungal strains: Penicillium citrinum, Aspergillus fumigatus, Rhodotorula gracilis. Several techniques of cell wall disintegration were studied, including ultrasound disintegration, homogenization in bead mill, application of chemicals of various types, and osmotic shock. The release of proteins from fungal cells and the activity of a cytosolic enzyme, glucose-6-phosphate dehydrogenase, in the crude extracts were assayed to determine and compare the efficacy of each method. The presented studies allowed adjusting the particular method to a particular strain. The mechanical methods of disintegration appeared to be the most effective for the disintegration of yeast, R. gracilis, and filamentous fungi, A. fumigatus and P. citrinum. Ultrasonication and bead milling led to obtaining fungal cell-free extracts containing high concentrations of soluble proteins and active glucose-6-phosphate dehydrogenase systems

Expanding the knowledge about Leishmania species in wild mammals and dogs in the Brazilian savannah

Background: Wild, synanthropic and domestic mammals act as hosts and/or reservoirs of several Leishmania spp. Studies on possible reservoirs of Leishmania in different areas are fundamental to understand host-parasite interactions and develop strategies for the surveillance and control of leishmaniasis. In the present study, we evaluated the Leishmania spp. occurrence in mammals in two conservation units and their surroundings in Brasília, Federal District (FD), Brazil. Methods: Small mammals were captured in Brasília National Park (BNP) and Contagem Biological Reserve (CBR) and dogs were sampled in residential areas in their vicinity. Skin and blood samples were evaluated by PCR using different molecular markers (D7 24Sα rRNA and rDNA ITS1). Leishmania species were identified by sequencing of PCR products. Dog blood samples were subjected to the rapid immunochromatographic test (DPP) for detection of anti-Leishmania infantum antibodies. Results: 179 wild mammals were studied and 20.1% had Leishmania DNA successfully detected in at least one sample. Six mammal species were considered infected: Clyomys laticeps, Necromys lasiurus, Nectomys rattus, Rhipidomys macrurus, Didelphis albiventris and Gracilinanus agilis. No significant difference, comparing the proportion of individuals with Leishmania spp., was observed between the sampled areas and wild mammal species. Most of the positive samples were collected from the rodent N. lasiurus, infected by L. amazonensis or L. braziliensis. Moreover, infections by Trypanosoma spp. were detected in N. lasiurus and G. agilis. All 19 dog samples were positive by DPP; however, only three (15.8%) were confirmed by PCR assays. DNA sequences of ITS1 dog amplicons showed 100% identity with L. infantum sequence. Conclusions: The results suggest the participation of six species of wild mammals in the enzootic transmission of Leishmania spp. in FD. This is the first report of L. amazonensis in N. lasiurus

Brucella abortus Uses a Stealthy Strategy to Avoid Activation of the Innate Immune System during the Onset of Infection

To unravel the strategy by which Brucella abortus establishes chronic infections, we explored its early interaction with innate immunity. Methodology/Principal Findings Brucella did not induce proinflammatory responses as demonstrated by the absence of leukocyte recruitment, humoral or cellular blood changes in mice. Brucella hampered neutrophil (PMN) function and PMN depletion did not influence the course of infection. Brucella barely induced proinflammatory cytokines and consumed complement, and was strongly resistant to bactericidal peptides, PMN extracts and serum. Brucella LPS (BrLPS), NH-polysaccharides, cyclic glucans, outer membrane fragments or disrupted bacterial cells displayed low biological activity in mice and cells. The lack of proinflammatory responses was not due to conspicuous inhibitory mechanisms mediated by the invading Brucella or its products. When activated 24 h post-infection macrophages did not kill Brucella, indicating that the replication niche was not fusiogenic with lysosomes. Brucella intracellular replication did not interrupt the cell cycle or caused cytotoxicity in WT, TLR4 and TLR2 knockout cells. TNF-α-induction was TLR4- and TLR2-dependent for live but not for killed B. abortus. However, intracellular replication in TLR4, TLR2 and TLR4/2 knockout cells was not altered and the infection course and anti-Brucella immunity development upon BrLPS injection was unaffected in TLR4 mutant mice. Conclusion/Significance We propose that Brucella has developed a stealth strategy through PAMPs reduction, modification and hiding, ensuring by this manner low stimulatory activity and toxicity for cells. This strategy allows Brucella to reach its replication niche before activation of antimicrobial mechanisms by adaptive immunity. This model is consistent with clinical profiles observed in humans and natural hosts at the onset of infection and could be valid for those intracellular pathogens phylogenetically related to Brucella that also cause long lasting infections

Effects of Soil Water and Nitrogen on Growth and Photosynthetic Response of Manchurian Ash (Fraxinus mandshurica) Seedlings in Northeastern China

Soil water and nitrogen (N) are considered to be the main environmental factors limiting plant growth and photosynthetic capacity. However, less is known about the interactive effects of soil water and N on tree growth and photosynthetic response in the temperate ecosystem. seedlings. The seedlings were exposed to three water regimes including natural precipitation (CK), higher precipitation (HW) (CK +30%) and lower precipitation (LW) (CK −30%), and both with and without N addition for two growing seasons. We demonstrated that water and N supply led to a significant increase in the growth and biomass production of the seedlings. LW treatment significantly decreased biomass production and leaf N content, but they showed marked increases in N addition. N addition could enhance the photosynthetic capability under HW and CK conditions. Leaf chlorophyll content and the initial activity of Rubisco were dramatically increased by N addition regardless of soil water condition. The positive relationships were found between photosynthetic capacity, leaf N content, and SLA in response to water and N supply in the seedling. Rubisco expression was up-regulated by N addition with decreasing soil water content. Immunofluorescent staining showed that the labeling for Rubisco was relatively low in leaves of the seedlings under LW condition. The accumulation of Rubisco was increased in leaf tissues of LW by N addition. seedlings, which may provide novel insights on the potential responses of the forest ecosystem to climate change associated with increasing N deposition