The effect of some micronutrients and heavy metals on phosphate absorption by maize root cortex segments

The effect of salts (nitrates, chlorides, and sulfates) of microelements, Cd²⁺, Ni²⁺, and Co²⁺ and the effect of boric acid and ammonium molybdate on phosphate uptake by maize root cortex segments were tested. Higher concentration (0.1 mM) of Cu²⁺ salts caused enhancement of phosphate efflux to the extent that efflux was higher than influx. Inhibitory action on phosphate uptake by maize root cortex segments was exerted by following salts: 0.01 mM Cu²⁺ salts (20–30% inhibition), 0.5 mM ZnSO₄ (9.7%), 0.5 and 0.05 mM ZnCl₂ (34.3% and 20.8%), 0.1 mM salts of Cd²⁺, Ni²⁺, Co²⁺ (35–78%). 1 mM FeS_O4 had significant stimulatory effect (92%) on phosphate uptake. Much weaker stimulatory effect was exerted by 1 mM FeCl₃ (14%), 0.05 mM ZnS_O4 (9.6%), 0.005 mM ZnCla and ZnS_O4 (8.4 and 18.5%) and 0.001 mM CdCl2 and CdS_O4 (20.8 and 12.4%). All other tested salts-salts of Mn²⁺ (0.1 and 0.01 mM), 0.01 and 0.001 mM salts of Co²⁺ and Ni²⁺, 0.001 mM salts of Cu²⁺, 0.001–10 mM boric acid, and 0.001–0.1 mM ammonium molybdate left phosphate uptake unaffected.     

Nesting behaviour and foraging characteristics of Andrena cineraria (Hymenoptera: Andrenidae)

The current studies were carried out in the three experimental locations of Kashmir valley during 2013 to 2016. The species Andrena cineraria formed the dense nest aggregations in plan grounds, barren lands and hilly areas near the fruit orchards and other landscapes with clay loam soil type. The species start flying and foraging in the orchards from April till July. The nests were allodalous, 29–36 cm in depth, with cells located obliquely around the main barrow. The nests were dense with a maximum density of 11.09 nests/m² observed in landscapes of Budgam. The barrow diameters were found varying with depth from main entrance. The maximum barrow diameter recorded was 2.05 mm. At certain depth, the female constructs the first cell and the upper nest burrow is vertical and lower is oblique. The nest entrance is generally hidden under the tumulus. In the depth of average 30.48 cm, each cell directly opens to main burrow either alternately or unilaterally. The cell number, diameter, and length varied with depth. Foraging behaviour of A. cineraria on various fruit crops and other shrubs and social forestry trees were determined and the abundance, visitation rate, total visits and time spend per flower were found significant, especially on fruit crops. The significance of the studies is important for the melittologists, as it will help in the conservation of bee fauna. The study is also important in using this species for pollination purpose and would also help to detect and understand the possible pre-adaptation of species in temperate region of Kashmir valley.     

Transcriptomic analysis and molecular docking reveal genes involved in the response of Aedes aegypti larvae to an essential oil extracted from Eucalyptus

BackgroundAedes aegypti (L.) is an urban mosquito, vector of several arboviruses that cause severe diseases in hundreds of million people each year. The resistance to synthetic insecticides developed by Ae. aegypti populations worldwide has contributed to failures in vector control campaigns, increasing the impact of arbovirus diseases. In this context, plant-derived essential oils with larvicidal activity could be an attractive alternative for vector control. However, the mode of action and the detoxificant response of mosquitoes to plant derived compounds have not been established, impairing the optimization of their use.Methods and findingsHere we compare gene expression in Ae. aegypti larvae after 14 hrs of exposure to Eucalyptus camaldulensis essential oil with a control group exposed to vehicle (acetone) for the same lapse, by using RNA-Seq. We found differentially expressed genes encoding for cuticle proteins, fatty-acid synthesis, membrane transporters and detoxificant related gene families (i.e. heat shock proteins, cytochromes P450, glutathione transferases, UDP-glycosyltransferases and ABC transporters). Finally, our RNA-Seq and molecular docking results provide evidence pointing to a central involvement of chemosensory proteins in the detoxificant response in mosquitoes.Conclusions and significanceOur work contributes to the understanding of the physiological response of Ae. aegypti larvae to an intoxication with a natural toxic distilled from Eucalyptus leafs. The results suggest an involvement of most of the gene families associated to detoxification of xenobiotics in insects. Noteworthy, this work provides important information regarding the implication of chemosensory proteins in the detoxification of a natural larvicide. Understanding the mode of detoxification of Eucalyptus distilled compounds could contribute to their implementation as a tool in mosquito control.     

Proteolytic and N-Glycan Processing of Human α1-Antitrypsin Expressed in Nicotiana benthamiana

Plants are increasingly being used as an expression system for complex recombinant proteins. However, our limited knowledge of the intrinsic factors that act along the secretory pathway, which may compromise product integrity, renders process design difficult in some cases. Here, we pursued the recombinant expression of the human protease inhibitor α1-antitrypsin (A1AT) in Nicotiana benthamiana. This serum protein undergoes intensive posttranslational modifications. Unusually high levels of recombinant A1AT were expressed in leaves (up to 6 mg g⁻¹ of leaf material) in two forms: full-length A1AT located in the endoplasmic reticulum displaying inhibitory activity, and secreted A1AT processed in the reactive center loop, thus rendering it unable to interact with target proteinases. We found that the terminal protein processing is most likely a consequence of the intrinsic function of A1AT (i.e. its interaction with proteases [most likely serine proteases] along the secretory pathway). Secreted A1AT carried vacuolar-type paucimannosidic N-glycans generated by the activity of hexosaminidases located in the apoplast/plasma membrane. Notwithstanding, an intensive glycoengineering approach led to secreted A1AT carrying sialylated N-glycan structures largely resembling its serum-derived counterpart. In summary, we elucidate unique insights in plant glycosylation processes and show important aspects of postendoplasmic reticulum protein processing in plants.Recombinant protein-based drugs are among the fastest growing areas of development in the pharmaceutical industry. Consequently, there is a demand for exploring new production systems. Plants are increasingly being used for the expression of recombinant proteins, primarily because of their remarkable production speed and yield (for review, see Gleba et al., 2014). The highly conserved secretory pathway between human and plant cells allows similar, if not identical, protein folding, assembly, and posttranslational modifications. Importantly, plants are able to synthesize complex N-glycans, a prerequisite for the in vivo activity of many therapeutically interesting proteins. Despite substantial differences in N-glycan diversity, we and others have shown that plants are highly amendable to glycan engineering and allow proteins with controlled human-type N-glycosylation profiles to be generated (Castilho and Steinkellner, 2012). Moreover, it has even been possible to reconstruct entire human glycosylation pathways, which was shown by the introduction of the human sialylation and O-glycosylation processes in Nicotiana benthamiana (Castilho et al., 2010, 2012). These accomplishments render plants suitable for the production of human proteins that require a complex glycosylation profile.Notwithstanding, to use plants as a versatile expression host for complex human proteins, it is important to fully understand intracellular processes. Particularly detailed knowledge about constraints along the plant cell secretory pathway, including proteolytic processing, is required, because these constraints may compromise protein integrity and quality. Despite major achievements in controlling protein-bound oligosaccharide formation, some plant glycosylation peculiarities are not entirely understood. For example, plant cells synthesize so-called paucimannosidic N-glycans, a type of truncated glycans usually absent in mammals (Lerouge et al., 1998). The biosynthesis and physiological significance of this N-glycan formation has yet to be completely explained (Strasser et al., 2007; Liebminger et al., 2011). Another process not fully understood in plants is subcellular localization of proteins. Aberrant intracellular deposition and as a consequence, incorrect glycosylation of recombinant proteins are often reported. For example, recombinant proteins designed for secretion are frequently also located in the endoplasmic reticulum (ER) and as a consequence, carry oligomannosidic carbohydrates instead of the desired complex-type glycans (Loos et al., 2011; Schneider et al., 2014a). By contrast, KDEL-tagged proteins designed for ER retention are sometimes partially secreted (Van Droogenbroeck et al., 2007; Niemer et al., 2014). How and at which biosynthetic stage these plant-specific peculiarities arise are largely unpredictable, which makes controlled expression of recombinant proteins with features authentically to their natural counterparts a difficult task.One human protein that is pharmaceutically interesting, and thus needed in large amounts at high quality, is α1-antitrypsin (A1AT). This highly glycosylated protease inhibitor from the serpin superfamily interacts with a wide variety of proteases (Gettins, 2002). Like other serpins, A1AT is characterized by an exposed and mobile reactive center loop (RCL) with a Met (³⁵⁸M) residue acting as bait for specific target proteinases (Travis and Salvesen, 1983). The main biological role of plasma A1AT is to prevent excessive action of leukocyte-derived Ser proteinases, especially neutrophil elastase, in the circulatory system (Blank and Brantly, 1994). Therapeutic A1AT used in augmentation therapies is currently purified from pooled human serum, and the treatment can cost up to $100,000 per year per patient (Alkins and O’Malley, 2000). Concerns over the supply and safety of the products have urged searches for alternative recombinant sources of A1AT. Recombinant A1AT has been produced in human and nonhuman cell production systems with limited success (Blanchard et al., 2011; Brinkman et al., 2012; Ross et al., 2012; Lee et al., 2013). The production suffers from two major drawbacks: low expression levels and/or incorrect glycosylation (Garver et al., 1987; Chang et al., 2003; McDonald et al., 2005; Hasannia et al., 2006; Karnaukhova et al., 2006; Plesha et al., 2007; Agarwal et al., 2008; Nadai et al., 2009; Huang et al., 2010; Arjmand et al., 2011; Jha et al., 2012). The mature plasma-derived 52-kD protein has three N-linked glycosylation sites that are mainly decorated with disialylated structures (Kolarich et al., 2006). Sialylated N-glycans are a well-known requisite for the plasma half-life of A1AT (Mast et al., 1991; Lindhout et al., 2011; Lusch et al., 2013); the difficulties associated with obtaining them hamper the generation of biologically active A1AT in many expression systems.Here, we pursued the expression of recombinant human A1AT in glycoengineered N. benthamiana and investigated the system’s ability to generate active sialylated variants. Unusually high amounts of A1AT were obtained using a plant viral-based transient expression system. The inhibitor was efficiently secreted to the intercellular space (IF); however, peptide mapping showed that the secreted A1AT was truncated at both the N and C termini. Mass spectrometry (MS) -based N-glycan analysis of IF-derived A1AT showed that vacuolar typical paucimannosidic N-glycans were present. By expressing A1AT in Arabidopsis (Arabidopsis thaliana) knockout plants lacking β-N-acetylhexosaminidase (HEXO) activity (Liebminger et al., 2011), we showed that paucimannosidic structures are generated by the action of HEXO3 located at the plasma membrane.Coexpression with the mammalian genes necessary for in planta sialylation allowed the synthesis of disialylated A1AT, and sialylation levels could be increased by the synthesis of multiantennary glycans. By contrast, full-length A1AT purified from total soluble extracts exhibited ER-typical oligomannosidic carbohydrates. Using live-cell imaging, a GFP-tagged A1AT fusion did, indeed, exhibit aberrant ER-associated deposition of full-length A1AT. Elastase inhibition assays showed that ER-retained A1AT exhibits inhibitory activity, whereas the IF-derived truncated form was rendered inactive by cleavage within its RCL.     

Aggregation Factor as an Inhibitor of Bacterial Binding to Gut Mucosa

Modern research in the area of probiotics is largely devoted to discovering factors that promote the adherence of probiotic candidates to host mucosal surfaces. The aim of the present study was to test the role of aggregation factor (AggL) and mucin-binding protein (MbpL) from Lactococcus sp. in adhesion to gastrointestinal mucosa. In vitro, ex vivo, and in vivo experiments in rats were used to assess the adhesive potential of these two proteins expressed in heterologous host Lactobacillus salivarius BGHO1. Although there was no influence of MbpL protein expression on BGHO1 adhesion to gut mucosa, expression of AggL had a negative effect on BGHO1 binding to ileal and colonic rat mucosa, as well as to human HT29-MTX cells and porcine gastric mucin in vitro. Because AggL did not decrease the adhesion of bacteria to intestinal fragments in ex vivo tests, where peristaltic simulation conditions were missing, we propose that intestinal motility could be a crucial force for eliminating aggregation-factor-bearing bacteria. Bacterial strains expressing aggregation factor could facilitate the removal of pathogens through the coaggregation mechanism, thus balancing gut microbial ecosystems in people affected by intestinal bacteria overgrowth.