Month: January 2018

Research featuring genetically engineered cyanobacteria for the production of liquid biofuels including ethanol , isobutyraldehyde and isobutanol , and free fatty acids has recently flourished. Although using cyanobacteria as cell factories has become more common, studies are still carried out with standard laboratory model organisms rather than with potential production strains. For their desirable growth qualities, much consideration has been given to Axitinib strains of the genus Arthrospira , which emerged from a screen of cyanobacterial strains for superior growth traits, and show that it is amenable to genetic manipulation. Leptolyngbya BL0902 has good growth characteristics when compared to two common outdoor production strains of the genus Arthrospira. We show that Leptolyngbya BL0902 can receive and maintain conjugal shuttle vectors, express an antibiotic resistance gene and a yemGFP reporter gene, and be subjected to transposon-tagging mutagenesis. Conjugation from E. coli donor cells has been used to introduce DNA into a wide variety of cyanobacteria, and broad-host-range plasmids derived from RSF1010 have been shown to replicate in many strains . To determine whether these methods could be used with Leptolyngbya BL0902, we performed biparental matings with Leptolyngbya BL0902 and a conjugal E. coli donor strain that contained the cargo plasmid pRL1383a, the conjugal plasmid pRL443, and the helper plasmid pRL623. Transconjugant colonies became apparent on selective mating plates after about one week and showed robust growth after transfer to fresh selective plates. Control conjugations without the cargo plasmid never showed any antibiotic resistant colonies. The ability to genetically modify Leptolyngbya BL0902 was further demonstrated by the heterologous expression of the yemGFP gene. The recombinant plasmid pAM4413 carrying the yemGFP gene was electroporated into AM1359, and the resulting strain was conjugated with Leptolyngbya BL0902. After one week, Nutlin-3 isolated transconjugant colonies were restreaked on fresh selective plates, and isolated colonies were then patched to fresh selective plates. Liquid cultures were grown in selective BG-11 medium. Expression of yemGFP was observed by fluorescence microscopy . Our initial conjugation experiments were performed with donor strains carrying the helper plasmid pRL623, which carries 3 methylase genes. The methylase genes are required for efficient conjugation into Anabaena recipient strains . To assess the necessity of these genes for Leptolyngbya BL0902 conjugation, we determined the efficiency of conjugal transfers in biparental and triparental matings with and without the helper plasmid pRL623 and with two different conjugal plasmids: pRL443 and pRK2013 . The conjugation protocol was modified as described in the methods to yield more reproducible data for transconjugant colony forming units .

Within the developing ovule primordia, much is known about molecular patterning events along the proximal to distal axis and the mechanisms of integument development. Also dramatic progress has been made with respect to understanding the subsequent development of the female gametophyte within the maturing ovule. However, considerably less is known about the earliest steps in ovule development: the mechanisms of ovule initiation, and in the establishment and maintenance of the meristematic tissues of the carpel margin meristem that generate the ovule primordia. Gynoecial development in Arabidopsis initiates at stage 6 of floral development. The gynoecial primordium is first morphologically recognizable as a dome or mound of cells, oval in cross section, that forms from the cells of the central most portion of floral meristem. During stage 6 the different spatial domains of the gynoecial tube are already discernable based on the differential expression of genes within the medial portion of the gynoecium versus the lateral domains, as well as along the inner to outer axis. During floral stages 6 and 7 the proliferation of cells along the perimeter of the gynoecial dome leads to the formation of a tube-shaped structure. The single gynoecium primordium likely represents a composite of two congenitally-fused carpel organs in a phylogenetic sense. In this scenario, the medial portions of the gynoecium represent the fused margins of the two component carpels. The adaxial portions of the medial/marginal domain maintain meristematic potential throughout the elongation of the gynoecial tube and these regions have been termed carpel margin meristems. Each Arabidopsis gynoecium contains two CMMs that are positioned within the adaxial portions of the medial domain of the gynoecium. During floral stages 7 and 8 the CMM takes the shape of a ridge of tissue that extends along the apical basal extent of the gynoecial tube. During mid to late stage 8 each CMM gives rise to two rows of ovule primordia from the peripheral portions of the meristematic ridge. Later, the CMM also gives rise to the gynoecial septum and transmitting tract and likely generates portions of the stigmatic and stylar tissues. A variety of data CUDC-907 supply suggests that the proper specification of adaxial and medial/ marginal positional identities are important for the development of the CMM and subsequent ovule initiation. A number of genes have been suggested to play a role in the maintenance of meristematic potential in the CMM and for the subsequent initiation of ovule primordia from the flanks of the CMM. While no single mutant has been reported to strongly Tubulin Acetylation Inducer disrupt ovule initiation, several higher order mutant combinations have been reported to disrupt the initiation of ovule primordia from the CMM. The seuss aintegumenta double mutant is one such genetic mutant combination.

Part of the neuraminidase effect is to unmask Siglec receptors on the cell surface thus increasing their binding to viral sialic acids. Siglec-1 transfected CHO cells bound to gp120 without neuraminidase treatment, suggesting that most of Siglec-1 exists in an unmasked state, likely due to its large size with 17 predicted immunoglobulin domains. In contrast, Siglec-9, although a high affinity receptor for sialic acid, displayed significant adhesion only after neuraminidase treatment, suggesting most of the receptors are masked on cell surface. Consistent with the binding results, HIV-1 R5-pseudovirus KU-0059436 molecular weight infections of MDM were preferentially inhibited by sialo- but not their asialo-compounds and by sodium periodate treatment of the viruses. However, the effects of the sialyllated compounds in X4 infections were less conclusive as X4-viruses infect macrophages at a very low level due to the preferential usage of CXCR4. The results of Siglec blocking antibodies on R5-pseudovirus infection further defined the contribution of individual Siglec receptors in HIV-1 infection of macrophages. The preferential involvement of Siglec-1 and, to a lesser extent, Siglec-3 was observed in JRFL pseudovirus infections blocked by Siglec antibodies, as well as sialyllactose-inhibited HIV-1BaL infections. These results are also consistent with gp120 binding experiments using Siglec-transfected CHO cells. Thus, the contribution of Siglec-1 in HIV-1 infection reflects its high binding affinity to sialic acid and favorable availability for viral attachment. While the exact distributions of viral sialic acids on HIV-1 isolates remain unknown, their predicted N-glycan sites vary significantly both in positions and numbers. In contrast to conserved CD4 binding site on gp120, half of the glycosylation sites in gp120 variable loop regions are not conserved among different strains. The variations in viral envelope sialyloglycan distribution may very well result in variations in Siglec-dependent viral attachment, consistent with the observed variations in sialic acid-dependent infections among JRFL, DH125, AD8 and HW1 strains of HIV-1. In addition, host factors could influence HIV-1 attachment. Interestingly, the expression of Siglec-1 is up-regulated by inflammation and infection, including HIV-1 infection. This raises the possibility that increased Siglec-1 expression on macrophages at inflammatory sites may further enhance HIV replication and thus contribute to viral dissemination. This is also consistent with a recent finding of an association between immune HhAntag691 activation and progressive SIV infection in pigtail macaques. Thus, viral envelope glycan heterogeneities together with host Siglec expressions could lead to a wide range of sialic acid-dependent host susceptibilities to HIV infection. In conclusion, we suggest that HIV-1 viruses utilize their sialoglycans on the envelope protein to facilitate viral attachment to macrophages through binding to Siglec receptors, in particular Siglec-1, and thus enhancing their binding to CD4 for productive entry.

It is important to treat electrostatic interactions with accurate methods, such as PME, to avoid potential serious artifacts. It has been shown that choosing simulation parameters, including thermostat and electrostatic treatment, is a subtle issue and that the choice of charge-groups may lead to unphysical effects. Baumketner et al. also reported that charge-group based truncation with reaction-field electrostatics may cause artificial repulsions between charged residues, identified as the microscopic reason behind artificial unfolding of INCB28060 protein in some simulations. Here, charge-groups were chosen to be small to avoid artifacts. Periodic boundary conditions were applied in all directions. To determine whether the binding motifs of ProTa and Neh2 have tendencies to adopt bound-state-like structures in their free states, coordinates from the MD trajectories were compared with the corresponding PDB crystal structures. The Kelch domain of mouse Keap1 was expressed in Escherichia coli BL21 grown in minimal M9 medium. The N-terminally His-tagged protein was purified by affinity chromatography using Ni SepharoseTM 6 Fast Flow beads. The tag was then cleaved by incubation with His-tagged tobacco etch virus protease overnight at 25uC. Cholesterol is an intriguing molecule linked to numerous fundamental physiological functions; thus, alterations in cholesterol metabolism are associated with a range of disorders that include significant current and future healthcare burdens. At least one reason for this extensive linkage of cholesterol to physiological states is that, in contrast to other lipidic membrane components, there are a number of reasonably selective pharmacological tools available with which to modulate the amount and/ or effects of cholesterol in cellular membranes. Indeed, this ability to target native cholesterol with reasonable selectivity, coupled with the availability of sensitive quantitative assays, has led to cholesterol being the first molecule identified as having a direct role in the essential membrane merger steps of fast, calcium-triggered exocytosis, as occurs under physiological conditions, in depolarized nerve terminals. In addition, as a microdomain organizer, cholesterol also plays a critical role in maintaining the localized presence of other components critical to targeting, priming, docking, and subsequent fusion. Among the tools used to study cholesterol, perhaps the most widely applied has been methyl-?-cyclodextrin, a cyclic oligosaccharide that can deplete cellular membranes of cholesterol by increasing the water solubility of the sterol. MbCD has thus been used with increasing frequency to examine the physiological roles of cholesterol. Studies utilizing MbCD have implicated cholesterol in mediating or modulating a wide range of membrane-associated cell BMN673 inquirer properties and functions in a range of secretory cell types, including neurons.

The Keap1-binding motif of ProTa shares a similar sequence with that of the Neh2. Crystal structures of ProTa and Neh2 peptides bound to the Kelch domain of Keap1 further reveal that these two proteins bind to the same site on the Kelch domain and form similar b-turn conformations. The Kelch domain adopts a six-bladed Gefitinib bpropeller structure with each blade composed of four anti-parallel b-strands. Both ProTa and Neh2 bind to the positively charged face of the b-propeller where the inter-blade loops are located and the electrostatic interactions are crucial for the stability of the complexes. Interestingly, despite the high sequence identity and structural similarity of the binding motifs, ProTa seems to have a lower binding affinity to Keap1 compared to Neh2. Atomistic microsecond scale MD simulations were used to investigate the molecular mechanisms by which the intrinsically disordered ProTa and Neh2 interact with Keap1. In particular, we focused on whether their XEEXGE motifs bind to Kelch domain through coupled folding and binding, PSEs or a combination of both mechanisms. Our results show that in their free states, both the Keap1-binding motifs of ProTa and Neh2 display intrinsic propensities to form bound-state-like b-turns, and that the CT99021 residues outside of the motifs may also contribute to the stability of the structural elements. We found that the Keap1- binding motif of Neh2 adopted a b-turn conformation that more closely resembled its bound-state structure than that of ProTa. Based on these results, we propose that binding occurs synergistically via a combination of PSEs and coupled folding and binding with a heavy bias towards PSEs, especially for Neh2. The better understanding of the binding mechanisms may provide insight into developing of therapeutics that can be used to promote cellular response to oxidative stress. All simulations were performed using GROMACS version 4, with the GROMOS96 53a6 united atom force-field parameter set. This force field has been shown to be reliable in simulating proteins, including b-peptide folding. Protonation states of ionizable residues were chosen based on their most probable state at pH 7. Protein and non-protein atoms were coupled to their own temperature baths, which were kept constant at 310 K using the weak coupling algorithm. Pressure was maintained isotropically at 1 bar using the Berendsen barostat. Prior to the production runs, the energy of each system was minimized using the steepest descents method. This was followed by 2 ps of positionrestrained dynamics with all non-hydrogen atoms restrained with a 1000 kJ mol21 force constant. The timestep was set to 2 fs. Initial atom velocities were taken from a Maxwellian distribution at 310 K. All bond lengths were constrained using the LINCS algorithm. Cut-off of 1.0 nm was used for Lennard-Jones interactions and the real part of the long-range electrostatic interactions, which were calculated using the Particle-Mesh Ewald method. 0.12 nm grid-spacing was used for PME.