Site-specific N-Linked Glycosylation of Receptor Guanylyl Cyclase C Regulates Ligand Binding, Ligand-mediated Activation and Interaction with Vesicular Integral Membrane Protein 36, VIP36

Guanylyl cyclase C (GC-C) is a multidomain, membrane-associated receptor guanylyl cyclase. GC-C is primarily expressed in the gastrointestinal tract, where it mediates fluid-ion homeostasis, intestinal inflammation, and cell proliferation in a cGMP-dependent manner, following activation by its ligands guanylin, uroguanylin, or the heat-stable enterotoxin peptide (ST). GC-C is also expressed in neurons, where it plays a role in satiation and attention deficiency/hyperactive behavior. GC-C is glycosylated in the extracellular domain, and differentially glycosylated forms that are resident in the endoplasmic reticulum (130 kDa) and the plasma membrane (145 kDa) bind the ST peptide with equal affinity. When glycosylation of human GC-C was prevented, either by pharmacological intervention or by mutation of all of the 10 predicted glycosylation sites, ST binding and surface localization was abolished. Systematic mutagenesis of each of the 10 sites of glycosylation in GC-C, either singly or in combination, identified two sites that were critical for ligand binding and two that regulated ST-mediated activation. We also show that GC-C is the first identified receptor client of the lectin chaperone vesicular integral membrane protein, VIP36. Interaction with VIP36 is dependent on glycosylation at the same sites that allow GC-C to fold and bind ligand. Because glycosylation of proteins is altered in many diseases and in a tissue-dependent manner, the activity and/or glycan-mediated interactions of GC-C may have a crucial role to play in its functions in different cell types.

Regulation of lipid biosynthesis, sliding motility, and biofilm formation by a membrane-anchored nucleoid-associated protein of mycobacterium tuberculosis

Bacteria use a number of small basic proteins for organization and compaction of their genomes. By their interaction with DNA, these nucleoid-associated proteins (NAPs) also influence gene expression. Rv3852, a NAP of Mycobacterium tuberculosis, is conserved among the pathogenic and slow-growing species of mycobacteria. Here, we show that the protein predominantly localizes in the cell membrane and that the carboxy-terminal region with the propensity to form a transmembrane helix is necessary for its membrane localization. The protein is involved in genome organization, and its ectopic expression in Mycobacterium smegmatis resulted in altered nucleoid morphology, defects in biofilm formation, sliding motility, and change in apolar lipid profile. We demonstrate its crucial role in regulating the expression of KasA, KasB, and GroEL1 proteins, which are in turn involved in controlling the surface phenotypes in mycobacteria.

Euglena sp as a suitable source of lipids for potential use as biofuel and sustainable wastewater treatment

Prolific algal growth in sewage ponds with high organic loads in the tropical regions can provide cost-effective and efficient wastewater treatment and biofuel production. This work examines the ability of Euglena sp. growing in wastewater ponds for biofuel production and treatment of wastewater. The algae were isolated from the sewage treatment plants and were tested for their nutrient removal capability. Compared to other algae, Euglena sp. showed faster growth rates with high biomass density at elevated concentrations of ammonium nitrogen (NH4-N) and organic carbon (C). Profuse growth of these species was observed in untreated wastewaters with a mean specific growth rate (mu) of 0.28 day(-1) and biomass productivities of 132 mg L-1 day(-1). The algae cultured within a short period of 8 days resulted in the 98 % removal of NH4-N, 93 % of total nitrogen 85 % of ortho-phosphate, 66 % of total phosphate and 92 % total organic carbon. Euglenoids achieved a maximum lipid content of 24.6 % (w/w) with a biomass density of 1.24 g L-1 (dry wt.). Fourier transform infrared spectra showed clear transitions in biochemical compositions with increased lipid/protein ratio at the end of the culture. Gas chromatography and mass spectrometry indicated the presence of high contents of palmitic, linolenic and linoleic acids (46, 23 and 22 %, respectively), adding to the biodiesel quality. Good lipid content (comprised quality fatty acids), efficient nutrient uptake and profuse biomass productivity make the Euglena sp. as a viable source for biofuel production in wastewaters.

Role of outer membrane exopolymers of Acidithiobacillus ferrooxidans in adsorption of cells onto pyrite and chalcopyrite

Bacterial surface polymers play a major role in the adhesion of bacterial cells to solid surfaces. Lipopolysaccharides (LPS) are essential constituents of the cell walls of almost all Gram-negative bacteria. This paper reports the results of the investigations on the role of outer membrane exopolymers (LPS) of the chemolithotroph, Acidithiobacillus ferrooxidans, in adsorption of the cells onto pyrite and chalcopyrite. Optimization of EDTA treatment for removal of LPS from cell surface and the surface characterization of EDTA-treated cells are outlined. There was no change in cell morphology or loss in cell motility upon treatment with upto 0.04 mM EDTA for 1 h. Partial removal of LPS by EDTA treatment resulted in reduced adsorption of the cells on both pyrite and chalcopyrite. The protein profile of the EDTA-extractable fraction showed presence of certain outer membrane proteins indicating that EDTA treatment results in temporary gaps in the outer membrane. Also, specificity towards pyrite compared to chalcopyrite that was exhibited by untreated cells was lost when their exopolymer layers were stripped off, which could be attributed to the role of outer membrane proteins in the mineral-specificity exhibited by the bacteria. (C) 2013 Elsevier B.V. All rights reserved.

Gene Transfection in High Serum Levels: Case Studies with New Cholesterol Based Cationic Gemini Lipids

Background: Six new cationic gemini lipids based on cholesterol possessing different positional combinations of hydroxyethyl (-CH2CH2OH) and oligo-oxyethylene -(CH2CH2O)(n)- moieties were synthesized. For comparison the corresponding monomeric lipid was also prepared. Each new cationic lipid was found to form stable, clear suspensions in aqueous media. Methodology/Principal Findings: To understand the nature of the individual lipid aggregates, we have studied the aggregation properties using transmission electron microscopy (TEM), dynamic light scattering (DLS), zeta potential measurements and X-ray diffraction (XRD). We studied the lipid/DNA complex (lipoplex) formation and the release of the DNA from such lipoplexes using ethidium bromide. These gemini lipids in presence of a helper lipid, 1, 2-dioleoyl phophatidyl ethanol amine (DOPE) showed significant enhancements in the gene transfection compared to several commercially available transfection agents. Cholesterol based gemini having -CH2-CH2-OH groups at the head and one oxyethylene spacer was found to be the most effective lipid, which showed transfection activity even in presence of high serum levels (50%) greater than Effectene, one of the potent commercially available transfecting agents. Most of these geminis protected plasmid DNA remarkably against DNase I in serum, although the degree of stability was found to vary with their structural features. Conclusions/Significance: -OH groups present on the cationic headgroups in combination with oxyethylene linkers on cholesterol based geminis, gave an optimized combination of new genera of gemini lipids possessing high transfection efficiency even in presence of very high percentage of serum. This property makes them preferential transfection reagents for possible in vivo studies.

Effects of a Delocalizable Cation on the Headgroup of Gemini Lipids on the Lipoplex-Type Nanoaggregates Directly Formed from Plasmid DNA

Lipoplex-type nanoaggregates prepared from pEGFP-C3 plasmid DNA (pDNA) and mixed liposomes, with a gemini cationic lipid (CL) 1,2-bis(hexadecyl imidazolium) alkanes], referred as (C(16)Im)(2)C-n (where C-n is the alkane spacer length, n = 2, 3, 5, or 12, between the imidazolium heads) and DOPE zwitterionic lipid, have been analyzed by zeta potential, gel electrophoresis, SAXS, cryo-TEM, fluorescence anisotropy, transfection efficiency, fluorescence confocal microscopy, and cell viability/cytotoxicity experiments to establish a structure-biological activity relationship. The study, carried out at several mixed liposome compositions, alpha, and effective charge ratios, rho(eff), of the lipoplex, demonstrates that the transfection of pDNA using CLs initially requires the determination of the effective charge of both. The electrochemical study confirms that CLs with a delocalizable positive charge in their headgroups yield an effective positive charge that is 90% of their expected nominal one, while pDNA is compacted yielding an effective negative charge which is only 10-25% than that of the linear DNA. SAXS diffractograms show that lipoplexes formed by CLs with shorter spacer (n = 2, 3, or 5) present three lamellar structures, two of them in coexistence, while those formed by CL with longest spacer (n = 12) present two additional inverted hexagonal structures. Cryo-TEM micrographs show nanoaggregates with two multilamellar structures, a cluster-type (at low alpha value) and a fingerprint-type, that coexist with the cluster-type at moderate alpha composition. The optimized transfection efficiency (TE) of pDNA, in HEK293T, HeLa, and H1299 cells was higher using lipoplexes containing gemini CLs with shorter spacers at low a value. Each lipid formulation did not show any significant levels of toxicity, the reported lipoplexes being adequate DNA vectors for gene therapy and considerably better than both Lipofectamine 2000 and CLs of the 1,2-bis(hexadecyl ammnoniun) alkane series, recently reported.

Lysis dynamics and membrane oligomerization pathways for Cytolysin A (ClyA) pore-forming toxin

Pore-forming toxins are known for their ability to efficiently form transmembrane pores which eventually leads to cell lysis. The dynamics of lysis and underlying self-assembly or oligomerization pathways leading to pore formation are incompletely understood. In this manuscript the pore-forming kinetics and lysis dynamics of Cytolysin-A (ClyA) toxins on red blood cells (RBCs) are quantified and compared with experimental lysis data. Lysis experiments are carried out on a fixed mass of RBCs, under isotonic conditions in phosphate-buffered saline, for different initial toxin concentrations ranging from 2.94-14.7 nM. Kinetic models which account for monomer binding, conformation and oligomerization to form the dodecameric ClyA pore complex are developed and lysis is assumed to occur when the number of pores per RBC (n(p)) exceeds a critical number, n(pc). By analysing the model in a sublytic regime (n(p) < n(pc)) the number of pores per RBC to initiate lysis is found to lie between 392 and 768 for the sequential oligomerization mechanism and between 5300 and 6300 for the non-sequential mechanism. Rupture rates which are first order in the number of RBCs are seen to provide the best agreement with the lysis experiments. The time constants for pore formation are estimated to lie between 1 and 20 s and monomer conformation time scales were found to be 2-4 times greater than the oligomerization times. Cell rupture takes places in 100s of seconds, and occurs predominantly with a steady number of pores ranging from 515 to 11 000 on the RBC surface for the sequential mechanism. Both the sequential irreversible and non-sequential kinetics provide similar predictions of the hemoglobin release dynamics, however the hemoglobin released as a function of the toxin concentration was accurately captured only with the sequential model. Each mechanism develops a distinct distribution of mers on the surface, providing a unique experimentally observable fingerprint to identify the underlying oligomerization pathways. Our study offers a method to quantify the extent and dynamics of lysis which is an important aspect of developing novel drug and gene delivery strategies based on pore-forming toxins.

Biogenic nanosilver incorporated reverse osmosis membrane for antibacterial and antifungal activities against selected pathogenic strains: An enhanced eco-friendly water disinfection approach

Reverse osmosis (RO) membranes have been used extensively in water desalination plants, waste water treatment in industries, agricultural farms and drinking water production applications. The objective of this work is to impart antibacterial and antifungal activities to commercially available RO membrane used in water purification systems by incorporating biogenic silver nanoparticles (AgNPs) synthesized using Rosa indica wichuriana hybrid leaf extract. The morphology and surface topography of uncoated and AgNPs-coated RO membrane were studied using Scanning Electron Microscopy (SEM) and Atomic Force Microscopy (AFM). Elemental composition of the AgNPs-coated RO membrane was analyzed by energy-dispersive X-ray spectroscopy (EDAX). The functional groups were identified by Fourier Transform Infrared spectroscopy (FT-IR). Hydrophilicity of the uncoated and AgNPs-coated RO membrane was analyzed using water contact angle measurements. The thermal properties were studied by thermogravimetric analysis (TGA). The AgNPs incorporated RO membrane exhibited good antibacterial and antifungal activities against pathogenic bacterial strains such as E. coli, S. aureus, M. luteus, K. pneumoniae, and P. aeruginosa and fungal strains such as Candida tropicalis, C. krusei, C. glabrata, and C. albicans.

Cationic gemini lipids containing polyoxyethylene spacers as improved transfecting agents of plasmid DNA in cancer cells

Lipoplex nano-aggregates have been analyzed through biophysical characterization (electrostatics, structure, size and morphology), and biological studies (transfection efficiency and cell viability) in five cancer cell lines. Lipoplexes were prepared from pEGFP-C3 plasmid DNA (pDNA) and mixed liposomes, constituted by a zwitterionic lipid (DOPE) and a gemini cationic lipid (GCL) synthesized in this work, bis(hexadecyl dimethyl ammonium) oxyethylene], referred to as (C16Am)(2)(C2O)(n), (where n is the oxyethylene spacer length, n = 1, 2 or 3, between the ammonium heads). Cryo-TEM micrographs show nano-aggregates with two multilamellar structures, a cluster-type (at low-to-medium GCL composition) and a fingerprint-type that coexists with the cluster-type at medium GCL composition and appears alone at high GCL composition. SAXS diffractograms show that these lipoplexes present three lamellar structures, two of them coexisting at low and high GCL composition. The optimized transfection efficiency (TE) of pDNA was higher for lipoplexes containing GCLs with a longer (n = 3) or shorter (n = 1) polyoxyethylene spacer, at high GCL composition (alpha - 0.7) with low charge ratio (rho(eff) 2). In the all cancer cell lines studied, the TE of the optimized formulations was much better than those of both lipofectamine 2000 and lipoplexes with GCLs of the bis(hexadecyl dimethyl ammonium) alkane series recently reported. Probably, (a) the coexistence of two lamellar structures at high GCL composition synergizes the TE of these lipid vectors, (b) the orientation of the polyoxyethylene region in (C16Am)(2)(C2O)(3)/DOPE may occur in such a way that the spacing between two cationic heads becomes smaller than that in (C16Am)(2)(C2O)(2)/DOPE which is poor in terms of TE, and (c) the synergistic interactions between serum proteins and (C16Am)(2)(C2O)(n)/DOPE-pDNA lipoplexes containing a polyoxyethylene spacer improve TE, especially at high GCL content. Lipoplexes studied here show very low levels of toxicity, which confirm them as improved vectors of pDNA in gene therapy.

A new microscopic insight into membrane penetration and reorganization by PETIM dendrimers

Dendrimers are highly branched polymeric nanoparticles whose structure and topology, largely, have determined their efficacy in a wide range of studies performed so far. An area of immense interest is their potential as drug and gene delivery vectors. Realizing this potential, depending on the nature of cell surface-dendrimer interactions, here we report controlled model membrane penetration and reorganization, using a model supported lipid bilayer and poly(ether imine) (PETIM) dendrimers of two generations. By systematically varying the areal density of the lipid bilayers, we provide a microscopic insight, through a combination of high resolution scattering, atomic force microscopy and atomistic molecular dynamics simulations, into the mechanism of PETIM dendrimer membrane penetration, pore formation and membrane re-organization induced by such interactions. Our work represents the first systematic observation of a regular barrel-like membrane spanning pore formation by dendrimers, tunable through lipid bilayer packing, without membrane disruption.

Time resolved terahertz spectroscopy of low frequency electronic resonances and optical pump-induced terahertz photoconductivity in reduced graphene oxide membrane

Towards ultrafast optoelectronic applications of single and a few layer reduced graphene oxide (RGO), we study time domain terahertz spectroscopy and optical pump induced changes in terahertz conductivity of self-supported RGO membrane in the spectral window of 0.5-3.5 THz. The real and imaginary parts of conductivity spectra clearly reveal low frequency resonances, attributed to the energy gaps due to the van Hove singularities in the density of states flanking the Dirac points arising due to the relative rotation of the graphene layers. Further, optical pump induced terahertz conductivity is positive, pointing to the dominance of intraband scattering processes. The relaxation dynamics of the photo-excited carriers consists of three cooling pathways: the faster (similar to 450 fs) one due to optical phonon emission followed by disorder mediated large momentum and large energy acoustic phonon emission with a time constant of a few ps (called the super-collision mechanism) and a very large time (similar to 100 ps) arising from the deep trap states. The frequency dependence of the dynamic conductivity at different delay times is analyzed in term of Drude-Smith model. (C) 2014 Published by Elsevier Ltd.

Sequence and conformational preferences at termini of alpha-helices in membrane proteins: Role of the helix environment

-helices are amongst the most common secondary structural elements seen in membrane proteins and are packed in the form of helix bundles. These -helices encounter varying external environments (hydrophobic, hydrophilic) that may influence the sequence preferences at their N and C-termini. The role of the external environment in stabilization of the helix termini in membrane proteins is still unknown. Here we analyze -helices in a high-resolution dataset of integral -helical membrane proteins and establish that their sequence and conformational preferences differ from those in globular proteins. We specifically examine these preferences at the N and C-termini in helices initiating/terminating inside the membrane core as well as in linkers connecting these transmembrane helices. We find that the sequence preferences and structural motifs at capping (Ncap and Ccap) and near-helical (N' and C') positions are influenced by a combination of features including the membrane environment and the innate helix initiation and termination property of residues forming structural motifs. We also find that a large number of helix termini which do not form any particular capping motif are stabilized by formation of hydrogen bonds and hydrophobic interactions contributed from the neighboring helices in the membrane protein. We further validate the sequence preferences obtained from our analysis with data from an ultradeep sequencing study that identifies evolutionarily conserved amino acids in the rat neurotensin receptor. The results from our analysis provide insights for the secondary structure prediction, modeling and design of membrane proteins. Proteins 2014; 82:3420-3436. (c) 2014 Wiley Periodicals, Inc.