Advanced glycation end products in vitreous: Structural and functional implications for diabetic vitreopathy

PURPOSE. Advanced glycation end products (AGES) form irreversible cross- links with many macromolecules and have been shown to accumulate in tissues at an accelerated rate in diabetes. In the present study, AGE formation in vitreous was examined in patients of various ages and in patients with diabetes. Ex vivo investigations were performed on bovine vitreous incubated in glucose to determine AGE formation and cross-linking of vitreous collagen. METHODS. By means of an AGE-specific enzyme-linked immunosorbent assay (ELISA), AGE formation was investigated in vitreous samples obtained after pars plana vitrectomy in patients with and without diabetes. In addition, vitreous AGES were investigated in bovine vitreous collagen after incubation in high glucose, high glucose with aminoguanidine, or normal saline for as long as 8 weeks. AGEs and AGE cross-linking was subsequently determined by quantitative and qualitative assays. RESULTS. There was a significant correlation between AGEs and increasing age in patients without diabetes (r = 0.74). Furthermore, a comparison between age-matched diabetic and nondiabetic vitreous showed a significantly higher level of AGEs in the patients with diabetes (P < 0.005). Collagen purified from bovine vitreous incubated in 0.5 M glucose showed an increase in AGE formation when observed in dot blot analysis, immunogold labeling, and AGE ELISA. Furthermore, there was increased cross-linking of collagen in the glucose-incubated vitreous, when observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and protein separation. This cross-linking was effectively inhibited by coincubation with 10 mM aminoguanidine. CONCLUSIONS. This study suggests that AGEs may form in vitreous with increasing age. This process seems to be accelerated in the presence of diabetes and as a consequence of exposure to high glucose. Advanced glycation and AGE cross-linking of the vitreous collagen network may help to explain the vitreous abnormalities characteristic of diabetes.

Path-selective photoinduced electron transfer (PET) in a membrane-associated system studied by pH-dependent fluorescence

The pH-dependent fluorescence behavior of two regioisomeric 'receptor(1)-spacer(1)-fluorophore-spacer(2)-receptor(2)' systems 1 and 2 in micellar solutions of sodium dodecyl sulfate show that photoinduced electron transfer (PET) only occurs from the amine group connected to the 4-amino position of the aminonaphthalimide fluorophore in both cases. This demonstrates the directing influence of the photogenerated electric field within the aminonaphthalimide excited state on the electron transfer process. Since path-selectivity of PET is also known within the membrane-bound photosynthetic reaction center in bacteria, its origins may be illuminated by the simple experiments described here. (C) 2011 Elsevier B. V. All rights reserved.

A putative gene cluster for aminoarabinose biosynthesis is essential for Burkholderia cenocepacia viability

Using a conditional mutagenesis strategy we demonstrate here that a gene cluster encoding putative aminoarabinose (Ara4N) biosynthesis enzymes is essential for the viability of Burkholderia cenocepacia. Loss of viability is associated with dramatic changes in bacterial cell morphology and ultrastructure, increased permeability to propidium iodide, and sensitivity to sodium dodecyl sulfate, suggesting a general cell envelope defect caused by the lack of Ara4N.

Identification, expression, and DNA sequence of the GDP-mannose biosynthesis genes encoded by the O7 rfb gene cluster of strain VW187 (Escherichia coli O7:K1)

The O7-specific lipopolysaccharide (LPS) in strains of Escherichia coli consists of a repeating unit made of galactose, mannose, rhamnose, 4-acetamido-2,6-dideoxyglucose, and N-acetylglucosamine. We have recently cloned and characterized genetically the O7-specific LPS biosynthesis region (rfbEcO7) of the E. coli O7:K1 strain VW187 (C. L. Marolda, J. Welsh, L. Dafoe, and M. A. Valvano, J. Bacteriol. 172:3590-3599, 1990). In this study, we localized the gnd gene encoding gluconate-6-phosphate dehydrogenase at one end of the rfbEcO7 gene cluster and sequenced that end of the cluster. Three open reading frames (ORF) encoding polypeptides of 275, 464, and 453 amino acids were identified upstream of gndEcO7, all transcribed toward the gnd gene. ORF275 had 45% similarity at the protein level with ORF16.5, which occupies a similar position in the Salmonella enterica LT2 rfb region, and presumably encodes a nucleotide sugar transferase. The polypeptides encoded by ORFs 464 and 453 were expressed under the control of the ptac promoter and visualized in Coomassie blue-stained sodium dodecyl sulfate-polyacrylamide gels and by maxicell analysis. ORF464 expressed GDP-mannose pyrophosphorylase and ORF453 encoded a phosphomannomutase, the enzymes for the biosynthesis pathway of GDP-mannose, one of the nucleotide sugar precursors for the formation of the O7 repeating unit. They were designated rfbMEcO7 and rfbKEcO7, respectively. The RfbMEcO7 polypeptide was homologous to the corresponding protein in S. enterica LT2, XanB of Xanthomonas campestris, and AlgA of Pseudomonas aeruginosa, all GDP-mannose pyrophosphorylases. RfbKEcO7 was very similar to CpsG of S. enterica LT2, an enzyme presumably involved in the biosynthesis of the capsular polysaccharide colanic acid, but quite different from the corresponding RfbK protein of S. enterica LT2.

Outer membrane protein profiles and multilocus enzyme electrophoresis analysis for differentiation of clinical isolates of Proteus mirabilis and Proteus vulgaris

Outer membrane protein (MP) profiles and multilocus enzyme electrophoresis (MEE) analysis were used as tools for differentiating clinical isolates of Proteus spp. Fourteen distinct MP profiles were established by sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis in 54 clinical isolates of Proteus spp. (44 strains identified as P. mirabilis and 10 strains identified as P. vulgaris). Forty-one isolates of P. mirabilis and eight isolates of P. vulgaris were grouped within six and three MP profiles, respectively. The remaining P. mirabilis and P. vulgaris isolates had unique profiles. MEE analysis was used to further discriminate among the strains belonging to the same MP groups. Thirty-five distinct electrophoretic types (ETs) were identified among P. mirabilis isolates. The isolates of P. mirabilis from the four most common MP groups were subgrouped into 30 ETs. All of the P. vulgaris strains had unique ETs. The results suggest that upon biochemical classification of Proteus isolates as P. mirabilis or P. vulgaris, further differentiation among strains of the same species can be obtained by the initial determination of MP profiles followed by MEE analysis of strains with identical MPs.

Klebsiella pneumoniae increases the levels of Toll-like receptors 2 and 4 in human airway epithelial cells

Airway epithelial cells act as the first barrier against pathogens. These cells recognize conserved structural motifs expressed by microbial pathogens via Toll-like receptors (TLRs) expressed on the surface. In contrast to the level of expression in lymphoid cells, the level of expression of TLR2 and TLR4 in airway epithelial cells is low under physiological conditions. Here we explored whether Klebsiella pneumoniae upregulates the expression of TLRs in human airway epithelial cells. We found that the expression of TLR2 and TLR4 by A549 cells and human primary airway cells was upregulated upon infection with K. pneumoniae. The increased expression of TLRs resulted in enhancement of the cellular response upon stimulation with Pam3CSK4 and lipopolysaccharide, which are TLR2 and TLR4 agonists, respectively. Klebsiella-dependent upregulation of TLR expression occurred via a positive IkappaBalpha-dependent NF-kappaBeta pathway and via negative p38 and p44/42 mitogen-activated protein kinase-dependent pathways. We showed that Klebsiella-induced TLR2 and TLR4 upregulation was dependent on TLR activation. An isogenic capsule polysaccharide (CPS) mutant did not increase TLR2 and TLR4 expression. Purified CPS upregulated TLR2 and TLR4 expression, and polymyxin B did not abrogate CPS-induced TLR upregulation. Although no proteins were detected in the CPS preparation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and colloidal gold staining, we could not rule out the possibility that traces of protein in our CPS preparation could have been responsible, at least in part, for the TLR upregulation.

Comparison of a plant based natural surfactant with SDS for washing of As(V) from Fe rich soil

This study explores the possible application of a biodegradable plant based surfactant, obtained from Sapindus mukorossi, for washing low levels of arsenic (As) from an iron (Fe) rich soil. Natural association of As(V) with Fe(III) makes the process difficult. Soapnut solution was compared to anionic surfactant sodium dodecyl sulfate (SDS) in down-flow and a newly introduced suction mode for soil
column washing. It was observed that soapnut attained up to 86% efficiency with respect to SDS in removing As. Full factorial design of experiment revealed a very good fit of data. The suction mode generated up to 83 kPa pressure inside column whilst down-flow mode generated a much higher pressure of 214 kPa, thus making the suction mode more efficient. Micellar solubilisation was found to
be responsible for As desorption from the soil and it followed 1st order kinetics. Desorption rate coefficient of suction mode was found to be in the range of 0.005 to 0.01, much higher than down-flow mode values. Analysis of the FT-IR data suggested that the soapnut solution did not interact chemically with As, offering an option for reusing the surfactant. Soapnut can be considered as a soil washing
agent for removing As even from soil with high Fe content.

Glycation, glycoxidation, and cross-linking of collagen by glucose. Kinetics, mechanisms, and inhibition of late stages of the Maillard reaction

The Maillard or browning reaction between sugar and protein contributes to the increased chemical modification and cross-linking of long-lived tissue proteins in diabetes. To evaluate the role of glycation and oxidation in these reactions, we have studied the effects of oxidative and antioxidative conditions and various types of inhibitors on the reaction of glucose with rat tail tendon collagen in phosphate buffer at physiological pH and temperature. The chemical modifications of collagen that were measured included fructoselysine, the glycoxidation products N epsilon-(carboxymethyl)lysine and pentosidine and fluorescence. Collagen cross-linking was evaluated by analysis of cyanogen bromide peptides using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by changes in collagen solubilization on treatment with pepsin or sodium dodecylsulfate. Although glycation was unaffected, formation of glycoxidation products and cross-linking of collagen were inhibited by antioxidative conditions. The kinetics of formation of glycoxidation products proceeded with a short lag phase and were independent of the amount of Amadori adduct on the protein, suggesting that autoxidative degradation of glucose was a major contributor to glycoxidation and cross-linking reactions. Chelators, sulfhydryl compounds, antioxidants, and aminoguanidine also inhibited formation of glycoxidation products, generation of fluorescence, and cross-linking of collagen without significant effect on the extent of glycation of the protein. We conclude that autoxidation of glucose or Amadori compounds on protein plays a major role in the formation of glycoxidation products and cross-liking of collagen by glucose in vitro and that chelators, sulfhydryl compounds, antioxidants, and aminoguanidine act as uncouplers of glycation from subsequent glycoxidation and cross-linking reactions.

Application of colloidal gas aphron suspensions produced from Sapindus mukorossi for arsenic removal from contaminated soil

Colloidal gas aphron dispersions (CGAs) can be described as a system of microbubbles suspended homogenously in a liquid matrix. This work examines the performance of CGAs in comparison to surfactant solutions for washing low levels of arsenic from an iron rich soil. Sodium Dodecyl Sulfate (SDS) and saponin, a biodegradable surfactant, obtained from Sapindus mukorossi or soapnut fruit were used for generating CGAs and solutions for soil washing. Column washing experiments were performed in down-flow and up flow modes at a soil pH of 5 and 6 using varying concentration of SDS and soapnut solutions as well as CGAs. Soapnut CGAs removed more than 70% arsenic while SDS CGAs removed up to 55% arsenic from the soil columns in the soil pH range of 5–6. CGAs and solutions showed comparable performances in all the cases. CGAs were more economical since it contains 35% of air by volume, thereby requiring less surfactant. Micellar solubilization and low pH of soapnut facilitated arsenic desorption from soil column. FT-IR analysis of effluent suggested that soapnut solution did not interact chemically with arsenic thereby facilitating the recovery of soapnut solution by precipitating the arsenic. Damage to soil was minimal arsenic confirmed by metal dissolution from soil surface and SEM micrograph.

Functional comparison of the endothelial nitric oxide synthase Glu298Asp polymorphic variants in human endothelial cells

The G894T endothelial nitric oxide synthase (eNOS) polymorphism results in a Glu to Asp substitution at position 298. This position is located externally on the protein and as the regulation of eNOS is dependent on its subcellular localization and interaction with modulatory proteins, we aimed to address whether the substitution of Asp at 298 had any effect on these mechanisms. Initially, we developed a novel method to accurately determine molar quantities of each variant by expressing them as green fluorescent protein (GFP) fusion proteins and using recombinant adenoviruses to facilitate transient infection of human microvascular endothelial cells. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blotting of eNOS298Asp revealed a 135-kDa proteolytic fragment which was not present with eNOS298Glu. This proteolysis was prevented by using LDS buffer confirming that this differential cleavage is an artefact of sample preparation and unlikely to occur intracellularly. Nitric oxide was measured following stimulation with calcium ionophore or oestrogen in the presence of varying sepiapterin concentrations. GFP fluorescence was used to quantify the amount of fusion protein and calculate intracellular specific activity. There was no significant difference in intracellular specific activity between Glu298 and Asp298 eNOS in response to calcium ionophore or oestrogen. Tetrahydrobiopterin supplementation increased eNOS activity of both variants in an identical manner. The presence of the GFP also facilitated the visualization of the variants by confocal microscopy and demonstrated that both localized to the plasma membrane and the Golgi. These findings demonstrate that the Asp substitution at 298 does not have a major effect in modulating eNOS activity in vivo.

The application of colloidal gas aphrons in the recovery of fine cellulose fibres from paper mill wastewater

Colloidal gas aphrons (CGAs) are micron-sized gas bubbles of 25–30 µm in diameter produced by a high-speed stirrer in a vessel containing dilute surfactant solution. These bubbles, because of their small size, exhibit some colloidal properties. In this work, CGAs were used to separate fine fibres from a lean slurry of cellulosic pulp in a flotation column. The pulp fibres were recovered as foamate from the top. Sodium dodecyl sulphate at a concentration of 2.0 kg/m3 was used as a surfactant to generate the CGAs in a spinning disc apparatus. The results indicated that up to 70% flotation efficiency could be obtained within a short column height of 0.3–0.35 m. This technique can be applied to recover fine cellulosic pulp from paper-machine backwater.

LOCALIZATION OF ACTIN IN THE LIVER FLUKE, FASCIOLA-HEPATICA

Adult and 3-week-old juvenile Fasciola hepatica were examined for the presence of the cytoskeletal protein actin. Techniques of direct fluorescence using fluorescein isothiocyanate (FITC)-phalloidin and of indirect immunofluorescence using a monoclonal anti-actin antibody (MAA) demonstrated actin in the testes, sub-tegumental and gut musculature, tegumental cell bodies and tegumental spines. In contrast, polyclonal anti-actin antibody (PAA) revealed immunostaining only in the vitellaria. Effective removal of the tegument with 1 % (w/v) sodium dodecyl sulphate (SDS) was confirmed by scanning electron microscopy (SEM), and this enabled immunoblotting of whole fluke and tegumental fractions with and without spines. Whole fluke fractions produced three bands corresponding to molecules exhibiting relative molecular weights of 43, 28 and 15 kDa, respectively, whereas the tegumental fraction with spines revealed a single band corresponding to 15 kDa in size. The fraction without spines displayed no bands. The present study localised actin in a number of different tissue types within the liver fluke. Using MAA, three forms of actin have been identified in the whole fluke and a single one in the tegumental spines.

Electrically-responsive anti-adherent hydrogels for photodynamic antimicrobial chemotherapy

The loading of the photosensitisers meso-Tetra (N-methyl-4-pyridyl) porphine tetra tosylate (TMP), methylene blue (MB) and IMP with sodium dodecyl sulphate (SDS) into and release from hydrogels composed of the polyelectrolyte poly(methyl vinyl ether-co-maleic acid) crosslinked in a 2:1 ratio with PEG 10,000 were investigated as a potential rapid photodynamic antimicrobial chemotherapy (PACT) treatment for infected wounds using iontophoresis as a novel delivery method. Photosensitiser uptake was very high; (% TMP uptake; 95.53-96.72%) (% MB uptake; 90.58-93.26%) and was PMVE/MA concentration independent, whilst SDS severely limited TMP uptake (5.93-8.75%). Hydrogel hardness, compressibility and adhesiveness on the dermal surface of neonate porcine skin increased with PMVE/MA concentration and were significantly increased with SDS.

The ionic conductivities of the hydrogels increased with PMVE/MA concentration. Drug release was PMVE/MA concentration independent, except for drug release under iontophoteric conditions for MB and TMP (without SDS). In just 15 min, the mean% drug concentrations released of TMP, TMP (with SDS) and MB using an electric current ranged from 22.30 to 64.72 mu gml(-1), 6.37-4.59 mu gml(-1) and 11.73-36.57 mu gml(-1) respectively. These concentrations were in excess of those required to induce complete kill of clinical strains of meticillin-resistant Staphylococcus aureus and Burkholderia cepacia. Thus these results support our contention that the iontophoteric delivery of IMP and MB using anti-adherent, electrically-responsive, PEG-crosslinked PMVE/MA hydrogels are a potential option in the rapid PACT treatment of infected wounds. (c) 2012 Elsevier B.V. All rights reserved.

Localisation of actin in the liver fluke, Fasciola hepatica

Adult and 3-week-old juvenile Fasciola hepatica were examined for the presence of the cytoskeletal protein actin. Techniques of direct fluorescence using fluorescein isothiocyanate (FITC)-phalloidin and of indirect immunofluorescence using a monoclonal anti-actin antibody (MAA) demonstrated actin in the testes, sub-tegumental and gut musculature, tegumental cell bodies and tegumental spines. In contrast, polyclonal anti-actin antibody (PAA) revealed immunostaining only in the vitellaria. Effective removal of the tegument with 1% (w/v) sodium dodecyl sulphate (SDS) was confirmed by scanning electron microscopy (SEM), and this enabled immunoblotting of whole fluke and tegumental fractions with and without spines. Whole fluke fractions produced three bands corresponding to molecules exhibiting relative molecular weights of 43, 28 and 15 kDa, respectively, whereas the tegumental fraction with spines revealed a single band corresponding to 15 kDa in size. The fraction without spines displayed no bands. The present study localised actin in a number of different tissue types within the liver fluke. Using MAA, three forms of actin have been identified in the whole fluke and a single one in the tegumental spines.