Conversion of solid waste to activated carbon to improve landfill sustainability.

Heterogeneous composite wastes from landfills were evaluated as precursors for the generation of activated carbon (AC). A single-step chemical activation process was applied involving irradiation with microwave energy and impregnation with KOH. The average percentage yield of AC from active landfill precursor was higher than that from closed landfill for all depths sampled. Increase in impregnation ratio and irradiation power decreased the average percentage yield for both landfill precursors (active: 38.1 to 33.1%; closed: 42.1 to 33.3%). The optimum pH range for adsorption of methylene blue was pH 6-7, while adsorption increased with increase in temperature over the range 30 to 50°C. Carbonyl and hydroxyl groups were the major functional groups on the surface of AC. The properties of the AC are potentially suitable for the removal of cationic dyes and pollutants. AC generated from the landfill composite was comparable to that from other biomass being managed through AC generation. This is the first report to demonstrate the possible reuse of landfill composite as AC. The reuse option of landfill composite could provide a means of sustainable management of landfilled municipal waste.

Multivariate analysis of the effects of age, particle size and landfill depth on heavy metals pollution content of closed and active landfill precursors.

Multivariate analysis of a heavy metal pollution survey of closed and active landfill precursors was carried out in order to compare environmental risk levels in relation to age, particle size and depth of the precursors. Landfill precursors (77) were collected and analyzed for 15 USEPA toxic heavy metals using ICP-MS. Heavy metals concentrations in closed landfill precursors were significantly higher than those in the active landfill for 11 of 15 heavy metals investigated (closed landfill order: Fe > Al > Mn > Cu > Pb > Ba > Co > Cr > Ni > Cd > As > Se > Ti). Cluster analysis and correlation studies indicated the distribution of the metals was more influenced by landfill precursor size than by depth of the sample. Principal component analysis (PCA) showed that 10 of 15 of heavy metals of both landfill precursors were from similar anthropogenic sources. Heavy metals pollution indices (Igeo > 5, EF > 40 and CF > 7) of both active and closed landfill precursors exceeded limits in the order of Zn > Cd > Pb > Cu > Ag, indicating a major potential health risk influenced by age and particle size of precursor. Zn, Cd, Cu and Pb of both landfill precursors exceeded the USEPA set standard for assessment of human health risk for each of the metals (1 × 10-4 to 1 × 10-3). This study highlights the need for the integration of a clean-up process for precursors from both types of landfill to reduce possible environmental pollution during a reuse process.

Dietary intake of cadmium from Bangladeshi foods.

UNLABELLED: Human exposure to cadmium (Cd) is associated with various diseases and high levels of Cd have been detected in Bangladeshi population warranting further research to identify the source of this exposure. In this study, Cd levels in 327 and 94 samples of Bangladeshi food and non-food samples, respectively, were determined using inductively coupled plasma mass spectrometry. This is the largest number of Bangladeshi food and nonfood samples investigated for their Cd content. High Cd levels were detected in leafy vegetables (mean 31 [SD 29]µg/kg). Of these vegetables, lal shak (Amaranthus tricolor) contained the highest Cd level (303 µg/kg [wet weight]; mean 100.5 [SD 95]µg/kg). Bangladeshi rice also showed significant concentration of Cd (mean 37.2 [SD 30]µg/kg). Of particular concern is the very high level of Cd detected in some puffed rice, which we attribute to the illegal practice of using urea for whitening the puffed rice. Tobacco leaves, which are commonly consumed during betel quid chewing by Bangladeshis, contain significant levels of Cd (mean 95 [SD 87]µg/kg). The total daily intake (TDI) of Cd from foods for Bangladeshis was estimated to be 34.55 µg/d. This is rather high when compared to the TDI of Cd for other populations. Our analysis reveals that this is mainly due to the very high intake of rice and vegetables, and lower consumption of animal products (which are low in Cd), by the Bangladeshis. We also determined the provisional maximum tolerable daily intake and target hazard quotients values for Cd. Clearly a more balanced diet is necessary to reduce the Cd intake in the Bangladeshi population, especially by reducing the very high intake of rice and certain leafy vegetables. Food manufacturing and agricultural practices needs to be altered to reduce the entry of Cd into the food chain. PRACTICAL APPLICATION: Exposure to high levels of Cd can be harmful to human health and this study provides a comprehensive analysis of Cd levels in a variety of food items from Bangladesh. The findings are of particular importance to consumers of Bangladeshi foods in both Bangladesh and in other countries. Data obtained will be valuable resources for food safety and regulatory bodies as our study suggests entry of Cd in foods through use of illegal chemicals in food manufacturing processes.

Application of Fourier transform infrared spectroscopy for monitoring hydrolysis and synthesis reactions catalyzed by a recombinant amidase.

This study demonstrates the use of Fourier transform infrared (FTIR) spectroscopy for monitoring both synthesis and hydrolysis reactions catalyzed by a recombinant amidase (EC 3.5.1.4) from Pseudomonas aeruginosa. The kinetics of hydrolysis of acetamide, propionamide, butyramide, acrylamide, benzamide, phenylalaninamide, alaninamide, glycinamide, and leucinamide were determined. This revealed that very short-chain substrates displayed higher amidase activity than did branched side-chain or aromatic substrates. In addition, on reducing the polarity and increasing the substrates' bulkiness, a reduction of the amidase affinity for the substrates took place. Using FTIR spectroscopy it was possible to monitor and quantify the synthesis of several hydroxamic acid derivatives and ester hydrolysis products. These products may occur simultaneously in a reaction catalyzed by the amidase. The substrates used for the study of such reactions were ethyl acetate and glycine ethyl ester. Hydroxylamine was the nucleophile substrate used for the synthesis of acetohydroxamate compounds. Results presented in this article demonstrate the usefulness of FTIR spectroscopy as an important tool for understanding the enzyme structure-activity relationship because it provides a simple and rapid real-time assay for the detection and quantification of amidase hydrolysis and synthesis reactions in situ.

A survey of arsenic in foodstuffs on sale in the United Kingdom and imported from Bangladesh.

Arsenic is a highly toxic element and its presence in food composites is a matter of concern to the well being of both humans and animals. Arsenic-contaminated groundwater is often used in Bangladesh and West Bengal (India) to irrigate crops used for food and animal consumption, which could potentially lead to arsenic entering the human food chain. In this study, we used graphite furnace atomic absorption spectroscopy to determine the total arsenic concentrations in a range of foodstuffs, including vegetables, rice and fish, imported into the United Kingdom from Bangladesh. The mean and range of the total arsenic concentration in all the vegetables imported from Bangladesh were 54.5 and 5-540 microg/kg, respectively. The highest arsenic values found were for the skin of Arum tuber, 540 microg/kg, followed by Arum Stem, 168 microg/kg, and Amaranthus, 160 microg/kg. Among the other samples, freshwater fish contained total arsenic levels between 97 and 1318 microg/kg. The arsenic content of the vegetables from the UK was approximately 2- to 3-fold lower than those observed for the vegetables imported from Bangladesh. The levels of arsenic found in vegetables imported from Bangladesh in this study, in some cases, are similar to those previously recorded for vegetables grown in arsenic-affected areas of West Bengal, India, although lower than the levels reported in studies from Bangladesh. While the total arsenic content detected in our study in vegetables, imported from Bangladesh, is far less than the recommended maximum permitted level of arsenic, it does provide an additional source of arsenic in the diet. This raises the possibility that the level of arsenic intake by certain sectors of the UK population may be significantly higher then the general population and requires further investigations.

Measuring enzymatic activity of a recombinant amidase using Fourier transform infrared spectroscopy.

A method based on Fourier transform infrared spectroscopy (FT-IR) has been developed for assaying the Pseudomonas aeruginosa native amidase (E.C. 3.5.1.4), overproduced in an Escherichia coli strain. The kinetic of acetamide hydrolysis by the enzyme, in aqueous media, was monitored by measuring the intensity of the acetamide amide I band maximum at 1635 cm(-1) as a function of time. A value of 0.5mM(-1) cm(-1) was obtained for the extinction coefficient (epsilon) of acetamide at this frequency. The rate of the hydrolysis was found to be linear with the concentration of the enzyme up to 90 microM. The Michaelis-Menten kinetics parameters V and K(m) were determined as 30.7 U/mg and 4mM, respectively. These results were similar to those obtained using high-performance liquid chromatography analysis of the same hydrolytic reaction catalyzed by amidase either in water or in buffer. This suggests that the precision of the FT-IR method is suitable for the kinetic studies of amidase with the additional advantage of being able to perform a real-time measurement of the enzymatic activity.

Fourier transform infrared spectrometric analysis of protein conformation: effect of sampling method and stress factors.

Changes in the amide bands in Fourier transform infrared spectra of proteins are generally attributed to alterations in protein secondary structure. In this study spectra of five different globular proteins were compared in the solid and solution states recorded with several sampling techniques. Spectral differences for each protein were observed between the various sampling techniques and physical states, which could not all be explained by a change in protein secondary structure. For example, lyophilization in the absence of lyoprotectants caused spectral changes that could (partially) have been caused by the removal of hydrating water molecules rather than secondary structural changes. Moreover, attenuated total reflectance spectra of proteins in H2O were not directly comparable to transmission spectra due to the anomalous dispersion effect. Our study also revealed that the amide I, II, and III bands differ in their sensitivities to changes in protein conformation: For example, strong bands in the region 1620-1630 and 1685-1695 cm(-1) were seen in the amide I region of aggregated protein spectra. Surprisingly, absorbance of such magnitudes was not observed in the amide II and III region. It appears, therefore, that only the amide I can be used to distinguish between intra- and intermolecular beta-sheet formation. Considering the differing sensitivity of the different amide modes to structural changes, it is advisable to utilize not only the amide I band, but also the amide II and III bands, to determine changes in protein secondary structure. Finally, it is important to realize that changes in these bands may not always correspond to secondary structural changes of the proteins.

Structural model of a voltage-gated potassium channel based on spectroscopic data.

It is estimated that membrane proteins comprise as much as 30% of most genomes. Yet our knowledge of membrane-protein folding is still in its infancy. Consequently, there is a great need for developing approaches that can further advance our understanding of how peptides and proteins interact with membranes and thereby attain their folded structure. An approach that we have been exploring involves dissecting voltage-gated ion channels into simple peptide domains for the purpose of determining their structure in different media using physical techniques. We have synthesized peptides corresponding to the six membrane-spanning segments, as well as the pore domain, of the Shaker channel and characterized their secondary structures. From these studies we have developed a model for the transmembrane structure of the Shaker potassium channel that is constructed from alpha-helices. The hard structural data obtained from these studies lends support to the recent theoretical models of this channel protein that have been developed by others.

Effect of the disulfide bridge and the C-terminal extension on the oligomerization of the amyloid peptide ABri implicated in familial British dementia.

Familial British dementia (FBD) is a rare neurodegenerative disorder and shares features with Alzheimer's disease, including amyloid plaque deposits, neurofibrillary tangles, neuronal loss, and progressive dementia. Immunohistochemical and biochemical analysis of plaques and vascular amyloid of FBD brains revealed that a 4 kDa peptide named ABri is the main component of the highly insoluble amyloid deposits. In FBD patients, the ABri peptide is produced as a result of a point mutation in the usual stop codon of the BRI gene. This mutation produces a BRI precursor protein 11 amino acids longer than the wild-type protein. Mutant and wild-type precursor proteins both undergo furin cleavage between residues 243 and 244, producing a peptide of 34 amino acids in the case of ABri and 23 amino acids in the case of the wild-type (WT) peptide. Here we demonstrate that the intramolecular disulfide bond in ABri and the C-terminal extension are required to elongate initially formed dimers to oligomers and fibrils. In contrast, the shorter WT peptide did not aggregate under the same conditions. Conformational analyses indicate that the disulfide bond and the C-terminal extension of ABri are required for the formation of beta-sheet structure. Soluble nonfibrillar ABri oligomers were observed prior to the appearance of mature fibrils. A molecular model of ABri containing three beta-strands, and two beta-hairpins annealed by a disulfide bond, has been constructed, and predicts a hydrophobic surface which is instrumental in promoting oligomerization.

Vitamin D(2) modulates melittin-membrane interactions.

In the present work, mutual interaction of melittin, a pore forming hemolytic toxin from bee venom, and vitamin D(2), an antioxidant steroid, with dipalmitoyl phosphatidylcholine (DPPC) multilamellar liposomes has been investigated. Turbidity and Fourier transform infrared (FTIR) spectroscopic measurements, in combination with thermodynamic calculations, were used to monitor the modulating effect of vitamin D(2) on a melittin-DPPC membrane system. The results indicate that melittin on its own decreases the main phase transition to lower temperatures and also dramatically decreases the stability of the membrane. It has an overall disordering effect on the phospholipid membrane structures. Inclusion of vitamin D(2) at low concentrations (3, 6 mol%) into melittin containing DPPC liposomes slightly shifts the main phase transition to lower temperatures. High concentration of vitamin D(2) (9 mol%) has a more dramatic effect in shifting the main phase transition to lower temperature. It also causes a significant broadening in the phase transition curve. The present study also demonstrates that, with the addition of vitamin D(2) into melittin-DPPC system, absorbance value in turbidity study and the frequency of the CH(2) stretching band in FTIR study changes in a manner that are consistent with a reduction in the membrane perturbing effect of melittin on DPPC liposomes. Vitamin D(2) diminishes the disordering effect of melittin on DPPC lipids and produces a more ordered membrane system. These results were confirmed with thermodynamic calculations.

A synthetic peptide adhesion epitope as a novel antimicrobial agent.

The earliest step in microbial infection is adherence by specific microbial adhesins to the mucosa of the oro-intestinal, nasorespiratory, or genitourinary tract. We inhibited binding of a cell surface adhesin of Streptococcus mutans to salivary receptors in vitro, as measured by surface plasmon resonance, using a synthetic peptide (p1025) corresponding to residues 1025-1044 of the adhesin. Two residues within p1025 that contribute to binding (Q1025, E1037) were identified by site-directed mutagenesis. In an in vivo human streptococcal adhesion model, direct application of p1025 to the teeth prevented recolonization of S. mutans but not Actinomyces, as compared with a control peptide or saline. This novel antimicrobial strategy, applying competitive peptide inhibitors of adhesion, may be used against other microorganisms in which adhesins mediate colonization of mucosal surfaces.

Human complement factor I: its expression by insect cells and its biochemical and structural characterisation.

Factor I is a five-domain plasma serine protease which is essential for the regulation of the complement system. In order to express this, the factor I coding sequence was cloned into a recombinant baculovirus system, which was used to infect Trichoplusia ni cells. Using the native factor I leader sequence, recombinant factor I (rFI) was secreted into the culture medium. Purified rFI was recognised by polyclonal antisera and by the factor I-specific monoclonal antibody MRC-OX21. SDS PAGE showed that rFI was processed into two chains with molecular weights of 48,000 and 36,000. Amino acid sequence analysis showed that the N-terminal sequences of the rFI chains were the same as those of serum-derived factor I (sFI), confirming that processing was correct. Since both molecular weights were less than those observed for sFI, this is attributed to the replacement of complex-type oligosaccharides by high mannose ones in rFI. C3(NH,) cleavage assays showed that rFI had 55% the activity of sFI. Circular dichroism and Fourier transform infrared spectroscopy showed that the protein folding of rFI and sFI were very similar. Both had a secondary structure low in alpha-helix and high in beta-sheet, as expected from crystal structure and multiple sequence alignment analyses. It is inferred that the reduced activity of rFI is attributable to its changed glycosylation. The availability of rFI and structures for the domains in factor I makes possible new approaches to determine the molecular basis of its interactions with factor H and C3b.

Secondary structure analysis of the putative membrane-associated domains of the inward rectifier K+ channel ROMK1.

The inward rectifier K+ channels contain two putative membrane-spanning domains per subunit (M1, M2) and a 'pore' (P) region, which is similar to the H5 domain of voltage-gated K+ channels. Here we have used Fourier transform infrared (FTIR) and CD spectroscopy to analyse the secondary structures of synthetic peptides corresponding to the M1, M2 and P regions of ROMK1 in aqueous solution, in organic solvents and in phospholipid membranes. A previous CD study was unable to provide any structural data on a similar P peptide [Ben-Efraim and Shai (1997) Biophys. J. 72, 85-96]. However, our FTIR and CD spectroscopic analyses indicate that this peptide adopts an alpha-helical structure when reconstituted into dimyristoyl phosphatidylcholine vesicles and lysophosphatidyl choline (LPC) micelles as well as in trifluoroethanol (TFE) solvent. This result is in good agreement with a previous study on a peptide corresponding to the pore domain of a voltage-gated K+ channel [Haris, Ramesh, Sansom, Kerr, Srai and Chapman (1994) Protein Eng. 7, 255-262]. FTIR spectra of the M1 peptide in LPC micelles displayed a strong absorbance characteristic of an intermolecular beta-sheet structure, suggesting aggregation of the M1 peptide. Sucrose gradient centrifugation was used to separate aggregated peptide from peptide incorporated into micelles in an unaggregated manner; subsequent analysis by FTIR suggested that the M1 peptide adopted an alpha-helical structure when incorporated into phospholipid membranes. FTIR and CD spectra of the M2 peptide in phospholipids and high concentrations of TFE suggest that this peptide adopts an alpha-helical structure. The structural data obtained in these experiments have been used to propose a model for the structure of the membrane-associated core (M1-P-M2) of the inward rectifier K+ channel protein.

Synthetic peptide fragments as probes for structure determination of potassium ion-channel proteins.

Potassium channels are a diverse class of transmembrane proteins that are responsible for diffusion of potassium ion across cell membranes. The lack of large quantities of these proteins from natural sources, is a major hindrance in their structural characterization using biophysical techniques. Synthetic peptide fragments corresponding to functionally important domains of these proteins provide an attractive approach towards characterizing the structural organization of these ion-channels. Conformational properties of peptides from three different potassium channels (Shaker, ROMK1 and minK) have been characterized in aqueous media, organic solvents and in phospholipid membranes. Techniques used for these studies include FTIR, CD and 2D-NMR spectroscopy. FTIR spectroscopy has been a particularly valuable tool for characterizing the folding of the ion-channel peptides in phospholipid membranes; the three different types of potassium channels all share a common transmembrane folding pattern that is composed of a predominantly alpha-helical structure. There is no evidence to suggest the presence of any significant beta-sheet structure. These results are in excellent agreement with the crystal structure of a bacterial potassium channel (Doyle, D. A. et al. (1998) Science 280:69 77), and suggest that all potassium channel proteins may share a common folding motif where the ion-channel structure is constructed entirely from alpha-helices.

Synthetic putative transmembrane region of minimal potassium channel protein (minK) adopts an alpha-helical conformation in phospholipid membranes.

Minimal potassium channel protein (minK) is a potassium channel protein consisting of 130 amino acids, possessing just one putative transmembrane domain. In this study we have synthesized a peptide with the amino acid sequence RDDSKLEALYILMVLGFFGFFTLGIMLSYI, containing the putative transmembrane region of minK, and analysed its secondary structure by using Fourier-transform IR and CD spectroscopy. The peptide was virtually insoluble in aqueous buffer, forming intermolecular beta-sheet aggregates. On attempted incorporation of the peptide into phospholipid membranes with a method involving dialysis, the peptide adopted a predominantly intermolecular beta-sheet conformation identical with that of the peptide in aqueous buffer, in agreement with a previous report [Horvàth, Heimburg, Kovachev, Findlay, Hideg and Marsh, (1995) Biochemistry 34, 3893-3898]. However, by using an alternative method of incorporating the peptide into phospholipid membranes we found that the peptide adopted a predominantly alpha-helical conformation, a finding consistent with various proposed structural models. These observed differences in secondary structure are due to artifacts of aggregation of the peptide before incorporation into lipid.

Alterations in the structure of apolipoprotein B-100 determine the behaviour of LDL towards thromboplastin.

Apolipoprotein B-100 acts as an inhibitor of thromboplastin activity independently of the tissue factor pathway inhibitor (TFPI) associated with plasma lipoproteins. Analysis of the primary structure of Apo B-100 showed a higher than expected occurrence of lysine groups in the receptor-binding region. In order to demonstrate the participation of lysine groups of Apo B-100 in the inhibition of thromboplastin, thromboplastin and Apo B-100 were incubated together in the presence of poly-L-lysine, poly-L-arginine, lysine and arginine monomers. The inhibition of thromboplastin by Apo B-100 was completely suppressed in the presence of poly-L-lysine. Poly-L-arginine was found to be less effective and neither lysine or arginine monomers had any significant effect on the inhibitory effect of Apo B-100. Alterations in the structure of Apo B-100 reconstituted in lipid vesicles resembling LDL, brought about by lipid peroxidation and lipid loading were examined by means of Fourier transform infra-red spectroscopy. It was found that, upon oxidation without the addition of cupric ions, the apolipoprotein attains a more exposed conformation with an increase in alpha-helical structure. This increase occurred at the expense of beta-structure. On lipid loading, an increase in beta-structure at the expense of the alpha-helix, was demonstrated. It is therefore proposed that the variable action of LDL towards thromboplastin derives from alterations in the secondary structure of the Apo B-100, particularly the receptor-binding region.