This study investigates adsorption-desorption and the leaching potential of glyphosate and aminomethylphosphonic acid (AMPA) in control and amended-addition of cow dung or rice husk ash-acidic Malaysian soil with high oxide mineral content. The addition of cow dung or rice husk ash increased the adsorptive removal of AMPA. The isotherm data of glyphosate and AMPA best fitted the Freundlich model. The constant Kf for glyphosate was high in the control soil (544.873 mg g-1) followed by soil with cow dung (482.451 mg g-1) then soil with rice husk ash (418.539 mg g-1). However, for AMPA, soil with cow dung was high (166.636 mg g-1) followed by soil with rice husk ash (137.570 mg g-1) then the control soil (48.446 mg g-1). The 1/n values for both glyphosate and AMPA adsorptions were
1. This study was undertaken to examine the effect of feeding glycine (Gly)-fortified low protein (LP) diets on the growth performance, duodenal morphology and caecal microbial populations of broiler chickens raised under unheated, cyclic or constant heat stress environmental conditions. 2. From d 1 to 21 (starter phase), an equivalent number of birds were fed either a normal protein (NP) diet or a LP diet fortified with Gly. From d 22 to 42 (grower phase), an equivalent number of birds from each starter diet were distributed to one of the following dietary groups: (i) an NP diet during the starter and grower phases (NPNP), (ii) an NP diet during the starter phase and a LP diet during the grower phase (NPLP), (iii) an LP diet during the starter phase and an NP diet during the grower phase (LPNP) or (iv) LP diets during both phases (LPLP). 3. Commencing from d 22, an equivalent number of birds from each dietary group were exposed to (i) 23 ± 1°C throughout (unheated), (ii) 34 ± 1°C for 7 h each day from 10:00 to 17:00 (cyclic heat) or (iii) 34 ± 1°C throughout (constant heat). 4. Feeding the LP diet during the starter phase resulted in feed intake (FI), weight gain (WG), feed conversion ratios (FCR) and energy efficiency ratios (EER) similar to those for the NP diet. The birds fed the LP diet had a significantly higher protein efficiency ratio (PER) compared with the birds fed the NP diet. 5. During the grower phase, there were significant diet × temperature interactions for F, WG, FCR, PER, EER, villus height, crypt depth and caecal Clostridia. The birds fed the NPLP and LPLP diets had lower FI, WG and EER, higher FCR, shorter villus height and crypt depth and higher caecal Clostridia compared with the birds fed LPNP and NPNP diets under constant heat stress. However, feeding birds the NPLP and LPLP diets resulted in FI, WG, EER, FCR, morphology parameters and caecal Clostridia equivalent to the birds fed LPNP and NPNP diets, as well as improved PER, under unheated and cyclic heat stress conditions. 6. In conclusion, our results indicate that Gly-fortified LP diets can be fed to broilers under normal and acute heat stress environmental conditions without any adverse effects on performance. However, the use of such LP diets can be detrimental to broilers under chronic heat stress conditions.
Cancer is the second leading cause of death-1 in 6 deaths globally is due to cancer. Cancer metabolism is a complex and one of the most actively researched area in cancer biology. Metabolic reprogramming in cancer cells entails activities that involve several enzymes and metabolites to convert nutrient into building blocks that alter energy metabolism to fuel rapid cell division. Metabolic dependencies in cancer generate signature metabolites that have key regulatory roles in tumorigenesis. In this minireview, we highlight recent advances in the popular methods ingrained in biochemistry research such as stable and flux isotope analysis, as well as radioisotope labeling, which are valuable in elucidating cancer metabolites. These methods together with analytical tools such as chromatography, nuclear magnetic resonance spectroscopy and mass spectrometry have helped to bring about exploratory work in understanding the role of important as well as obscure metabolites in cancer cells. Information obtained from these analyses significantly contribute in the diagnosis and prognosis of tumors leading to potential therapeutic targets for cancer therapy.
The evolution of weed-resistant species threatens the sustainable use of glyphosate, which is the most important herbicide widely used in agriculture worldwide. Moreover, the high glyphosate resistance (>180-fold based on LD50) of Eleusine indica found in Malaysia, which carries a double mutation in its 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), made the control of this species more difficult. By contrast, the same species carrying the same double mutation in EPSPS (T102I+P106S) but found in China only shows a resistance level of not more than 14-fold based on GR50. The resistance level of this population is four times higher than that of the population carrying a single mutation (P106L). Although the members of this population survive under a high glyphosate dosage of 10,080gaeha-1, their growth was significantly inhibited by glyphosate under the recommend dose (840gaeha-1), where in the fresh weight was 85.4% of the control. EPSPS expression, relative copy number, and EPSPS activity in this population were similar to those of the susceptible population. In addition, the expression of two glutathione transferase (GST) genes (GST-U8 and GST-23) and the enzyme activity of the GST in this population did not significantly differ from those of the susceptible population. This finding is important in elucidating the resistance of the naturally evolved glyphosate-resistant (GR) weed species carrying a double mutation in EPSPS to glyphosate.
A series of ternary copper(II)-1,10-phenanthroline complexes with glycine and methylated glycine derivatives, [Cu(phen)(aa)(H(2)O)]NO(3)·xH(2)O 1-4 (amino acid (aa): glycine (gly), 1; DL: -alanine (DL: -ala), 2; 2,2-dimethylglycine (C-dmg), 3; sarcosine (sar), 4), were synthesized and characterized by FTIR, elemental analysis, electrospray ionization-mass spectra (ESI-MS), UV-visible spectroscopy and molar conductivity measurement. The determined X-ray crystallographic structures of 2 and 3 show each to consist of distorted square pyramidal [Cu(phen)(aa)(H(2)O)](+) cation, a nitrate counter anion, and with or without lattice water, similar to previously reported structure of [Cu(phen)(gly)(H(2)O)]NO(3)·1½H(2)O. It is found that 1-4 exist as 1:1 electrolytes in aqueous solution, and the cationic copper(II) complexes are at least stable up to 24 h. Positive-ion ESI-MS spectra show existence of only undissociated [Cu(phen)(aa)](+) species. Electron paramagnetic resonance, gel electrophoresis, fluorescence quenching, and restriction enzyme inhibition assay were used to study the binding interaction, binding affinity and selectivity of these complexes for various types of B-form DNA duplexes and G-quadruplex. All complexes can bind selectively to DNA by intercalation and electrostatic forces, and inhibit topoisomerase I. The effect of the methyl substituents of the coordinated amino acid in the above complexes on these biological properties are presented and discussed. The IC(50) values (24 h) of 1-4 for nasopharyngeal cancer cell line HK1 are in the range 2.2-5.2 μM while the corresponding values for normal cell line NP69 are greater than 13.0 μM. All complexes, at 5 μM, induced 41-60 % apoptotic cell death in HK1 cells but no significant cell death in NP69 cells.
A dynamic supported liquid membrane tip extraction (SLMTE) procedure for the effective extraction and preconcentration of glyphosate (GLYP) and its metabolite aminomethylphosphonic acid (AMPA) in water has been investigated. The SLMTE procedure was performed in a semi-automated dynamic mode and demonstrated a greater performance against a static extraction. Several important extraction parameters such as donor phase pH, cationic carrier concentration, type of membrane solvent, type of acceptor stripping phase, agitation and extraction time were comprehensively optimized. A solution of Aliquat-336, a cationic carrier, in dihexyl ether was selected as the supported liquid incorporated into the membrane phase. Quantification of GLYP and AMPA was carried out using capillary electrophoresis with contactless conductivity detection. An electrolyte solution consisting of 12 mM histidine (His), 8 mM 2-(N-morpholino)ethanesulfonic acid (MES), 75 microM cetyltrimethylammonium bromide (CTAB), 3% methanol, pH 6.3, was used as running buffer. Under the optimum extraction conditions, the method showed good linearity in the range of 0.01-200 microg/L (GLYP) and 0.1-400 microg/L (AMPA), acceptable reproducibility (RSD 5-7%, n=5), low limits of detection of 0.005 microg/L for GLYP and 0.06 microg/L for AMPA, and satisfactory relative recoveries (90-94%). Due to the low cost, the SLMTE device was disposed after each run which additionally eliminated the possibility of carry-over between runs. The validated method was tested for the analysis of both analytes in spiked tap water and river water with good success.
Many algae are rich sources of sulfated polysaccharides with biological activities. The physicochemical/rheological properties and biological activities of sulfated polysaccharides are affected by the pattern and number of sulfate moieties. Sulfation of carbohydrates is catalyzed by carbohydrate sulfotransferases (CHSTs) while modification of sulfate moieties on sulfated polysaccharides was presumably catalyzed by sulfatases including formylglycine-dependent sulfatases (FGly-SULFs). Post-translationally modification of Cys to FGly in FGly-SULFs by sulfatase modifiying factors (SUMFs) is necessary for the activity of this enzyme. The aims of this study are to mine for sequences encoding algal CHSTs, FGly-SULFs and putative SUMFs from the fully sequenced algal genomes and to infer their phylogenetic relationships to their well characterized counterparts from other organisms. Algal sequences encoding CHSTs, FGly-SULFs, SUMFs, and SUMF-like proteins were successfully identified from green and brown algae. However, red algal FGly-SULFs and SUMFs were not identified. In addition, a group of SUMF-like sequences with different gene structure and possibly different functions were identified for green, brown and red algae. The phylogeny of these putative genes contributes to the corpus of knowledge of an unexplored area. The analyses of these putative genes contribute toward future production of existing and new sulfated carbohydrate polymers through enzymatic synthesis and metabolic engineering.
The development of glyphosate-resistant genetically modified organisms (GMO) has increased the use of herbicide glyphosate by several magnitudes in recent years. It is now the most commonly used pesticide globally that affects aquatic habitats, especially fish. This study aims to add new knowledge on the effect of technical grade glyphosate on several toxicity parameters and to identify the most effective parameter in predicting technical grade glyphosate chronic toxicity (seven weeks) to fish, especially Malaysia's heavily farmed red tilapia. The results show that a relatively high concentration of technical grade glyphosate is needed to induce significant changes in all tested parameters. However, the results also indicate that the bodyweight index is the most sensitive toxicity parameter in that a reduction in body weight was observed at 25 mg/L of glyphosate. Negative correlations between the glyphosate concentration and toxicity parameters such as specific growth rate (SGR), hepato-somatic index (HIS), and gonado-somatic index (GSI) were observed. The fish condition factor and feed conversion ratio were found not to be affected at the highest glyphosate concentration tested (150 mg/L). To conclude, crossbred red tilapia (O. niloticus × O. mossambicus) is one potential species for evaluating the toxic effects of technical grade glyphosate on fish.
The ability to compare bovine and porcine skin gelatin based on their amino acid composition, polypeptides pattern, bloom strength, turbidity and foaming properties were investigated. Amino acid composition of both gelatin showed that the content of glycine, proline and arginine in porcine gelatin were higher than bovine gelatin. However, the polypeptides pattern between both gelatin is closely similar. The bloom strength of porcine gelatin was higher than bovine gelatin from pH 3 to pH 10. Both gelatin possessed highest bloom strength at pH 9. The lowest bloom strength of bovine gelatin was at pH 3 while porcine gelatin at pH 5. The highest turbidity of bovine gelatin obtained at pH 7 while porcine gelatin at pH 9. Foam expansion and foam stability of bovine gelatin were higher than porcine gelatin at all concentrations.
The inhibitory effect of onion extract on cassava leaf polyphenol oxidase was investigated. The polyphenol oxidase from cassava leaves was strongly inhibited by various anti-browning agents such as L-ascorbic acid and L-cysteine. The percentage of inhibition increased with the increased of anti-browning agents concentrations. The addition of heated onion extract exhibited a stronger inhibitory effect on cassava leaf polyphenol oxidase than the fresh onion extract. The highest percentage of inhibition was exhibited with heated onion extract in the presence of glucose and glycine, which was 87.18%. The onion extract inhibited the cassava leaf polyphenol oxidase non-competitively.
This study reported the extraction optimization and characterization of cobia (Rachycentron canadum) skin gelatin. Optimization study was carried out to determine the effect of CH3COOH concentration, skin to water ratio, extraction temperature and extraction time on gelatin yield (GY) and gel strength (GS) using Response Surface Methodology (RSM). The optimum conditions were 0.15mol/L for CH3COOH concentration, 82.4oC of extraction temperature, 6 h of extraction time and 1:6 of skin to water ratio, which produced cobia gelatin with GY of 20.10% and GS of 205.6 g. Characteristics of cobia skin gelatin (CG) were then compared to that of commercial bovine gelatin (BG). It was found that the most dominant amino acid in CG was glycine, proline and alanine. There was no difference in foaming and emulsifying properties of CG and BG at 1% concentration, but at 2% and 3% concentration, BG performed better. CG was found to have higher fat binding capacity but lower water holding capacity than BG. Least gelling concentration for CG was recorded at 2% while for BG at 1%. CG and BG had a pI at pH 6.05 and 4.82, respectively. This study shows that cobia skin gelatin has potential as halal alternative to bovine gelatin in food industry.
Corrosion is one of the major problems in petroleum mining and processing industry. The pipelines used to transport crude oil from reservoir to the processing installation were made from carbon steel that is susceptible towards corrosion. One of the best methods to prevent corrosion that occurred at the inner parts of carbon steel pipelines is to use organic corrosion inhibitor. One of the potent organic corrosion inhibitors is amino acids derivatives. In this study, dipeptide compound namely benzoylalanylglycine methyl ester and benzoylalanylglycine have been synthesized. The structure elucidation of the products was performed by IR, MS and NMR spectroscopy. The determination of corrosion inhibition activity utilized the Tafel method. The corrosion inhibition efficiency of glycine methyl ester, benzoylalanine, dipeptide benzoylalanylglycine methyl ester and dipeptide benzoylalanylglycine were 63.34%, 35.86%, 68.40% and 27.72%, respectively. These results showed that the formation of dipeptide benzoylalanylglycine methyl ester, derived from carboxylic protected glycine and amine protected alanine, increased the corrosion inhibition activity due to the loss of acidity center in the structure of glicine and L-alanine that would induce the corrosive environment towards carbon steel.
5-Enolpyruvylshikimate 3-phosphate synthase (EPSPS) is the primary target for the broad-spectrum herbicide, glyphosate. Improvement of EPSPS gene for high level of glyphosate tolerance is important to generate glyphosate-tolerant crops. In this study, we report the isolation and characterization of EPSPS genes of glyphosate-tolerant Pseudomonas nitroreducens strains FY43 and FY47. Both P. nitroreducens strains FY43 and FY47, which showed glyphosate tolerance up to 8.768% (518.4 mM, 32 × higher than field application), were isolated from soil samples collected from oil palm plantation with a long history of glyphosate application. The glyphosate tolerance property of EPSPS genes of strains FY43 and FY47 was functionally characterized by expressing the genes in Escherichia coli strain BL21(DE3). Error-prone PCR was performed to mutagenize native EPSPS gene of strains FY43 and FY47. Ten mutagenized EPSPS with amino acid changes (R21C, N265S, A329T, P71L, T258A, L184F, G292C, G292S, L35F and A242V) were generated through error-prone PCR. Both native and mutated EPSPS genes of strains FY43 and FY47 were introduced into Escherichia coli strain BL21(DE3) and transformants were selected on basal salt medium supplemented with 8.768% (518.4 mM) glyphosate. Mutants with mutations (R21C, N265S, A329T, P71L, T258A, L35F, A242V, L184F and G292C) showed sensitivity to 8.768% glyphosate, whereas glyphosate tolerance for mutant with G292S mutation was not affected by the mutation.
Thermostability remains one of the most desirable traits in many lipases. Numerous studies have revealed promising strategies to improve thermostability and random mutagenesis often leads to unexpected yet interesting findings in engineering stability. Previously, the thermostability of C-terminal truncated cold-adapted lipase from Staphylococcus epidermidis AT2 (rT-M386) was markedly enhanced by directed evolution. The newly evolved mutant, G210C, demonstrated an optimal temperature shift from 25 to 45 °C and stability up to 50 °C. Interestingly, a cysteine residue was randomly introduced on the loop connecting the two lids and accounted for the only cysteine found in the lipase. We further investigated the structural and mechanistic insights that could possibly cause the significant temperature shift. Both rT-M386 and G210C were modeled and simulated at 25 °C and 50 °C. The results clearly portrayed the effect of cysteine substitution primarily on the lid stability. Comparative molecular dynamics simulation analysis revealed that G210C exhibited greater stability than the wild-type at high temperature simulation. The compactness of the G210C lipase structure increased at 50 °C and resulted in enhanced rigidity hence stability. This observation is supported by the improved and stronger non-covalent interactions formed in the protein structure. Our findings suggest that the introduction of a single cysteine residue at the lid region of cold-adapted lipase may result in unexpected increased in thermostability, thus this approach could serve as one of the thermostabilization strategies in engineering lipase stability.
Two optimization strategies, codon usage modification and glycine supplementation, were adopted to improve the extracellular production of Bacillus sp. NR5 UPM β-cyclodextrin glycosyltransferase (CGT-BS) in recombinant Escherichia coli. Several rare codons were eliminated and replaced with the ones favored by E. coli cells, resulting in an increased codon adaptation index (CAI) from 0.67 to 0.78. The cultivation of the codon modified recombinant E. coli following optimization of glycine supplementation enhanced the secretion of β-CGTase activity up to 2.2-fold at 12 h of cultivation as compared to the control. β-CGTase secreted into the culture medium by the transformant reached 65.524 U/mL at post-induction temperature of 37 °C with addition of 1.2 mM glycine and induced at 2 h of cultivation. A 20.1-fold purity of the recombinant β-CGTase was obtained when purified through a combination of diafiltration and nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography. This combined strategy doubled the extracellular β-CGTase production when compared to the single approach, hence offering the potential of enhancing the expression of extracellular enzymes, particularly β-CGTase by the recombinant E. coli.
Glyphosate (GLY) is a major herbicide used throughout the world, and its continuous application has become an environmental issue. Adsorption is an important mechanism for removing organic contaminant in water. The present study characterized cow dung (CD) and rice husk ash (RHA), and determined the adsorption-desorption of GLY and its metabolite, aminomethylphoshonic acid (AMPA), on to them. The results revealed that both CD and RHA were alkaline and had no or low content of arsenic, cadmium, chromium and lead. The CD had lower surface area (13.104 mg2g-1) than RHA (21.500 m2g-1). The CD contained amines, phenol, ethers and carboxylic functional groups, while in addition to carboxylic and ether, RHA contains siloxane. Both CD and RHA had high affinities for GLY and AMPA. The Freundlich sorption coefficient (Kf) on AMPA were 2.915 and 2.660 for CD and RHA, respectively, while the values on GLY were 1.168 and 1.166 (mg g-1) for CD and RHA, respectively. Desorption of GLY only occurred at lower concentrations, while no desorption of AMPA was recorded, indicating their strong adsorption on CD and RHA. Considering their availabilities and affordable prices, both CD and RHA can be recommended as economical adsorbent for the removal of GLY and AMPA.
Glutamate receptors are the integral cellular components associated with excitotoxicity mechanism induced by the ischemic cascade events. Therefore the glutamate receptors have become the major molecular targets of neuroprotective agents in stroke researches. Recent studies have demonstrated that a Group I metabotropic glutamate receptor agonist, (S)-3,5-dihydroxyphenylglycine ((S)-3,5-DHPG) preconditioning elicits neuroprotection in the hippocampal slice cultures exposed to toxic level of N-methyl-d-aspartate (NMDA). We further investigated the preconditioning effects of (S)-3,5-DHPG on acute ischemic stroke rats. One 10 or 100μM of (S)-3,5-DHPG was administered intrathecally to Sprague-Dawley adult male rats, 2h prior to induction of acute ischemic stroke by middle cerebral artery occlusion (MCAO). After 24h, neurological deficits were evaluated by modified stroke severity scores and grid-walking test. All rats were sacrificed and infarct volumes were determined by 2,3,5-triphenyltetrazolium chloride staining. The serum level of neuron-specific enolase (NSE) of each rat was analyzed by enzyme-linked immunosorbent assay (ELISA). One and 10μM of (S)-3,5-DHPG preconditioning in the stroke rats showed significant improvements in motor impairment (P<0.01), reduction in the infarct volume (P<0.01) and reduction in the NSE serum level (P<0.01) compared to the control stroke rats. We conclude that 1 and 10μM (S)-3,5-DHPG preconditioning induced protective effects against acute ischemic insult in vivo.
Several Norovirus cases due to consumption of green onions have been reported during recent years but reports on red onions are not found. Onions are one of the major tastes in Malaysian food which are sometimes consuming raw especially the green onion. Viral contamination in onions can occur due to planting condition and not properly prepared food. This situation can pose the human health risk. A method was developed to detect the Norovirus that might present on different type of onions. In this study, 60 samples were collected from local market. Elution by Tryptose Phosphate Glycine broth and concentration steps using negatively charge filter were applied to enhance the detection of virus in food due to low copies of virus on food surface. The viral RNA was extracted using Qiagen Rneasy Mini kit before further detection using One-step RT-PCR. The total incidence of Norovirus in green onion and red onion was 13.33% (4/30) and 3.33 % (1/30) respectively. This is the first report of the detection of Norovirus in red and green onions in Malaysia. Based on the results, it is concluded that this method is reliable to detect Norovirus on onions and vegetables surface and hence can be applied in the laboratories for routine or food borne outbreak investigation.
Food labeling in accordance with Novel Food Regulation has been enforced in the European Community since 1997 with a series of updated legislations namely, EC/258/97, EC/1139/98, EC/49/2000, EC/50/2000 and EC/1829/2003. Guidelines and labeling regulations for the use of GMOs materials in food and feed products has also been introduced in Malaysia and Vietnam. Therefore, the demand for the establishment and development of a robust and rapid operation procedure for GMO detection has increased recently in both countries. The procedure of GMO detection emphasizes not only on detection tests but also on confirmation assays. This study employed PCR technology for detection and direct DNA sequencing for confirmation procedures respectively. The results demonstrated for the first time the presence of GM plants with glyphosate-resistant trait led by the control of P35S promoter and NOS terminator in either Malaysian or Vietnamese feed with high frequency (20 positive samples out of 24 analyzed samples). The P35S promoter, EPSPS gene and NOS terminator sequences obtained showed some mutations on single-stranded and double-stranded targeted sequences caused by single nucleotide insertion or single nucleotide changes. These results reinforce the need for development of detection procedures to comply with food/feed labeling system.