Fatty acid desaturase catalyzes the desaturation reactions by insertion of double bonds into the fatty acyl chain, producing unsaturated fatty acids. Though soluble fatty acid desaturases have been studied widely in advanced organisms, there are very limited studies of membrane fatty acid desaturases due to the difficulty of generating recombinant desaturase. Brassica napus is a rapeseed, which possesses a range of different membrane-bound desaturases capable of producing fatty acids including Δ3, Δ4, Δ8, Δ9, Δ12, and Δ15 fatty acids. The 1155 bp open reading frame of Δ12 fatty acid desaturase (FAD12) from Brassica napus codes for 383 amino acid residues with a molecular weight of 44 kDa. It was expressed in Escherichia coli at 37 °C in soluble and insoluble forms when induced with 0.5 mM IPTG. Soluble FAD12 has been purified using Ni2+-Sepharose affinity chromatography with a total protein yield of 0.728 mg/mL. Gas chromatography-mass spectrometry (GC-MS) analysis revealed that desaturase activity of FAD12 could produce linoleic acid from oleic acid at a retention time of 17.6 with a conversion rate of 47%. Characterization of purified FAD12 revealed the optimal temperature of FAD12 was 50 °C with 2 mM preferred substrate concentration of oleic acid. Analysis of circular dichroism (CD) showed FAD12 was made up of 47.3% and 0.9% of alpha-helix and β-sheet secondary structures. The predicted Tm value was 50.2 °C.
To investigate the suitability of chitosan films prepared using two different solvents, acetic acid (Chitosan-AA) and lactic acid (Chitosan-LA), for wound dressing, in comparison with a commercial preparation, Omiderm.
High-energy electron (2.0 MV) and gamma irradiation (60Co) has been used to modify polymeric silicone fluids of initial viscosities in the range, 90-700 cSt. Doses of electron and gamma radiation were delivered at rates of 0.246 kGy s(-1) and 15 kGy h(-1), respectively, exposure times being adjusted to ensure energy deposition in the range 30-360 kGy. Measurements were made using a differential viscometer based on a Bose Institute design. In line with expectation, samples of greater initial molecular weight (and hence greater viscosity), were observed to be more susceptible to radiation induced cross-linking than those of lower molecular weight. The role of dose rate and oxygen diffusion in determining the extent of change is discussed.
A modified potential of the sudden approximation, modified to include interactions among nuclei of different radii, is applied to explain the mass asymmetry of fission fragments in the thermal fission of Uranium-235. The results are encouraging in that the asymmetry feature in the fission yield is displayed. It appears that the mass asymmetry is a feature that can be explained without incorporating other effects. However, close correspondence requires addition of extra features.
The purpose of this study was to evaluate the effect of different cooking methods on the allergenicity of cockle and to identify proteins most frequently bound by IgE antibodies using a proteomics approach. Raw, boiled, fried and roasted extracts of the cockle were prepared. The protein profiles of the extracts were obtained by separation using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and 2-dimensional gel electrophoresis (2-DE). IgE-immunoblotting was then performed with the use of individual sera from patients with cockle allergy and the major IgE-binding proteins were analyzed by mass-spectrometry. SDS-PAGE of raw extract showed 13 protein bands. Smaller numbers of protein bands were detected in the boiled, fried and roasted extracts. The 2-DE gel profile of the raw extract further separated the protein bands to ~50 protein spots with molecular masses between 13 to 180 kDa and isoelectric point (pI) values ranging from 3 to 10. Immunoblotting of raw extract exhibited 11 IgE-binding proteins with two proteins of 36 and 40 kDa as the major IgE-binding proteins, while the boiled extract revealed 3 IgE-binding proteins. Fried and roasted extracts only showed a single IgE-binding protein at 36 kDa. 2-DE immunoblotting of raw extract demonstrated 5 to 20 IgE reactive spots. Mass spectrometry analysis led to identification of 2 important allergens, tropomyosin (36 kDa) and arginine kinase (40 kDa). Heated extracts showed a reduction in the number of IgE-reactive bands compared with raw extract, which suggest that thermal treatment can be used as a tool in attempting to reduce cockle allergenicity. The degree of allergenicity of cockle was demonstrated in the order raw > boiled > fried ≈ roasted. Two important allergens reacting with more than 50% of patients' sera identified using mass spectrometric approaches were tropomyosin and arginine kinase. Thus, allergens found in this study would help in component based diagnosis, management of cockle allergic patients and to the standardisation of allergenic test products as tools in molecular allergology.
A revision of a metal-on-ultra high molecular weight (UHMWPE) bearing couple for total hip replacement was performed due to aseptic loosening after 23 years in-vivo. It is a major long-term failure identified from wear generation. This study includes performing failure analysis of retrieved polyethylene acetabular cup from Zimmer Trilogy® Acetabular system. The UHMWPE acetabular cup was retrieved from a 61 years old male patient with ability to walk but limited leg movement when he presented to hospital in early 2016 with complaint left thigh pain. It was 23 years after his primary total hip replacement procedure. Surface roughness and morphology condition were measured using 3D laser microscope and Scanning Electron Microscope (SEM) to evaluate and characterize the wear features on polyethylene acetabular cup surface. ATR-Fourier Transform Infra-Red (ATR-FTIR), differential scanning calorimetry (DSC) and gel permeation chromatography (GPC) were used to characterize the chemical composition of carbon-oxygen bonding, crystallinity percentage and molecular weight of the polymer liner that might changes the mechanical properties of polyethylene. Nano indentation is to measure hardness and elasticity modulus where the ratio of hardness to elastic modulus value can be reflected as the degradation of mechanical properties. A prominent difference of thickness between two regions resulted from acentric loading concentration was observed and wear rate were measured. The linear wear rate for thin side and thick side were 0.33 mm/year and 0.05 mm/year respectively. Molecular weight reduction of 57.5% and relatively low ratio of hardness to elastic modulus (3.59 × 10-3) were the indicator of major mechanical properties degradation happened on UHMWPE acetabular cup. This major degradation was contributed by oxidation and polishing wear feature accompanied with delamination, craters, ripple and cracks were the indication of extensive usage of UHMWPE from the suggested life span of acetabular cup application.
Effects of aluminium hydroxide (ATH) addition on the properties of palm-based polyurethane composites were investigated. The hybrid composites were prepared by mixing 10 wt% of oil palm empty fruit bunch fiber (EFB) with ATH at varying amount of 2, 4 and 6 wt% of the overall mass of the resin. The compression stress and modulus gave the highest values of 575 and 2301 kPa, respectively at 2 wt% loading of ATH. The compression stress and modulus decreased drastically at 4 wt% (431 kPa and 1659 kPa, respectively) and further decreased at 6 wt% ATH (339 and 1468 kPa, respectively). However, the burning rate is inversely proportional to the loading percentage where the highest burning rate was observed at 2 wt% ATH. Sound absorption analysis indicated a large absorption coefficient at high frequency (4000 Hz) for all samples. The highest absorption coefficient was obtained from PU-EFB/ATH with 4 wt% ATH.
Solvent flow reactor system was introduced into the extraction system to increase the system efficiency and enhance the extraction yield by adding fresh solvent during the extraction processes. The liquefaction experiment was carried out at various flow-rates (1, 3 and 5 ml/min), reaction times (30, 45 and 60 min) and reaction temperatures (300ºC, 350ºC, 400ºC, 420ºC and 450ºC) with tetralin as solvent. Despite the ability of adding fresh solvent into the extraction process, the conversion of oil+gas was still considered to be low as there was ~25% of coal extracts left to be converted into low molecular weight compounds. One possible option to increase the oil yield is by applying catalyst that will further break up the coal extracts into small molecular weight compounds. In this study, a second reactor was introduced consisting of catalyst (NiSiO2) assuming that the catalyst would interact more effectively with coal extracts rather than the coal itself. In the
absence of catalyst, the oil yield was 55%. By introducing the Ni catalyst, the oil yield increased by 15%. Further analysis of GCMS showed that the oil from catalytic liquefaction gave out more low molecular weight compounds in comparison to the un-catalytic liquefaction oil.
Ferulic acid (FA) groups esterified to the arabinan side chains of pectic polysaccharides can be oxidatively cross-linked in vitro by horseradish peroxidase (HRP) catalysis in the presence of hydrogen peroxide (H(2)O(2)) to form ferulic acid dehydrodimers (diFAs). The present work investigated whether the kinetics of HRP catalyzed cross-linking of FA esterified to α-(1,5)-linked arabinans are affected by the length of the arabinan chains carrying the feruloyl substitutions. The kinetics of the HRP-catalyzed cross-linking of four sets of arabinan samples from sugar beet pulp, having different molecular weights and hence different degrees of polymerization, were monitored by the disappearance of FA absorbance at 316 nm. MALDI-TOF/TOF-MS analysis confirmed that the sugar beet arabinans were feruloyl-substituted, and HPLC analysis verified that the amounts of diFAs increased when FA levels decreased as a result of the enzymatic oxidation treatment with HRP and H(2)O(2). At equimolar levels of FA (0.0025-0.05 mM) in the arabinan samples, the initial rates of the HRP-catalyzed cross-linking of the longer chain arabinans were slower than those of the shorter chain arabinans. The lower initial rates may be the result of the slower movement of larger molecules coupled with steric phenomena, making the required initial reaction of two FAs on longer chain arabinans slower than on shorter arabinans.
Fruit wastes constituting up to half of total fruit weight represent a large pool of untapped resources for isolation of starch with diverse applications. In this work, the possibility of isolating starch from tropical fruit wastes and its extended application as a natural coagulant was elucidated. Amongst the 12 various parts of fruit wastes selected, only jackfruit seeds contained more than 50% of total starch content. Using alkaline extraction procedures, starch has been successfully isolated from local jackfruit seeds with a yield of approximately 18%. Bell-shaped starch granules were observed under SEM with a granule size ranging from 1.1 to 41.6 μm. Detailed starch characteristics were performed to provide a comparison between the isolated seed starch and also conventional starches. Among them, chemical properties such as the content of starch, amylose, amylopectin and the corresponding molecular weights are some of the key characteristics which governed their performance as natural coagulants. The potential use of isolated seed starch as an aid was then demonstrated in both suspensions of kaolin (model synthetic system) and Chlorella sp. microalga (real-time application) with plausible outcomes. At optimized starch dosage of 60 mg/L, the overall turbidity removal in kaolin was enhanced by at least 25% at a fixed alum dosage of 2.1 mg/L. Positive turbidity and COD removals were also observed in the treatment of Chlorella suspensions. Starches which served as bridging agents aided in the linkage of neighbouring microflocs and subsequently, forming macroflocs through a secondary coagulation mechanism: adsorption and bridging.
The major hemorrhagin from C. purpureomaculatus (mangrove pit viper) venom was purified to homogeneity and termed Maculatoxin. Maculatoxin has a molecular weight of 38 kDa as determined by SDS-PAGE. It is an acidic protein (pI= 4.2) and exhibited proteolytic and hemorrhagic activities (MHD10 = 0.84 microg in mice) but was not lethal to mice at a dose of 1 microg/g. The hemorrhagic activity of Maculatoxin was completely inactivated by EDTA and partially inhibited by ATP and citrate. The N-terminal sequence of Maculatoxin (TPEQQRFPPTYIDLGIFVDHGMYAT) shares a significant degree of homology with the metalloprotease domain of other venom hemorrhagins. Indirect ELISA showed anti-Maculatoxin cross reacted with protein components of many snake venoms. In the double-sandwich ELISA, however, anti-Maculatoxin cross-reacted only with venoms of certain species of the Trimeresurus (Asia lance-head viper) complex, and the results support the recent proposed taxonomy changes concerning the Trimeresurus complex.
Arterial/venous thrombosis is the major cardiovascular disorder accountable for substantial mortality; and the current demand for antithrombotic agents is extensive. Heparinases depolymerize unfractionated heparin (UFH) for the production of low molecular-weight heparins (LMWHs; used as anticoagulants against thrombosis). A microbial strain of Streptomyces sp. showing antithrombotic activity was isolated from the soil sample collected from north India. The strain was characterized by using 16S rRNA homology technique and identified as Streptomyces variabilis MTCC 12266 capable of producing heparinase enzyme. This is the very first communication reporting Streptomyces genus as the producer of heparinase. It was observed that the production of intracellular heparinase was [63.8 U/mg protein (specific activity)] 1.58 folds higher compared to extracellular heparinase [40.28 U/mg protein]. DEAE-Sephadex A-50 column followed by Sepharose-6B column purification of the crude protein resulted 19.18 folds purified heparinase. SDS-PAGE analysis of heparinase resulted an estimated molecular-weight of 42 kDa. It was also found that intracellular heparinase has the ability to depolymerize heparin to generate LMWHs. Further studies related to the mechanistic action, structural details, and genomics involved in heparinase production from Streptomyces variabilis are warranted for large scale production/purification optimization of heparinase for antithrombotic applications.
A wild-type, Gram-positive, rod-shaped, endospore-forming and motile bacteria has been isolated from palm oil mill sludge in Malaysia. Molecular identification using 16S rRNA gene sequence analysis indicated that the bacteria belonged to genus Paenibacillus. With 97 % similarity to P. alvei (AUG6), the isolate was designated as P. alvei AN5. An antimicrobial compound was extracted from P. alvei AN5-pelleted cells using 95 % methanol and was then lyophilized. Precipitates were re-suspended in phosphate buffered saline (PBS), producing an antimicrobial crude extract (ACE). The ACE showed antimicrobial activity against Salmonella enteritidis ATCC 13076, Escherichia coli ATCC 29522, Bacillus cereus ATCC 14579 and Lactobacillus plantarum ATCC 8014. By using SP-Sepharose cation exchange chromatography, Sephadex G-25 gel filtration and Tricine SDS-PAGE, the ACE was purified, which produced a ~2-kDa active band. SDS-PAGE and infrared (IR) spectroscopy indicated the proteinaceous nature of the antimicrobial compound in the ACE, and liquid chromatography electrospray ionization mass spectroscopy and de novo sequencing using an automatic, Q-TOF premier system detected a peptide with the amino acid sequence F-C-K-S-L-P-L-P-L-S-V-K (1,330.7789 Da). This novel peptide was designated as AN5-2. The antimicrobial peptide exhibited stability from pH 3 to 12 and maintained its activity after being heated to 90 °C. It also remained active after incubation with denaturants (urea, SDS and EDTA).
A thrombin-like enzyme, purpurase, was purified from the Cryptelytrops purpureomaculatus (mangrove pit viper) venom using high performance ion-exchange and gel filtration chromatography. The purified sample (termed purpurase) yielded a homogeneous band in SDS-polyacrylamide gel electrophoresis with a molecular weight of 35,000. The N-terminal sequence of purpurase was determined to be VVGGDECNINDHRSLVRIF and is homologous to many other venom thrombin-like enzymes. Purpurase exhibits both arginine ester hydrolase and amidase activities. Kinetic studies using tripeptide chromogenic anilide substrates showed that purpurase is not fastidious towards its substrate. The clotting times of fibrinogen by purpurase were concentration dependent, with optimum clotting activity at 3mg fibronogen/mL. The clotting activity by purpurase was in the following decreasing order: cat fibrinogen>human fibrinogen>dog fibrinogen>goat fibrinogen>rabbit fibrinogen. Reversed-phase HPLC analysis of the products of action of purpurase on bovine fibrinogen showed that only fibrinopeptide A was released. Indirect ELISA studies showed that anti-purpurase cross-reacted strongly with venoms of most crotalid venoms, indicating the snake venom thrombin-like enzymes generally possess similar epitopes. In the more specific double-sandwich ELISA, however, anti-purpurase cross-reacted only with venoms of certain species of the Trimeresurus complex, and the results support the recent proposed taxonomy changes concerning the Trimeresurus complex.
1. The major phospholipase A2 (PLA-DE4) of the venom of Trimeresurus purpureomaculatus (shore pit viper) has been purified to electrophoretic homogeneity. 2. The isoelectric point of the purified enzyme was determined to be 4.20, and the mol. wt was 31,700 as estimated by Sephadex G-75 gel filtration chromatography; and 14,000 as estimated by SDS-polyacrylamide gel electrophoresis. The purified enzyme hydrolyzed phosphatidylcholine (PC) faster than phosphatidylethanolamine (PE), whereas phosphatidylserine (PS) was not hydrolyzed at all (PC greater than PE greater than PS =0). However, in reaction system consisted of mixtures of PC and PS, phosphatidylserine was effectively hydrolyzed by the enzyme. 4. The phospholipase A2 exhibited edema-forming activity but not hemolytic, hemorrhagic or anticoagulant activities. It was not lethal to mice at a dosage of 10 micrograms/g by i.v. route.
Sea cucumber (Stichopus vastus) is considered an underutilized resource, since only its stomach and intestines are eaten raw as salad in a few countries and the remaining parts, especially the integument rich in collagen, is discarded. Hence a valuable by-product having potential nutraceutical and pharmaceutical applications is wasted. In the present investigation, pepsin-solubilized collagen (PSC) from the integument of S. vastus was isolated, purified and characterized.
An acidic, lethal phospholipase Az was purified to electrophoretic homogeneity from the venom of the Malayan cobra (Naja naja sputatrix). The enzyme has an isoelectric point of 5.58, a molecular weight of 12000, and a medium lethal dose (LD50) of 0.86 micrograms/g in mice by intravenous injection. The enzyme also exhibited weak anticoagulant and edema-forming activities. The amino acid composition of the enzyme is similar to those of other cobra venom phospholipases Az.
Klebsiella pneumoniae is a healthcare-associated bacterial pathogen which causes severe diseases in immunocompromised individuals. Concanavalin A (conA), a lectin which recognizes proteins with mannose or glucose residues, has been reported to agglutinate K. pneumoniae and hence, is postulated to have therapeutical potential for K. pneumoniae-induced liver infection. This study investigated the conA binding properties of a large collection of clinical isolates of K. pneumoniae. ConA agglutination reaction was demonstrated by 94 (51.4%) of 183 K. pneumoniae isolates using a microtiter plate assay. The conA agglutination reactions were inhibited in the presence of 2.5 mg/ml D-mannose and 2.5 mg/ml glucose, and following pretreatment of the bacterial suspension with protease and heating at 80ºC. Majority of the positive isolates originated from respiratory specimens. Isolation of conA-binding proteins from K. pneumoniae ATCC 700603 strain was performed using conA affinity column and the conA binding property of the eluted proteins was confirmed by western blotting analysis using conA-HRP conjugates. Proteins with molecular weights ranging from 35 to 60 kDa were eluted from the conA affinity column, of which four were identified as outer membrane protein precursor A (37 kDa), outer membrane protein precursor C (40 kDa), enolase (45 kDa) and chaperonin (60 kDa) using mass spectrometry analysis. Several conA binding proteins (including 45 and 60 kDa) were found to be immunogenic when reacted with rabbit anti-Klebsiella antibody. The function and interplay of the conA binding proteins in bacterium-host cell relationship merits further investigation.
The steady laminar combined convective flow with heat and mass transfer of a Newtonian viscous incompressible fluid over a permeable flat plate with linear hydrodynamic and thermal slips has been investigated numerically. The velocity of the external flow, the suction/injection velocity and the temperature of the plate surface are assumed to vary nonlinearly following the power law with the distance along the plate from the origin. Lie group analysis is used to develop the similarity transformations and the governing momentum, the energy conservation and the mass conservation equations are converted to a system of coupled nonlinear ordinary differential equations with the associated boundary conditions. The resulting equations are solved numerically using the Runge-Kutta-Fehlberg fourth-fifth order numerical method. The effects of hydrodynamic slip parameter (a), thermal slip parameter (b), suction/injection parameter (fw), power law parameter (m), buoyancy ratio parameter (N), Prandtl number (Pr) and Schmidt number (Sc) on the fluid flow, heat transfer and mass transfer characteristics are investigated and presented graphically. We have also shown the effects of the Reynolds number (Re) and the power law parameter (m) on the velocity slip and the thermal slip factors. Good agreement is found between the numerical results of the present paper and published results.
The effects of human cytomegalovirus (HCMV) infection on human fibroblast cell genomes were investigated using agarose gel electrophoresis. At selected intervals post-infection (PI), cellular D N A from mock-treated and HCMV-infected cells were prepared in low melting point agarose plugs. Results obtained following electrophoresis of the cellular D N A indicate that HCMV infection did not result in extensive degradation of the cellular DNA, even in samples obtained from cells which showed > 95% cytopathologic effects. High molecular weight DNA (> 23 Kb) comparable to that of the mock-treated samples were noted in a l l HCMV infected DNA samples. Digestion of the DNA samples with restriction endonucleases, EcoR I, Not I, Sfi I, and Nru I, however, resulted in the appearance of smaller DNA fragments (< 23 Kb) in samples obtained on day 3, 4, and 5 PI. Since these DNA bands appeared only in the infected cells, it was likely that these were the HCMV genomic DNA fragments. Findings presented in this study support the notion that the cellular DNA of HCMVinfected cells could remained intact and functiona. KEYWORDS: Cytomegalovirus, chromosomes, DNA, genomes, restriction enzymes