Studies on Plant Aspartate Transcarbamylase Purification and Properties of the Enzyme from Mung-Bean (Phaseolus aureus) Seedlings

Aspartate transcarbamylase is purified from mung bean seedlings by a series of steps involving manganous sulphate treatment, ammonium sulphate fractionation, DEAE-cellulose chromatography, followed by a second ammonium sulphate fractionation and finally gel filtration on Sephadex-G 100. The enzyme is homogeneous on ultracentrifugation and on polyacrylamide gel electrophoresis. It functions optimally at 55°C. It has two pH optima, one at 8.0 and the other at 10.2. The enzyme follows Michaelis-Menten kinetics with l-aspartate as the variable substrate. However, it exhibits sigmoid saturation curves at both the pH optima when the concentration of carbamyl phosphate is varied. The enzyme is allosterically inhibited by UMP at both the pH optima. Increasing phosphorylation of the uridine nucleotide decreases the inhibitory effect. The enzyme is desensitized to inhibition by UMP on treatment with p-hydroxymercuribenzoate, gel electrophoresis indicating that the enzyme is dissociated by this treatment; the dissociated enzyme can be reassociated by treatment with 2-mercaptoethanol. The properties of the mung bean enzyme are compared with the enzyme from other sources.

Postglacial climate changes and vegetation responses in northern Europe

Postglacial climate changes and vegetation responses were studied using a combination of biological and physical indicators preserved in lake sediments. Low-frequency trends, high-frequency events and rapid shifts in temperature and moisture balance were probed using pollen-based quantitative temperature reconstructions and oxygen-isotopes from authigenic carbonate and aquatic cellulose, respectively. Pollen and plant macrofossils were employed to shed light on the presence and response rates of plant populations in response to climate changes, particularly focusing on common boreal and temperate tree species. Additional geochemical and isotopic tracers facilitated the interpretation of pollen- and oxygen-isotope data. The results show that the common boreal trees were present in the Baltic region (~55°N) during the Lateglacial, which contrasts with the traditional view of species refuge locations in the south-European peninsulas during the glacial/interglacial cycles. The findings of this work are in agreement with recent paleoecological and genetic evidence suggesting that scattered populations of tree species persisted at higher latitudes, and that these taxa were likely limited to boreal trees. Moreover, the results demonstrate that stepwise changes in plant communities took place in concert with major climate fluctuations of the glacial/interglacial transition. Postglacial climate trends in northern Europe were characterized by rise, maxima and fall in temperatures and related changes in moisture balance. Following the deglaciation of the Northern Hemisphere and the early Holocene reorganization of the ice-ocean-atmosphere system, the long-term temperature trends followed gradually decreasing summer insolation. The early Holocene (~11,700-8000 cal yr BP) was overall cool, moist and oceanic, although the earliest Holocene effective humidity may have been low particularly in the eastern part of northern Europe. The gradual warming trend was interrupted by a cold event ~8200 cal yr BP. The maximum temperatures, ~1.5-3.0°C above modern values, were attained ~8000-4000 cal yr BP. This mid-Holocene peak warmth was coupled with low lake levels, low effective humidity and summertime drought. The late Holocene (~4000 cal yr BP-present) was characterized by gradually decreasing temperatures, higher lake levels and higher effective humidity. Moreover, the gradual trends of the late Holocene were probably superimposed by higher-frequency variability. The spatial variability of the Holocene temperature and moisture balance patterns were tentatively attributed to the differing heat capacities of continents and oceans, changes in atmospheric circulation modes and position of sites and subregions with respect to large water bodies and topographic barriers. The combination of physical and biological proxy archives is a pivotal aspect of this work, because non-climatic factors, such as postglacial migration, disturbances and competitive interactions, can influence reshuffling of vegetation and hence, pollen-based climate reconstructions. The oxygen-isotope records and other physical proxies presented in this work manifest that postglacial climate changes were the main driver of the establishment and expansion of temperate and boreal tree populations, and hence, large-scale and long-term vegetation patterns were in dynamic equilibrium with climate. A notable exception to this pattern may be the postglacial invasion of Norway spruce and the related suppression of mid-Holocene temperate forest. This salient step in north-European vegetation history, the development of the modern boreal ecosystem, cannot be unambiguously explained by current evidence of postglacial climate changes. The results of this work highlight that plant populations, including long-lived trees, may be able to respond strikingly rapidly to changes in climate. Moreover, interannual and seasonal variation and extreme events can exert an important influence on vegetation reshuffling. Importantly, the studies imply that the presence of diffuse refuge populations or local stands among the prevailing vegetation may have provided the means for extraordinarily rapid vegetation responses. Hence, if scattered populations are not provided and tree populations are to migrate long distances, their capacity to keep up with predicted rates of future climate change may be lower than previously thought.

Transport of retinol in the duck plasma

Retinol-binding protein and prealbumin were isolated from duck plasma by chromatography on DEAE-cellulose-and DEAE-Sephadex A-50, gel filtration on Sephadex G- 100 and preparative Polyacrylamide gel electrophoresis. The molecular weights of the retinolbinding protein-prealbumin complex, prealbumin and retinol-binding protein were found to be 75,000, 55,0000 and 20,000, respectively. On sodium dodecyl sulphate Polyacrylamide gel electrophoresis, prealbumin dissociated into identical subunits exhibiting a molecular weight of 13,500. Retinol-binding protein exhibited microheterogeneity on electrophoresis, whereas prealbumin moved as a single band unlike the multiple bands observed in chicken and rat.The ultraviolet and fluorescence spectra of the two proteins were similar to those isolated from other species. No carbohydrate moiety was detected in either retinol-binding protein or prealbumin. Duck retinol-binding protein and prealbumin showed cross-reactivity with their counterparts in chicken but differed immunologically from those of goat and man. Retinolbinding protein and prealbumin could be dissociated at low ionic strength, in 2M urea, by CMsephadex chromatography or on preparative electrophoresis. Although the transport of retinol in duck plasma is mediated by carrier proteins as in other species, it is distinguished by the absence of microheterogeneity in prealbumin and of an apo-retinol-binding protein form that could be transported in the plasma.

Fate of pathogen indicators in a domestic blend of food waste and wastewater through a two-stage anaerobic digestion system

Anaerobic digestion is a viable on-site treatment technology for rich organic waste streams such as food waste and blackwater. In contrast to large-scale municipal wastewater treatment plants which are typically located away from the community, the effluent from any type of on-site system is a potential pathogenic hazard because of the intimacy of the system to the community. The native concentrations of the pathogen indicators Escherichia coli, Clostridium perfringens and somatic coliphage were tracked for 30 days under stable operation (organic loading rate (OLR) = 1.8 kgCOD m(-3) day(-1), methane yield = 52% on a chemical oxygen demand (COD) basis) of a two-stage laboratory-scale digester treating a mixture of food waste and blackwater. E. coli numbers were reduced by a factor of 10(6.4) in the thermophilic stage, from 10(7.5+/-0.3) to 10(1.1+/-0.1) cfu 100 mL(-1), but regenerated by a factor of 10(4) in the mesophilic stage. Neither the thermophilic nor mesophilic stages had any significant impact on C. perfringens concentrations. Coliphage concentrations were reduced by a factor of 10(1.4) across the two stages. The study shows that anaerobic digestion only reduces pathogen counts marginally but that counts in effluent samples could be readily reduced to below detection limits by filtration through a 0.22 microm membrane, to investigate membrane filtration as a possible sanitation technique.

Nature of the thiamin-binding protein from chicken egg yolk

A simple, rapid and efficient procedure for the purification of thiamin-binding protein from chicken egg yolk was developed. The method involved removal, by exclusion, of lipoproteins from DEAE-cellulose and subsequent elution of water-soluble proteins held on the ion-exchanger with 1 M-NaCl, followed by treatment of the eluted protein fraction with an aqueous suspension of dextran/charcoal to generate apoprotein from the holoprotein. The resultant protein fraction was subjected to bioaffinity chromatography on thiamin pyrophosphate--AE (aminoethyl)-Sepharose. The protein eluted specifically with 10 microM-thiamin at pH 7.0, was homogeneous by the criteria of polyacrylamide-gel disc electrophoresis, had a mol.wt. of 38 000 +/- 2000 and was not a glycoprotein. The purified thiamin-binding protein specifically interacted with riboflavin-binding protein with no detectable deleterious affect on its (14C)thiamin-binding capacity. The protein bound [14C]thiamin with a molar ratio of 1.0, with dissociation constant (Kd) 0.41 microM. This protein-ligand interaction was inhibited by thiamin analogues and antagonists. The absorption spectrum of the protein in the presence of thiamin exhibited significant hypochromism at the 278 nm band, indicating the involvement of aromatic amino acid residues of the protein, during its binding to the ligand. The protein cross-reacted with the monospecific antiserum to egg-white thiamin-binding protein, showing thereby that thiamin-binding proteins present in chicken egg yolk and white are the products of the same structural gene.

Catalyzed air oxidation for treatment of thiosalt effulents

Continuous slurry reactor runs of two to four weeks duration were carried out for catalyzed air oxidation of thiosalts under a variety of conditions using poly (4-vinylpyridine) - Cu (II) and quaternized poly (4-vinylpyridine) - Cu (II) catalysts. Results obtained indicate that these catalysts have high activity and relatively long-term catalyst stability for thiosalt waste streams of < 1000 ppm thiosalt level. Using 2% (w/w) slurries of the poly (4-vinylpyridine) Cu (II) catalyst, effective oxidation of 700 ppm S2O32− influent to an effluent of < 100 ppm total thio-salts can be carried out continuously for at least one month when operating at 20 to 30°C with solution flow rates of$˜1l/h and aeration of 1300 XXX/h using a two-stage reactor system comprised of 12 l reactors. At higher thiosalt influent levels (i.e. > 1600 ppm) increased reaction temperatures enable depletion to < 100 ppm thiosalt effluent levels for up to one week of continuous operation. The catalysts deactivate much more readily at these higher influent levels as a result of greater copper losses and appreciable adsorption of S2O32− and S4O62−. The behaviour of continuous slurry reactors employed in the experimental studies, by use of batch reaction data for the poly (4-vinylpyridine) Cu (II) catalyzed oxidation of thiosalts, can be modelled successfully. Quaternized poly (4-vinylpyridine) Cu (II) catalyst has good long-term stability and copper losses are very low. The poly (4-vinylpyridine) Cu (II) catalyst, however, is susceptible to appreciable oxidation of the polymer matrix on long-term usage. This oxidation of the polymer matrix results in a substantial loss in the activity of the regenerated catalyst.

Isoaccepting Lysine Transfer Ribonucleic Acid Species of Pseudomonas Aeruginosa

Pseudomonas aeruginosa tRNA was treated with iodine, CNBr and N-ethylmaleimide,three thionucleotide-specific reagents. Reaction with iodine resulted in extensive loss of acceptor activity by lysine tRNA, glutamic acid tRNA, glutamine tRNA, serine tRNA and tyrosine tRNA. CNBr treatment resulted in high loss of acceptor ability by lysine tRNA, glutamic acid tRNA and glutamine tRNA. Only the acceptor ability of tyrosine tRNA was inhibited up to 66% by N-ethylmaleimide treatment, a reagent specific for 4-thiouridine. By the combined use of benzoylated DEAE-cellulose and DEAESephadex columns, lysine tRNA of Ps. aeruginosa was resolved into two isoaccepting species, a major, tRNAL'y and a minor, tRNA'Ys. Co-chromatography of 14C-labelled tRNALYS and 3H-labelled tRNALy, on benzoylated DEAE-cellulose at pH4.5 gave two distinct, non-superimposable profiles for the two activity peaks, suggesting that they were separate species. The acceptor activity of these two species was inhibited by about 95% by iodine and CNBr. Both the species showed equal response to codons AAA and AAG and also for poly(A) and poly(A1,Gl) suggesting that the anticodon of these species was UUU. Chemical modification of these two species by iodine did not inhibit the coding response. The two species of lysine of Ps. aeruginosa are truly redundant in that they are indistinguishable either by chemical modification or by their coding response.

Oxidase-peroxidase enzymes of Datura innoxia. Oxidation of formylphenylacetic acid ethyl ester

An enzyme system from Datura innoxia roots oxidizing formylphenylacetic acid ethyl ester was purified 38-fold by conventional methods such as (NH4)2SO4 fractionation, negative adsorption on alumina Cy gel and chromatography on DEAE-cellulose. The purified enzyme was shown to catalyse the stoicheiometric oxidation of formylphenylacetic acid ethyl ester to benzoylformic acid ethyl ester and formic acid, utilizing molecular O2. Substrate analogues such as phenylacetaldehyde and phenylpyruvate were oxidized at a very low rate, and formylphenylacetonitrile was an inhilating agents, cyanide, thiol compounds and ascorbic acid. This enzyme was identical with an oxidase-peroxidase isoenzyme. Another oxidase-peroxidase isoenzyme which separated on DEAE-chromatography also showed formylphenylacetic acid ethyl ester oxidase activity, albeit to a lesser extent. The properties of the two isoenzymes of the oxidase were compared and shown to differ in their oxidation and peroxidation properties. The oxidation of formylphenylacetic acid ethyl ester was also catalysed by horseradish peroxidase. The Datura isoenzymes exhibited typical haemoprotein spectra. The oxidation of formylphenylacetic acid ethyl ester was different from other peroxidase-catalysed reactions in not being activated by either Mn2+ or monophenols. The oxidation was inhibited by several mono- and poly-phenols and by catalase. A reaction mechanism for the oxidation is proposed.

Effect Of Human Chorionic Gonadotrophin And Ovine Luteinizing Hormone On Rat Ovarian Macromolecular Metabolism

Administration of human chorionic gonadotrophin (HCG) or ovine LH to immature rats primed with pregnant mare serum gonadotrophin (PMSG) stimulated the rate of synthesis of polyadenylic acid (poly A)-rich RNA in the ovaries. The rate of total RNA synthesis was not affected significantly by hormone treatment, whereas protein synthesis was enhanced. The increase in the rate of synthesis of poly(A)-rich RNA in the ovaries could be inferred as induction of messenger RNA synthesis after the hormone treatment. The poly(A)-rich nature of the isolated RNA was established by oligo(dT)–cellulose chromatography, binding to Millipore filter disks and hydridization with [3H]polyuridylic acid. The level of cyclic AMP in the ovaries of such rats was also raised after administration of LH, the increase coincided with the increase in the rate of synthesis of poly(A)-rich RNA. The implications of these results are discussed in the light of the biochemical basis of luteinization and the action of LH.

Harvest: A biotextile future

- Description of the work Harvest: A biotextile future consists of four bags constructed from kombucha, each utilizing a different approach to this material. The kombucha material is a byproduct of the fermented green tea, kombucha, and is comprised of a symbiotic culture of bacteria and yeast (SCOBY) that forms a fast growing curd or pellicle on the surface of the tea. This pellicle is harvested, washed, and dried to make a material with characteristics that can range between leather and paper in handle. The pellicle is one hundred per cent cellulose, with the individual fibres growing together to produce a durable and strong non-woven textile. Techniques explored with the dry kombucha material include folding, stitching, and laser etching. The final bags were designed with reference to classic tropes of fashion accessories: the briefcase, the clutch, the valise and the handbag. The valise included three jars in which the kombucha was displayed as ‘growing’ within the bag. - Research Background This work sits within an emerging field of practice in which fashion design intersects with biotechnology. Designers such as Suzanne Lee have explored constructing garments from bacteria byproducts, and bio-artists Oron Catts and Ionat Zurr have created ‘victimless leather’ grown from cultured cells. Although still speculative, these collaborations between science and design point to new material applications for fashion. Our work contributes to this area through testing both the growing of the textile and its application to construct durable fashion artefacts. - Research Contribution Harvest: A biotextile future makes two contributions to new knowledge in the area of design for sustainability within fashion. The first contribution lies in extending the technical experimentation required to grow and manipulate the textile. For the briefcase, the pattern shape was ‘grown’ into the required shape, using a shaped container. Other techniques used in the bags included weaving, folding and laser etching the material to extend its functional and decorative properties. Experimentation with the growing and drying of the material led to the production of a wide range of physical properties, in which the material was more brittle or flexible as required. The second research contribution lies in the proposal of this material for use in durable fashion accessories. The material is still speculative and small-scale in production, however the four bags illustrate the potential for kombucha as a biodegradable alternative to leather or synthetic materials. - Research Significance This interplay of science and design research opens up an exploration for a speculative future of sustainable, biodegradable textiles using live bacteria to enable ‘homegrown’ vegan apparel. The collaborators on this project include scientist Peter Musk and fashion designers Alice Payne and Dean Brough. Harvest: A biotextile future was exhibited at the State Library of Queensland’s Asia Pacific Design Library, 1-5 November 2015, as part of The International Association of Societies of Design Research’s (IASDR) biannual design conference. The work was chosen for display by a panel of experts, based on the criteria of design innovation and contribution to new knowledge in design.

A simple method for large-scale preparation of chick embryo tRNA

A simple method for preparing bulk quantities of tRNA from chick embryo has been developed. In this method chick embryos were homogenized in a buffer of pH 4.5, followed by deproteinization with phenol. The aqueous layer was allowed to separate under gravity. The resulting aqueous layer, after two more phenol treatments, was directly passed through a DEAE-cellulose column and the tRNA eluted therefrom with 1 Image NaCl. The tRNA prepared by this method was as active as the one prepared at neutral pH.

In vitro micropropagation of elite rosewood (Dalbergia latifolia Roxb

Induction of single and multiple shoots was obtained from nodal expiants of 60–80 year-old elite trees of rosewood on Murashige and Skoog's basal medium supplemented with 6-benzylaminopurine (1.0 mg 1-1) and delta -Naphthalene acetic acid (0.05 mg 1-1) or indole acetic acid (0.5 mg 1-1). Multiplication of shoots was obtained on MS (reduced major elements) or Woody Plant Medium supplemented with 6-benzylaminopurine (1.0 mg 1-1) and kinetin (0.5–1.0 mg 1-1). Excised shoots were rooted on half-strength MS with IBA (2.0 mg 1-1) to obtain complete plantlets. The regenerated plantlets have been acclimatized and successfully transferred to the soil.

A new mixed-matrix membrane for DMFCs

A new mixed-matrix membrane based on stabilized phosphotungstic acid (PTA) incorporated to chitosan (CS)-hydroxy ethyl cellulose (HEC) for application in direct methanol fuel cells (DMFCs) is reported. Membranes are characterised using Fourier Transform Spectroscopy (FTIR), Thermo-Gravimetric Analysis (TGA), Scanning Electron Microscopy (SEM) and their mechanical properties are evaluated. The PTA content in the CS-HEC blend and its influence on proton conductivity, water/methanol sorption, and methanol cross-over in the DMFC is studied. The DMFC with 3 wt. % stabilized PTA-CS-HEC mixed-matrix membrane delivers peak power-density of 58 mW/cm(2) at a load current-density of 210 mA/cm(2) with a lower methanol cross-over than that observed for a DMFC operating with a Nafion membrane electrolyte.

Microneurovascular free muscle transfer with cross-over nerve grafts in facial reanimation

Microneurovascular free muscle transfer with cross-over nerve grafts in facial reanimation Loss of facial symmetry and mimetic function as seen in facial paralysis has an enormous impact on the psychosocial conditions of the patients. Patients with severe long-term facial paralysis are often reanimated with a two-stage procedure combining cross-facial nerve grafting, and 6 to 8 months later with microneurovascular (MNV) muscle transfer. In this thesis, we recorded the long-term results of MNV surgery in facial paralysis and observed the possible contributing factors to final functional and aesthetic outcome after this procedure. Twenty-seven out of forty patients operated on were interviewed, and the functional outcome was graded. Magnetic resonance imaging (MRI) of MNV muscle flaps was done, and nerve graft samples (n=37) were obtained in second stage of the operation and muscle biopsies (n=18) were taken during secondary operations.. The structure of MNV muscles and nerve grafts was evaluated using histological and immunohistochemical methods ( Ki-67, anti-myosin fast, S-100, NF-200, CD-31, p75NGFR, VEGF, Flt-1, Flk-1). Statistical analysis was performed. In our studies, we found that almost two-thirds of the patients achieved good result in facial reanimation. The longer the follow-up time after muscle transfer the weaker was the muscle function. A majority of the patients (78%) defined their quality of life improved after surgery. In MRI study, the free MNV flaps were significantly smaller than originally. A correlation was found between good functional outcome and normal muscle structure in MRI. In muscle biopsies, the mean muscle fiber diameter was diminished to 40% compared to control values. Proliferative activity of satellite cells was seen in 60% of the samples and it tended to decline with an increase of follow-up time. All samples showed intramuscular innervation. Severe muscle atrophy correlated with prolonged intraoperative ischaemia. The good long-term functional outcome correlated with dominance of fast fibers in muscle grafts. In nerve grafts, the mean number of viable axons amounted to 38% of that in control samples. The grafted nerves characterized by fibrosis and regenerated axons were thinner than in control samples although they were well vascularized. A longer time between cross facial nerve grafting and biopsy sampling correlated with a higher number of viable axons. P75Nerve Growth Factor Receptor (p75NGFR) was expressed in every nerve graft sample. The expression of p75NGFR was lower in older than in younger patients. A high expression of p75NGFR was often seen with better function of the transplanted muscle. In grafted nerve Vascular Endothelial Growth Factor (VEGF) and its receptors were expressed in nervous tissue. In conclusion, most of the patients achieved good result in facial reanimation and were satisfied with the functional outcome. The mimic function was poorer in patients with longer follow-up time. MRI can be used to evaluate the structure of the microneurovascular muscle flaps. Regeneration of the muscle flaps was still going on many years after the transplantation and reinnervation was seen in all muscle samples. Grafted nerves were characterized by fibrosis and fewer, thinner axons compared to control nerves although they were well vascularized. P75NGFR and VEGF were expressed in human nerve grafts with higher intensity than in control nerves which is described for the first time.

Effect of pretreatment on the formation of 5-chloromethyl furfural derived from sugarcane bagasse

Chloromethylfurfural (CMF), a valuable intermediate for the production of chemicals and fuel, can be derived in high yields from the cellulose component of biomass. This study examined the effect of sugar cane bagasse components and biomass architecture on CMF/bio-oil yield using a HCl/dichloroethane biphasic system. The type of pretreatment affected bio-oil yield, as the CMF yield increased with increasing glucan content. CMF yield reached 81.9% with bagasse pretreated by acidified aqueous ionic liquid, which had a glucan content of 81.6%. The lignin content of the biomass was found to significantly reduce CMF yield, which was only 62.3% with acid-catalysed steam exploded sample having a lignin content of 29.6%. The change of CMF yield may be associated with fibre surface changes as a result of pretreatment. The hemicellulose content also impacted negatively on CMF yield. Storage of the bio-oil in chlorinated solvents prevented CMF degradation.