Identification of expressed HSP`s in blacklip abalone (Haliotis rubra Leach) during heat and salinity stresses

Both prokaryotes and eukaryotes express a set of highly conserved proteins in response to external and internal stress. The stressors include tissue trauma,anoxia, heavy metal toxicity, infection, changed salinity, and the mmost characterized, heat shock. The result is an expression of stress proteins or heat shock proteins (HSP's) which lead to protection of protein integrity, and also to tolerance under continued heat stress conditions. The Australian backflip abalone (Haliotis rubra) is found principally in southern coastal water and also in estuarine/bay environments. Esturaine/bay environments have greater fluctuations in environmental conditions, especially those of salinity and water temperature, than they are found along oceanic coasts. Abalone from esturaine/bay and oceanic coastal environments were subjected to either increased temperature (2° C/day for a total of 10°C) or hyposalinity (80% seawater). Esturaine/bay abolone were less affectes than the oceanic animals by temperature increase and also demonstrated the ability to volume regualte 3 h after the initial salinity shock. SDS-PAGE and Western blotting techniques, together with dot blots of total protein, using HSP70 specific antibodies, were used to detect HSP70s in the foot muscle of the animals and indicated an expression of HSP70 in response to heat shock in abalone, but not following hyposalinity shock. RT-PCR yeilded a partial cDNA clone of HSP70 from the foot muscle.

Characterisation of proteins in the milk of fur seals

Milk protein composition was investigated throughout the lactation periods of the Australian fur seal (Arctocephalus pusillus doriferus) and Antarctic fur seal (Arctocephalus gazella). The mean protein content of the milk was found to be 10.9% and 10.6% respectively. The concentration of total protein did not change during lactation, although a decline in casein content of the milk in late lactation was apparent. Milk protein concentration during a foraging/suckling cycle of the Antarctic fur seal analysed at the time of arrival on shore, and 24 h and 72 h after arrival was 12.8%, 11.4% and 12.5% respectively. Re-feeding animals at 72 h resulted in a significant increase in milk protein content to 14.9%. Characterisation of milk protein by SDS-PAGE analysis revealed 5 casein and 10 major whey protein bands. Amino-terminal sequencing indicated that the majority of the whey fraction of the milk is β-lactoglobulin (β-LG). The limited amino acid sequence indicated 3 different β-LGs were secreted in the milk. Subsequently, RT-PCR was used to extend the sequence of one of the β-LGs and translation of the 464 bp fragment indicated that it shared 79% sequence identity with feline β-LG II.

Copper/zinc superoxide dismutase is phosphorylated and modulated specifically by granulocyte-colony stimulating factor in myeloid cells.

Using two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (2-D SDS-PAGE) of 32P-labeled cytosolic and membrane extracts, we identified a 21.5 kDa phosphoprotein with an isoelectric point of 6.0 in NFS-60 cells that was phosphorylated maximally at 15 min by treatment with granulocyte-colony stimulating factor (G-CSF) but not with interlevkin-3 (IL-3) or colony-stimulating factor-1 (macrophage-colony stimulating factor (CSF-1 (M-CSF)). The phosphorylation of this protein, designated 21.5/6.0, was unaffected by a series of antiproliferative agents [32]. These findings suggested that the 21.5/6.0 phosphoprotein may be involved in specific G-CSF-mediated biological responses such as activation and/or differentiation. We sought to characterize this 21.5/6.0 by a novel combination of 2-D SDS-PAGE and hydroxyapatite (HTP)-chromatography. Amino acid sequence determination of 21.5/6.0 revealed it to share a high level of homology with copper/zinc superoxide dismutase (Cu/Zn-SOD), indicating that a Cu/Zn-SOD is phosphorylated following treatment with G-CSF. This is the first report of the phosphorylation and possible involvement of Cu/Zn-SOD protein in granulocyte activation/differentiation events. In addition, Cu/Zn-SOD levels and activity were diminished by G-CSF but not IL-3 treatment. This new protocol combining 2-D SDS-PAGE and HTP-chromatography allows the characterization of low abundance phosphoproteins involved in the cellular responses to G-CSF and presumably to other cytokines/growth factors.

Purification and characterisation of endo-β-1,4-glucanase and laminarinase enzymes from the gecarcinid land crab Gecarcoidea natalis and the aquatic crayfish Cherax destructor

Laminarinase and endo-β-1,4-glucanase were purified and characterised from the midgut gland of the herbivorous land crab Gecarcoidea natalis and the crayfish Cherax destructor. The laminarinase isolated from G. natalis was estimated to have a molecular mass of 41 kDa by SDS-PAGE and 71 kDa by gel filtration chromatography. A similar discrepancy was noted for C. destructor. Possible reasons for this are discussed. Laminarinase (EC 3.2.1.6) from G. natalis had a V_max of 42.0 µmol reducing sugars produced min–¹ mg protein–¹, a K_m of 0.126% (w/v) and an optimum pH range of 5.5–7, and hydrolysed mainly β-1,3-glycosidic bonds. In addition to the hydrolysis of β-1,3-glycosidic bonds, laminarinase (EC 3.2.1.39) from C. destructor was capable of significant hydrolysis of β-1,4-glycosidic bonds. It had a V_max of 19.6 µmol reducing sugars produced min^–1 mg protein^–1, a K_m of 0.059% (w/v) and an optimum pH of 5.5. Laminarinase from both species produced glucose and other short oligomers from the hydrolysis of laminarin. Endo-β-1,4-glucanase (EC 3.2.1.4) from G. natalis had a molecular mass of 52 kDa and an optimum pH of 4–7. It mainly hydrolysed β-1,4-glycosidic bonds, but was also capable of significant hydrolysis of β-1,3-glycosidic bonds. Two endo-β-1,4-glucanases, termed 1 and 2, with respective molecular masses of 53±3 and 52 kDa, were purified from C. destructor. Endo-β-1,4-glucanase 1 was only capable of hydrolysing β-1,4-glycosidic bonds and had an optimum pH of 5.5. Endo-β-1,4-glucanases from both species produced some glucose, cellobiose and other short oligomers from the hydrolysis of carboxymethyl cellulose.

Purification and characterization of naringinase from a newly isolated strain of Aspergillus niger 1344 for the transformation of flavonoids

An extracellular naringinase (an enzyme complex consisting of α-L-rhamnosidase and β-D-glucosidase activity, EC 3.2.1.40) that hydrolyses naringin (a trihydroxy flavonoid) for the production of rhamnose and glucose was purified from the culture filtrate of Aspergillus niger 1344. The enzyme was purified 38-fold by ammonium sulphate precipitation, ion exchange and gel filtration chromatography with an overall recovery of 19% with a specific activity of 867 units per mg of protein. The molecular mass of the purified enzyme was estimated to be about 168 kDa by gel filtration chromatography on a Sephadex G-200 column and the molecular mass of the subunits was estimated to be 85 kDa by sodium dodecyl sulphate-Polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme had an optimum pH of 4.0 and temperature of 50 °C, respectively. The naringinase was stable at 37 °C for 72 h, whereas at 40 °C the enzyme showed 50% inactivation after 96 h of incubation. Hg2+, SDS, p-chloromercuribenzoate, Cu2+ and Mn2+ completely inhibited the enzyme activity at a concentration of 2.5–10 mM, whereas, Ca2+, Co2+ and Mg2+ showed very little inactivation even at high concentrations (10–100 mM). The enzyme activity was strongly inhibited by rhamnose, the end product of naringin hydrolysis. The enzyme activity was accelerated by Mg2+ and remained stable for one year after storage at −20 °C. The purified enzyme preparation successfully hydrolysed naringin and rutin, but not hesperidin.

Structure and properties of biomedical films prepared from aqueous and acidic silk fibroin solutions

Silk fibroin films are promising materials for a range of biomedical applications. To understand the effects of casting solvents on film properties, we used water (W), formic acid (FA), and trifluoroacetic acid (TFA) as solvents. We characterized molecular weight, secondary structure, mechanical properties, and degradation behavior of cast films. Significant degradation of fibroin was observed for TFA-based film compared to W and TA-based films when analyzed by SDS-PAGE. Fibroin degradation resulted in a significant reduction in tensile strength and modulus of TFA-based films. Compared to water, TFA-based films demonstrated lower water solubility (19.6% vs. 62.5% in 12 h) despite having only a marginal increase in their ß-sheet content (26.9% vs. 23.7%). On the other hand, FA-based films with 34.3% ß-sheet were virtually water insoluble. Following solubility treatment, ß-sheet content in FA-based films increased to 50.9%. On exposure to protease XIV, water-annealed FA-based films lost 74% mass in 22 days compared to only 30% mass loss by ethanol annealed FA films. This study demonstrated that a small variation in the ß-sheet percentage and random coil conformations resulted in a significant change in the rates of enzymatic degradation without alteration to their tensile properties. The film surface roughness changed with the extent of enzymatic hydrolysis.

Characterization of Type 1 ribosome inactivating proteins in edible plants

The ribosome inactivating proteins (RIPs) from plants possess RNA N-glycosidase activity that depurinates the major rRNA, thus damaging ribosome in an irreversible manner and arresting protein synthesis. RIPs occur in fungi, bacteria and plants and are abundant in angiosperms, where they appear to have defensive role. RIPs are presently classified as rRNA N-glycosidase in the enzyme nomenclature (EC 3.2.2.22) and do exhibit other enzymatic activities such as ribonuclease and deoxyribonuclease activities. RIPs are classified into two groups based on their difference in their primary structure. Type I RIPs consist of a single polypeptide chain of approximately 26–35 kDa that possess an RNA N-glycosidase activity. These proteins have attracted a great deal of attention because of their anti-viral, anti-tumor, and anti-microbial activities, which is useful in medical research and development. Here, we describe isolation of a novel protein from Momordica sp, a highclimbing vine from family Cucurbitaceae which is native to the tropical regions of Africa, Asia, Arabia and Caribbean. The purified protein has been verified by SDS-PAGE and mass spectrometry to contain only single chain Type-1 ribosome inactivating proteins (RIPs). With present experiments, we determined the presence of RIPs in edible plant materials, including some that are eaten raw by human beings. The novel protein is further characterized to validate its therapeutic potential.

Balsamin, a novel ribosome-inactivating protein from the seeds of Balsam apple Momordica balsamina

Plant seeds, a rich source of proteins, are considered important for their application as functional ingredients in a food system. A novel ribosome-inactivating protein (RIP), balsamin was purified from the seeds of Balsam apple, Momordica balsamina. Balsamin was purified by ion exchange chromatography on CM Sepharose and gel filtration on superdex-75. It has a molecular weight of 28 kDa as shown by SDS-PAGE analysis. Balsamin inhibits protein synthesis in a rabbit reticulocyte lysate-based cell free translation assay with an IC50 of 90.6 ng ml⁻¹. It has RNA N-glycosidase activity and releases a 400-base long fragment termed the Endo fragment from 28S rRNA in the same manner as does saporin-6 from Saponaria officinalis. The N-terminal sequence analysis of the first 12 amino acids of balsamin revealed that it shares 83% similarity with type I RIP α-MMC from Momordica charantia and 50% similarity with β-MMC (from Momordica charantia), bryodin I (from Bryonia dioica) and luffin a (from Luffa cylindrica). Balsamin was further characterized by mass spectrometry. CD spectroscopic studies indicate that secondary structure of balsamin contains helix (23.5%), β-strand (24.6%), turn (20%) and random coil (31.9%). Thus RIPs activity expressed in vegetables like Momordica sp. advocates its usage in diet.

Arginase II inhibition prevents nitrate tolerance

BACKGROUND AND PURPOSE Nitrate tolerance, the loss of vascular responsiveness with continued use of nitrates, remains incompletely understood and is a limitation of these therapeutic agents. Vascular superoxide, generated by uncoupled endothelial NOS (eNOS), may play a role. As arginase competes with eNOS for L-arginine and may exacerbate the production of reactive oxygen species (ROS), we hypothesized that arginase inhibition might reduce nitrate tolerance.

EXPERIMENTAL APPROACH Vasodilator responses were measured in aorta from C57Bl/6 and arginase II knockout (argII –/–) mice using myography. Uncoupling of eNOS, determined as eNOS monomer : dimer ratio, was assessed using low-temperature SDS-PAGE and ROS levels were measured using L-012 and lucigenin-enhanced chemiluminescence.

KEY RESULTS Repeated application of glyceryl trinitrate (GTN) on aorta isolated from C57Bl/6 mice produced a 32-fold rightward shift of the concentration–response curve. However this rightward shift (or resultant tolerance) was not observed in the presence of the arginase inhibitor (s)-(2-boronethyl)-L-cysteine HCl (BEC; 100 µM) nor in aorta isolated from argII –/– mice. Similar findings were obtained after inducing nitrate tolerance in vivo. Repeated administration of GTN in human umbilical vein endothelial cells induced uncoupling of eNOS from its dimeric state and increased ROS levels, which were reduced with arginase inhibition and exogenous L-arginine. Aortae from GTN tolerant C57Bl/6 mice exhibited increased arginase activity and ROS production, whereas vessels from argII –/– mice did not.

CONCLUSION AND IMPLICATIONS Arginase II removal prevents nitrate tolerance. This may be due to decreased uncoupling of eNOS and consequent ROS production.

Functionality of nano and 3D-microhierarchical TiO₂ particles as coagulants for sericin extraction from the silk degumming wastewater

Amine-functionalised TiO2 particles were employed as coagulants to remove sericin from the silk degumming wastewater. Two types of TiO2 particles including 3D-microhierarchical TiO2 (Micron-size) and TiO2 nanoparticles (Degussa P-25) were used in this study. The surfaces of both types of TiO2 particles were functionalised using 3-aminopropyl trimethoxysilane (APTMS). The impacts of TiO2 type, pH, TiO2 concentration, and settling time on sericin removal efficiency were investigated. The efficiency of TiO2 particles in sericin removal was evaluated by measuring the residual turbidity and UV-vis spectra of the solutions before and after the treatment. Moreover, the COD, SDS-PAGE and protein assay tests were conducted to further analyse the treated solutions. The results demonstrated that the sericin removal efficiency of around 67% and turbidity reduction of 95% were achieved at the optimum conditions of 0.04 g TiO2, pH = 5, and 60 min settling time. Nano and micron-size TiO2 particles showed similar efficiency for sericin removal, but micron-size particles outweighed due to their higher efficiency in inducing greater turbidity reduction after 60 min. The obtained sericin/TiO2 composites were stable after several cycles of wash and could be useful in different fields including cosmetic, and textile finishing, among others.

Making use of IT: web page

The use of the Web has become an essential part of teaching and learning in Australian schools. Nevertheless, many students lack knowledge of how to properly evaluate and cite Web-sourced information. This paper presents criteria, by which students can judge the reliability of Web resources, and guidelines on the citation of Web information. These strategies have been implemented successfully by the authors in their classes.

Matrix model for web page community construction

The World Wide Web is now a huge information source with its own characteristics. In most cases, traditional database-based technologies are no longer suitable for web information processing and management. For effectively processing and managing web. information, it is necessary to reveal intrinsic relationships/structures among concerned web information objects such as web pages. In this work, a set of web pages that has its own intrinsic structure is called a web page community. This paper proposes a matrix model to describe relationships among concerned web pages. Based on this model, intrinsic relationships among pages could be revealed, and in turn a web page community could be constructed. The issues that are related to this model in its application are deeply investigated and studied. Some applications based on this model are presented, which demonstrate the potential of this matrix model in different kinds of web page community construction and information processing.

Web page clustering : a hyperlink-based similarity and matrix-based hierarchical algorithms

This paper proposes a hyperlink-based web page similarity measurement and two matrix-based hierarchical web page clustering algorithms. The web page similarity measurement incorporates hyperlink transitivity and page importance within the concerned web page space. One clustering algorithm takes cluster overlapping into account, another one does not. These algorithxms do not require predefined similarity thresholds for clustering, and are independent of the page order. The primary evaluations show the effectiveness of the proposed algorithms in clustering improvement.

Utilizing hyperlink transitivity to improve web page clustering

The rapid increase of web complexity and size makes web searched results far from satisfaction in many cases due to a huge amount of information returned by search engines. How to find intrinsic relationships among the web pages at a higher level to implement efficient web searched information management and retrieval is becoming a challenge problem. In this paper, we propose an approach to measure web page similarity. This approach takes hyperlink transitivity and page importance into consideration. From this new similarity measurement, an effective hierarchical web page clustering algorithm is proposed. The primary evaluations show the effectiveness of the new similarity measurement and the improvement of web page clustering. The proposed page similarity, as well as the matrix-based hyperlink analysis methods, could be applied to other web-based research areas..

Mining more gold from web page hyperlinks

The development of the Internet has boosted prosperity of the World Wide Web, which is now a huge information source. Because of characteristics of the web, in most cases, traditional databasebased technologies are no longer suitable for web information retrieval and management. To effectively manage web information, it is necessary to reveal intrinsic relationships/structures among web information objects by eliminating noise factors. This paper proposes a mechanism that could be widely used in information processing, including web information processing and noise factor elimination for getting more intrinsic relationships. As an application case of this mechanism, one relevant web page finding algorithm is proposed to uncover intrinsic relationship among web pages from their hyperlink patterns, and find more semantic relevant web pages. The experimental evaluation shows the feasibility and effectiveness of the algorithm and demonstrates the potential of the proposed mechanism in web applications.