Cloning,over-expression,purification, and characterisation of N-acetylneuraminate synthase from Streptococcus agalactiae

N-acetylneuraminate synthase (NeuAc-synthase; E.C. 4.1.3.19) is one of the two enzymes responsible for sialic acid (N-acetylneuraminic acid) synthesis in bacteria. Potential genes encoding NeuAc synthase in Streptococcus agalactiae and Bacillus subtilis were identified from a BLAST search of the EMBL/GenBank/DDBJ database using the E. coli neuB gene sequence as a probe and the genes cloned and expressed at high level in Escherichia coli. The neuB gene of S. agalactiae was shown to encode an active NeuAc synthase, whereas the spsE gene product from B. subtilis did not have this activity. Expression of the native S. agalactiae neuB gene product enzyme in E. coli resulted in a product that was prone to proteolysis during purification so the protein was tagged with a hexa-histidine tag at its N-terminus and the enzyme was rapidly purified to homogeneity by ammonium sulphate fractionation and Ni-chelating affinity chromatography in two steps. Measurement of the subunit molecular mass by electrospray ionisation mass spectrometry (M(r) = 38, 987 +/- 3) and of the native molecular mass by gel filtration chromatography (M(r) = 78,000) clearly demonstrated that the enzyme is dimeric. The effects of EDTA, temperature, and pH on the activity of the S. agalactiae NeuAc synthase were examined. Enzyme activity was maximal at pH 7 and was dependent on the presence of metal ions such as Mg(2+), Mn(2+) or Co(2+). The purified enzyme was inhibited by the reagent phenylglyoxal and the substrates N-acetyl mannosamine or phosphoenol pyruvate afforded protection against this inhibition, suggesting that one or more arginine residues are involved in substrate recognition and binding. The ease of expression and the properties of the enzyme should now permit a thorough study of the specificity of the enzyme and provide the prerequisites for attempts to alter this specificity by directed evolution for the production of novel sialic acid analogues.     

In vitro propagation of temperate Australian terrestrial orchids: revisiting asymbiotic compared with symbiotic germination

Using a common temperate herbaceous terrestrial orchid from Australia (Caladenia latifolia) this study investigated 19 asymbiotic media variations comprising four commonly used basal media [half‐strength Murashige and Skoog (½MS), Knudson C (KC), Pa5, and Vacin and Went (VW), with combinations of the plant growth regulators 6‐benzylaminopurine (BA) and α‐naphthalene acetic acid (NAA) or coconut water (CW) and compared their performance with germination on a standard symbiotic germination medium, oatmeal agar (OMA). Percentage germination of seeds every 2 weeks for a total of 8 weeks (five replicates per treatment), time to germination, and growth and development phases in seedlings were recorded. ½MS with 5% (v/v) fresh CW delivered 93% germination, with seedling vigour and development indistinguishable from OMA (95% germination). The same protocol was applied to a further ten species (including the endangered Caladenia huegelii), demonstrating high asymbiotic germination performance (60–93%) across a wide phylogenetic range of terrestrial orchid species. © 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 176 , 556–566.     

Selection of optimal stage for protocorm-like bodies and production of artificial seeds for direct regeneration on different media and short term storage of Dendrobium Shavin White

Micropropagation is currently the most popular method for orchid propagation through the production of protocorm-like bodies (PLBs). It is suggested that converting the PLBs into artificial seeds by encapsulation with sodium alginate can be useful for short-term preservation and distribution to the laboratories and commercial nurseries. Prior to the production of artificial seeds, the best developmental stage of PLBs based on sizes for increased conversion to plantlet was determined. PLBs were categorized based on size and presence of shoot namely ≤2 mm (S1), >2–4 mm (S2), >4–6 mm (S3), >2–4 mm with shoot (S4) and >4–6 mm with shoot (S5). S4 and S5 gave significantly higher conversion percentage (85 and 90 %, respectively) as compared to the PLBs without shoot (S1, S2 and S3). Thus, for uniformity PLBs of 3–5 mm with shoot were used for encapsulation with sodium alginate to form artificial seeds. The feasibility of germinating artificial seeds of Dendrobium Shavin White in different substrates namely; M1 (semi-solid ½ Murashige and Skoog (1962) basal medium), M2 (cotton irrigated with sterilized liquid ½ MS basal medium), M3 (cotton irrigated with sterilized distilled water) and M4 (cotton irrigated with non-sterilized distilled water) was tested. The encapsulated PLBs regenerated well in M1 where 96 % of encapsulated PLBs germinated after 12 days of inoculation and 76 % of them converted into plantlet after 37 days of inoculation while PLBs subjected to sterile distilled water gave 56 % germination and 44 % conversion after 42 and 167 days of inoculation respectively. The ability to store encapsulated PLBs would be advantageous for transport of planting materials. Encapsulated PLBs survived longer when stored at 25 ± 2 °C compared to 4 °C, 10 °C and 30 ± 2 °C whereby storage up to 75 days retained 80–92 % survival. Further storage up to 135 days retained 52 % survival. All plantlets survived after acclimatization when transferred to charcoal media under shade.     

Alginate-encapsulation, short-term storage and plant regeneration from protocorm-like bodies of Aranda Wan Chark Kuan ‘Blue’?×?Vanda coerulea Grifft. ex. Lindl. (Orchidaceae)

This article demonstrates the single bead alginate-encapsulation and conversion (complete plantlet regeneration) from protocorm-like bodies (PLBs) of Aranda Wan Chark Kuan ??Blue???×?Vanda coerulea Grifft. ex. Lindl. (AV) (a monopodial orchid hybrid) for the first time. PLBs, induced from leaf segments of AV were isolated from in vitro proliferating PLB clusters. Individual PLBs (4?±?1?mm diameter) were encapsulated in calcium alginate beads to manage mass propagation, short-term storage and germplasm sharing. The superior gel matrix for encapsulation was obtained using 3?% sodium alginate and 75?mM calcium chloride (CaCl₂·2H₂O). Highest percentage of germination (98.1?%) and conversion (96.2?%) of encapsulated PLBs (capsules) was obtained on plant growth regulator-free half-strength MS (Murashige and Skoog, Physiol Plant 15:473?C497, 1962) medium. Successful storage of capsules, until 180?days, was achieved at 25?°C under zero-irradiance with germination and conversion frequency of 76.9 and 70.2?%, respectively. Plantlets regenerated from capsules were acclimatized successfully with 92?% survival rate.     

An oral cancer biobank initiative: a platform for multidisciplinary research in a developing country

Identification of diagnostic markers for early detection and development of novel and therapeutic agents for effective patient management are the main motivation for cancer research. Biological specimens from large cohort and case-control studies which are crucial in providing successful research outcomes are often the limiting factor that hinders research efforts, especially in developing countries. Therefore, the Malaysian Oral Cancer Database and Tissue Bank System (MOCDTBS) were established to systematically collect large number of samples with comprehensive sociodemographic, clinicopathological, management strategies, quality of life and associated patient follow-up data to facilitate oral cancer research in Malaysia. The MOCDTBS also promotes sharing among researchers and the development of a multidisciplinary research team. The following article aims to describe the process of setting-up and managing the MOCDTBS.