Effects of equilibration time, precooling and developmental stage on the survival of mouse embryos cryopreserved by vitrification

Factorial experiments were carried out to examine the effects of equilibration time, precooling and developmental stage on the postthaw in vitro survival of vitrified mouse embryos. Eight-cell embryos, compacted morulae, or blastocysts were cryopreserved using vitrification Solution 1 (VS1; 10% glycerol + 20% propylene glycol), and vitrification Solution 2 (VS2; 25% glycerol + 25% propylene glycol) in phosphate buffered saline + 10% calf serum. Each embryo stage group was first equilibrated in VS1 for 5, 10 or 20 min and then exposed to either a precooled ( approximately 4 degrees C) or nonprecooled ( approximately 20 degrees C) VS2 in a 0.25-ml straw before they were plunged directly into liquid nitrogen. Results of this study showed an interaction between precooling, equilibration time and developmental stage which affect significantly post-thaw embryo survival (P< 0.05). High survival rates were obtained after 10 min equilibration in VS1 irrespective of the embryo developmental stage. Precooling of the VS2 significantly improved the survival mainly of blastocysts. However, eight-cell and morula-stage embryos also showed high survival rates when they were exposed to precooled VS2 after 5 min equilibration in VS1. It was further observed that morulae usually exhibit high survival rates, and vitrification conditions are more critical for early and advanced stage embryo development.     

Rhinitis,Ocular, Throat and Dermal Symptoms,Headache and Tiredness among Students in Schools from Johor Bahru,Malaysia: Associations with Fungal DNA and Mycotoxins in Classroom Dust

There are few studies on rhinitis and sick building syndrome (SBS) among students in tropical countries. We studied associations between levels of five fungal DNA sequences, two mycotoxins (sterigmatocystin and verrucarol) and cat allergen (Fel d 1) levels in schools and rhinitis and other weekly SBS symptoms in the students. Fungal DNA was measured by quantitative PCR and cat allergen by ELISA. Pupils (N = 462) from eight randomly selected schools in Johor Bahru, Malaysia participated (96%). Dust samples were collected by cotton swabs and Petri dishes exposed for one week. None of the schools had a mechanical ventilation system, but all classrooms had openable windows that were kept open during lectures and indoor CO₂ levels were low (mean 492 ppm; range 380–690 ppm). Weekly nasal symptoms (rhinitis) (18.8%), ocular (11.6%), throat (11.1%), dermal symptoms, headache (20.6%) and tiredness (22.1%) were common. Total fungal DNA in swab samples was associated with rhinitis (p = 0.02), ocular symptoms (p = 0.009) and tiredness (p = 0.001). There were positive associations between Aspergillus versicolor DNA in Petri dish samples, ocular symptoms (p = 0.02) and tiredness (p = 0.001). The level of the mycotoxin verrucarol (produced by Stachybotrys chartarum) in swab samples was positively associated with tiredness (p = 0.04). Streptomyces DNA in swab samples (p = 0.03) and Petri dish samples (p = 0.03) were negatively associated with tiredness. In conclusion, total fungal contamination, measured as total fungal DNA) in the classrooms, Aspergillus versicolor and verrucarol can be risk factors for rhinitis and SBS symptoms among students in the tropical country Malaysia.     

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.     

Imaging techniques for the detection of stored product pests

Stored grains are subject to deterioration and losses through various factors, but mainly insects and fungi. Various techniques are employed to detect stored product pests; however, there is an urgent need for an industrial-scale on-line detection technique. Near-infrared hyperspectroscopic imaging and soft X-rays have shown the potential for real-time application. These techniques are particularly effective for detecting internal infestations of stored grains. The digital images of the scanned objects are analyzed for various spectral and image features using statistical techniques such as complex multivariate tools. Classification accuracies as high as 80–100 % have been achieved for various pest and grain combinations. Dual-energy X-rays have been shown to detect the concealed eggs of stored product insect pests. The main threats to stored cereals come from Aspergillus spp., Penicillium spp., and Fusarium spp., which may produce mycotoxins. These imaging techniques have shown good results in the detection of fungal infections of stored grain.     

Firefly distribution and abundance on mangrove vegetation assemblages in Sepetang estuary,Peninsular Malaysia

Pteroptyx fireflies are commonly reported to congregate in large numbers in mangroves. Not much is known about the relationships between firefly distribution and abundance with specific mangrove vegetation assemblages. We conducted a study to investigate the vegetation assemblages that structure the distribution and abundance of Pteroptyx tener in Peninsular Malaysia. The distribution and abundance of fireflies were assessed along an 8 km stretch of mangroves in Sepetang estuary using visual assessment. Statistical analysis was carried out to test the correlation between length of display section and percentage cover of P. tener colonies and the relationship between percentage cover of fireflies with different vegetation assemblages. Five distinct vegetation assemblages were identified comprising different combination of four mangrove species. It was found that shorter display sections had higher percentage cover of P. tener colonies. In addition, vegetation assemblage which consisting of mainly Sonneratia caseolaris and Nypa fruticans was the most preferred type. The results of this study point to the necessity to consider not only a single mangrove species but the entire vegetation assemblage for firefly conservation.     

Characterisation of recombinant Hevea brasiliensis allene oxide synthase: effects of cycloxygenase inhibitors, lipoxygenase inhibitors and salicylates on enzyme activity.

Mechanical wounding and jasmonic acid (JA) treatment have been shown to be important factors in controlling laticifer differentiation in Hevea brasiliensis (rubber tree). With the long-term aim of potentially modifying the endogenous levels of JA in H. brasiliensis by gene transfer, we describe in this paper the molecular cloning of a H. brasiliensis allene oxide synthase (AOS) cDNA and biochemical characterisation of the recombinant AOS (His(6)-HbAOS) enzyme. The AOS cDNA encodes a protein with the expected motifs present in CYP74A sub-group of the cytochrome P450 super-family of enzymes that metabolise 13-hydroperoxylinolenic acid (13-HPOT), the intermediate involved in JA synthesis. The recombinant H. brasiliensis AOS enzyme was estimated to have a high binding affinity for 13-HPOT with a K(m) value of 4.02+/-0.64 microM. Consistent with previous studies, mammalian cycloxygenase (COX) and lipoxygenase (LOX) inhibitors were shown to significantly reduce His(6)-HbAOS enzyme activity. Although JA had no effect on His(6)-HbAOS, salicylic acid (SA) was shown to significantly inhibit the recombinant AOS enzyme activity in a dose dependent manner. Moreover, it was demonstrated that SA, and various analogues of SA, acted as competitive inhibitors of His(6)-HbAOS when 13-HPOT was used as substrate. We speculate that this effect of salicylates on AOS activity may be important in cross-talking between the SA and JA signalling pathways in plants during biotic/abiotic stress.     

A dominant negative mutant of protein kinase CK2 exhibits altered auxin responses in Arabidopsis

Protein kinase CK2 is a pleiotropic Ser/Thr kinase, evolutionary conserved in eukaryotes. Studies performed in different organisms, from yeast to humans, have highlighted the importance of CK2 in cell growth and cell-cycle control. However, the signalling pathways in which CK2 is involved have not been fully identified. In plants, the phytohormone auxin is a major regulator of cell growth. Recent discoveries have demonstrated that differential distribution of within auxin plant tissues is essential for developmental processes, and that this distribution is dependent on polar auxin transport. We report here that a dominant-negative mutant of CK2 (CK2mut) in Arabidopsis thaliana shows phenotypic traits that are typically linked to alterations in auxin-dependent processes. However, CK2mut plants exhibit normal responses to exogenous indole-3-acetic acid (IAA) indicating that they are not affected in the perception of the hormone but upstream in the pathway. We demonstrate that mutant plants are not deficient in IAA but are impaired in its transport. Using genetic and pharmacological tools we show that CK2 activity depletion hinders correct formation of auxin gradients and leads to widespread changes in the expression of auxin-related genes. In particular, members of the auxin efflux carrier family (PINs), and the protein kinase PINOID, both key regulators of auxin fluxes, were misexpressed. PIN4 and PIN7 were also found mislocalized, with accumulation in endosomal bodies. We propose that CK2 functions in the regulation of auxin-signalling pathways, particularly in auxin transport.     

A fluorescence-based alkaline phosphatase-coupled polymerase assay for identification of inhibitors of dengue virus RNA-dependent RNA polymerase

The flaviviral RNA-dependent RNA polymerase (RdRp) is an attractive drug target. To discover new inhibitors of dengue virus RdRp, the authors have developed a fluorescence-based alkaline phosphatase-coupled polymerase assay (FAPA) for high-throughput screening (HTS). A modified nucleotide analogue (2'-[2-benzothiazoyl]-6'-hydroxybenzothiazole) conjugated adenosine triphosphate (BBT-ATP) and 3'UTR-U(30) RNA were used as substrates. After the polymerase reaction, treatment with alkaline phosphatase liberates the BBT fluorophore from the polymerase reaction by-product, BBT(PPi), which can be detected at excitation and emission wavelengths of 422 and 566 nm, respectively. The assay was evaluated by examining the time dependency, assay reagent effects, reaction kinetics, and signal stability and was validated with 3'dATP and an adenosine-nucleotide triphosphate inhibitor, giving IC(50) values of 0.13 μM and 0.01 μM, respectively. A pilot screen of a diverse compound library of 40,572 compounds at 20 μM demonstrated good performance with an average Z factor of 0.81. The versatility and robustness of FAPA were evaluated with another substrate system, BBT-GTP paired with 3'UTR-C(30) RNA. The FAPA method presented here can be readily adapted for other nucleotide-dependent enzymes that generate PPi.     

Atrovirisidone B, a new prenylated depsidone with cytotoxic property from the roots of Garcinia atroviridis

A new prenylated depsidone, atrovirisidone B (2), together with naringenin (3) and 3,8"-binaringenin (4) were isolated from the roots of Garcinia atroviridis. Their structures were determined on the basis of spectral data interpretation. Compound 2 showed cytotoxic activity against human breast (MCF-7), human prostate (DU-145) and human lung (H-460) cancer cells.