Lovastatin and (+)‐geodin production by Aspergillus terreus from crude glycerol

The use of pure substrate represents a significant proportion of the cost of manufacturing a drug such as lovastatin. This study explores the production of lovastatin and (+)‐geodin by Aspergillus terreus ATCC 20542 using biodiesel‐derived crude glycerol (CG) as a feedstock. Shake flask experiments showed reduced lovastatin production and glycerol consumption in the presence of 10–50 g/L CG with respect to pure glycerol controls. At 50 g/L, lovastatin and (+)‐geodin production was significantly reduced by 82 and 73%, respectively. The lowest lovastatin inhibition was detected in 30 g/L of CG (48%), which was accompanied by a significant rise in (+)‐geodin production (338%). Further investigation was performed on three major impurities found in CG, namely methanol (MeOH), sodium chloride (NaCl), and fatty acids (oleic acid and palmitic acid (PA), soap). None was particularly inhibitory for lovastatin, except soap and PAs, which reduced its production by more than 50% at all concentrations tested. In contrast, (+)‐geodin was inhibited in the presence of MeOH and PA by up to 46 and 91%, respectively. These observations indicate that partial purification of CG would be potentially useful in improving production of lovastatin and (+)‐geodin by A. terreus.     

Glutamate oxidative injury to RGC-5 cells in culture is necrostatin sensitive and blunted by a hydrogen sulfide (H2S)-releasing derivative of aspirin (ACS14)

Oxidative stress to RGC-5 cells in culture was delivered by exposure to a combination of glutamate (Glu) and buthionine-S,R-sulfoximine (BSO). The effect of the insult on cell survival was quantified by the resazurin-reduction and a dead/live assays. Moreover, breakdown of DNA, the localisation of phosphatidylserine and reactive radical species (ROS) and its quantification were determined. In addition, various proteins and mRNAs were studied using Western blot, real time PCR and immunocytochemistry. ACS14, its sulfurated moiety ACS1 and aspirin were tested for their ability to blunt the negative effects of Glu/BSO on RGC-5 cells. In addition assays were carried out to see whether any of these substances influenced glutathione (GSH). Glu/BSO dose-dependently kills RGC-5 cells by a mechanism that involves an elevation of ROS accompanied by a breakdown of DNA, expression of phosphatidylserine and the activation of p38 MAPK. The process is unaffected by the pan caspase inhibitor z-VAD-fmk, does not involve the activation of apoptosis inducing factor (AIF) but is sensitive to active necrostatin-1. In cell viability studies (resazurin-reduction assay), ACS1 and ACS14 equally counteracted the negative effects of 5mM Glu/BSO to RGC-5 cells but aspirin was only effective with a milder oxidative stress (1 mM Glu/BSO). In all other assays ACS14 was very much more effective than aspirin at counteracting the influence of 5mM Glu/BSO. Moreover, ACS14 and ACS1 directly stimulated GSH while aspirin was ineffective. In addition the neuroprotecive effect of ACS14 was specifically blunted by the non-specific potassium channel blocker glibenclamide. Also the up-regulation of Bcl-2, HO-1 and XIAP induced by 5mM Glu/BSO were all attenuated to a greater extent by ACS14 (20 μM) than aspirin (20 μM). These data show that ACS14 is a very effective neuroprotectant when compared with aspirin. ACS14 maintains its aspirin characteristics and has the ability to release H(2)S. The combined multiple actions of aspirin and H(2)S in the form of ACS14 is worthy to consider for possible use in the treatment of glaucoma.     

Effect of Feeding Palm Oil By-Products Based Diets on Muscle Fatty Acid Composition in Goats

The present study aims to evaluate the effects of feeding palm oil by-products based diets on different muscle fatty acid profiles in goats. Thirty-two Cacang × Boer goats were randomly assigned to four dietary treatments: (1) control diet (CD), (2) 80% decanter cake diet (DCD), (3) 80% palm kernel cake diet (PKCD) and (4) CD plus 5% palm oil (PO) supplemented diet (CPOD). After 100 days of feeding, four goats from each group were slaughtered and longissimus dorsi (LD), infraspinatus (IS) and biceps femoris (BF) were sampled for analysis of fatty acids. Goats fed the PKCD had higher (P<0.05) concentration of lauric acid (C12:0) than those fed the other diets in all the muscles tested. Compared to the other diets, the concentrations of palmitic acid (C16:0) and stearic acid (C18:0) were lower (P<0.05) and that of linoleic acid (C18:2 n-6) was higher (P<0.05) in the muscles from goats fed the CD. It was concluded that palm kernel cake and decanter cake can be included in the diet of goats up to 80% with more beneficial than detrimental effects on the fatty acid profile of their meat.     

Differential responses in water use efficiency in two varieties of Catharanthus roseus under drought stress

Two varieties, rosea and alba, of Catharanthus roseus (L.) G. Don. were screened for their water use efficiency under two watering regimes, viz. 60 and 100% filed capacity in the present study. Drought stress was imposed at 60% filed capacity from 30 to 70 days after sowing, while the control pots were maintained at 100% filed capacity throughout the entire growth period. Leaf area duration, cumulative water transpired, water use efficiency, net assimilation rate, mean transpiration rate, harvest index, biomass and yield under the water deficit level were measured from both stressed and well-watered control plants. Water use efficiency significantly increased in both varieties under water stress. Drought stress decreased leaf area duration, cumulative water transpired, net assimilation rate, mean transpiration rate, harvest index, and biomass yield in both varieties studied. Among the varieties, rosea variety showed the best results.     

Biological attributes of Ooencyrtus nezarae Ishii (Hymenoptera: Encyrtidae) reared on refrigerated eggs of Riptortus pedestris (= clavatus) Fabricius (Hemiptera: Alydidae)

Storing host eggs at low temperatures has been used to mass rear parasitoids of stink bugs, including Riptortus pedestris Fabricius (Hemiptera: Alydidae), a major soybean pest in Korea and Japan. However, no information on the effect of cold storage on parasitization by Ooencyrtus nezarae Ishii (Hymenoptera: Encyrtidae), one of the major parasitoids of R. pedestris, has been published. In this study, we examined biological attributes, including parasitism rate, development time, sex ratio, adult size, and longevity, of O. nezarae when the adult parasitoids were provided with host eggs refrigerated for 0, 15, 30, 60, and 120 days at 2.0 °C. None of the attributes of the first or second generation of O. nezarae was negatively affected by host egg refrigeration up to 30 days. In addition, O. nezarae could parasitize refrigerated host eggs successfully for the first four days of post-refrigeration period when they were kept at 26.3 °C and 78.7% RH conditions. Therefore, refrigeration of R. pedestris eggs can be a good method to mass rear O. nezarae.     

Tobacco mutants with reduced microtubule dynamics are less susceptible to TMV

A panel of seven SR1 tobacco mutants (ATER1 to ATER7) derived via T‐DNA activation tagging and screening for resistance to a microtubule assembly inhibitor, ethyl phenyl carbamate, were used to study the role of microtubules during infection and spread of tobacco mosaic virus (TMV). In one of these lines, ATER2, α‐tubulin is shifted from the tyrosinylated into the detyrosinated form, and the microtubule plus‐end marker GFP–EB1 moves significantly slower when expressed in the background of the ATER2 mutant as compared with the SR1 wild type. The efficiency of cell‐to‐cell movement of TMV encoding GFP‐tagged movement protein (MP‐GFP) is reduced in ATER2 accompanied by a reduced association of MP‐GFP with plasmodesmata. This mutant is also more tolerant to viral infection as compared with the SR1 wild type, implying that reduced microtubule dynamics confer a comparative advantage in face of TMV infection.     

Exploring the collagen-binding site of the DDR1 tyrosine kinase receptor

Discoidin domain receptors 1 and 2 (DDR1 and DDR2) are tyrosine kinase receptors activated by triple-helical collagens. Aberrant expression and signaling of these receptors have been implicated in several human diseases linked to accelerated matrix degradation and remodeling including tumor invasion, atherosclerosis and liver fibrosis. The objective of this study is to characterize the collagen-binding sites in the discoidin domains of DDR1 and DDR2 at a molecular level. We expressed glutathione S-transferase fusion proteins containing the discoidin and extracellular domains of DDR1 and DDR2 in insect cells and subjected them to a solid-phase collagen-binding assay. We found high affinity binding of the DDR extracellular domains to immobilized type I collagen and confirmed the discoidin-collagen interaction with an enzyme-linked immunosorbent assay-based read-out. Furthermore, we created a three-dimensional model of the DDR1 discoidin domain based on the related domains of blood coagulation factors V and VIII. This model predicts the presence of four neighboring, surface-exposed loops that are topologically equivalent to a major phospholipid-binding site in factors V and VIII. To test the involvement of these loops in collagen binding, we mutated individual amino acid residues to alanine or deleted short sequence stretches within these loops. We found that several residues within loop 1 (Ser-52-Thr-57) and loop 3 (Arg-105-Lys-112) as well as Ser-175 in loop 4 are critically involved in collagen binding. Our structure-function analysis of the DDR discoidin domains provides new insights into this non-integrin-mediated collagen-signaling mechanism and may ultimately lead to the design of small molecule inhibitors that interfere with aberrant DDR function.     

Mutations affecting somite formation in the Medaka (Oryzias latipes)

The metameric structure of the vertebrate trunk is generated by repeated formation of somites from the unsegmented presomitic mesoderm (PSM). We report the initial characterization of nine different mutants affecting segmentation that were isolated in a large-scale mutagenesis screen in Medaka (Oryzias latipes). Four mutants were identified that show a complete or partial absence of somites or somite boundaries. In addition, five mutations were found that cause fused somites or somites with irregular sizes and shapes. In situ hybridization analysis using specific markers involved in the segmentation clock and antero-posterior (A-P) polarity of somites revealed that the nine mutants can be compiled into two groups. In group 1, mutants exhibit defects in tailbud formation and PSM prepatterning, whereas A-P identity in the somites is defective in group 2 mutants. Three mutants (planlos, pll; schnelles ende, sne; samidare, sam) have characteristic phenotypes that are similar to those in zebrafish mutants affected in the Delta/Notch signaling pathway. The majority of mutants, however, exhibit somitic phenotypes distinct from those found in zebrafish, such as individually fused somites and irregular somite sizes. Thus, these Medaka mutants can be expected to provide clues to uncovering novel components essential for somitogenesis.