Constructions of expression vectors of polyhydroxybutyrate-co-hydroxyvalerate (PHBV) and transient expression of transgenes in immature oil palm embryos

Polyhydroxybutyrate-co-hydroxyvalerate (PHBV) is a polyhydroxyalkanoate (PHA) bioplastic group with thermoplastic properties is thus high in quality and can be degradable. PHBV can be produced by bacteria, but the process is not economically competitive with polymers produced from petrochemicals. To overcome this problem, research on transgenic plants has been carried out as one of the solutions to produce PHBV in economically sound alternative manner. Four different genes encoded with the enzymes necessary to catalyze PHBV are bktB, phaB, phaC and tdcB. All the genes came with modified CaMV 35S promoters (except for the tdcB gene, which was promoted by the native CaMV 35S promoter), nos terminator sequences and plastid sequences in order to target the genes into the plastids. Subcloning resulted in the generation of two different orientations of the tdcB, pLMIN (left) and pRMIN (right), both 17.557 and 19.967 kb in sizes. Both plasmids were transformed in immature embryos (IE) of oil palm via Agrobacterium tumefaciens. Assays of GUS were performed on one-week-old calli and 90% of the calli turned completely blue. This preliminary test showed positive results of integration. Six-months-old calli were harvested and RNA of the calli were isolated. RT-PCR was used to confirm the transient expression of PHBV transgenes in the calli. The bands were 258, 260, 315 and 200 bp in size for bktB, phaB, phaC and tdcB transgenes respectively. The data obtained showed that the bktB, phaB, phaC and tdcB genes were successfully integrated and expressed in the oil palm genome.     

Significant enhancement of lignan accumulation in hairy root cultures of Linum album using biotic elicitors

Linum album has been shown to accumulate some lignans with antiviral and anticancer properties such as podophyllotoxin (PTOX) and 6-methoxy podophyllotoxin (MPTOX). In this research, we examined the effects of fungal elicitors on the production of lignans in L. album hairy root cultures. The biosynthesis of lignans was differentially affected by fungal elicitors. Fusarium graminearum extract induced the highest increase of PTOX, 190 μg g^?1 dry weight (DW), and lariciresinol, 260 μg g^?1 DW, which was two-fold and three-fold greater than the untreated control, respectively, while Trichoderma viride extract enhanced the accumulation of MPTOX, instead of PTOX, up to 160 µg g^?1 DW, which was 2.4-fold greater than the control. The enhancing effects of fungal elicitors on lignans production was correlated with the increased expression of some key genes involved in the biosynthesis of these compounds, phenylalanine ammonia-lyase, cinnamoyl-CoA reductase, cinnamyl-alcohol dehydrogenase and pinoresinol-lariciresinol reductase.     

Adipocyte lineage differentiation potential of MSCs isolated from reaming material

Mesenchymal stem cells (MSCs) obtained from various sources have been used for different therapeutic applications including tissue regeneration. Reamer/irrigator/aspirator (RIA) has been increasingly used in recent years for the derivation of MSCs. Here in this investigation we have comparatively analyzed MSCs obtained from iliac crest bone marrow (ICBM) and RIA for their morphology, cluster determinant (CD) markers, and adipogenic differentiation capacity. MSCs were isolated, cultured, and purified from both sources and then flow cytometric studies were performed to study their characteristics. The differentiation potential of RIA and ICBM was examined by an Oil Red O staining protocol. Moreover, the tissue-specific markers related to adipogenesis were analyzed by real-time polymerase chain reaction (RT-PCR). The cells were cultured in the relevant induction medium and then adipogenic lineage differentiation was tested and confirmed for all MSC preparations. Additionally, analysis by flow cytometer was indicative of RIA derived MSCs (RIA-MSCs) having a more homogenous population than ICBM derived MSCs. The RIA-MSCs differentiation toward adipogenic lineage was more efficient compared with ICBM-MSCs. Direct comparative analysis of RIA to ICBM-MSCs indicated that the RIA-MSCs had a higher potential toward adipocyte lineage differentiation compared with ICBM-MSCs.     

Assessment of IL-10, IL-1ß and TNF-α gene polymorphisms in patients with peri-implantitis and healthy controls

Molecular Biology Reports - Peri-implantitis (PI) is a multifactorial condition caused by the interactions of pathogens and the host immune response. Previous studies have demonstrated a...     

Rapid upregulation of endothelial P-selectin expression via reactive oxygen species generation

Endothelial cell ICAM-1 upregulation in response to TNF-alpha is mediated in part by reactive oxygen species (ROS) generated by the endothelial membrane-associated NADPH oxidase and occurs maximally after 4 h as the synthesis of new protein is required. However, thrombin-stimulated P-selectin upregulation is bimodal, the first peak occurring within minutes. We hypothesize that this early peak, which results from the release of preformed P-selectin from within Weibel-Palade bodies, is mediated in part by ROS generated from the endothelial membrane-associated xanthine oxidase. We found that this rapid expression of P-selectin on the surface of endothelial cells was accompanied by qualitatively parallel increases in ROS generation. Both P-selectin expression and ROS generation were inhibited, dose dependently, by the exogenous administration of disparate cell-permeable antioxidants and also by the inhibition of either of the known membrane-associated ROS-generating enzymes NADPH oxidase or xanthine oxidase. This rapid, posttranslational cell signaling response, mediated by ROS generated not only by the classical NADPH oxidase but also by xanthine oxidase, may well represent an important physiological trigger of the microvascular inflammatory response.     

Assessment of Heavy Metal Pollution and Human Health Risks Assessment in Soils Around an Industrial Zone in Neyshabur,Iran

Biological Trace Element Research - Heavy metal pollution of soils in industrial zones continues to attract attention because of its potential human health risks. The present research is an attempt...     

Transmission of RNA silencing signal through grafting confers virus resistance from transgenically silenced tobacco rootstocks to non-transgenic tomato and tobacco scions

We examined the transmission of RNA silencing signal in non-transgenic tomato and tobacco scions grafted onto the tobacco Sd1 rootstocks, which is silenced in both NtTOM1 and NtTOM3 required for tobamovirus multiplication. When the non-transgenic tomato scions were grafted onto the Sd1 rootstocks, RT-PCR analysis of the scions showed the reduced level of mRNA compared with that before grafting in both LeTH3 and LeTH1, tomato homologs of NtTOM1 and NtTOM3, respectively. siRNAs from both genes were detected in the scions after grafting but not before grafting. Further tomato scions were inoculated with Tomato mosaic virus (ToMV) and used for virus infection. They showed very low level of virus accumulation. Necrotic responding tobacco to tobamovirus was grafted onto the rootstock of Sdl. RT-PCR analysis showed low level expression of both NtTOM1 and NtTOM3 in the scions but siRNA was detected after grafting. When the leaves of scions were inoculated with ToMV or Tobacco mosaic virus, they produced very few local necrotic lesions (LNLs) while the control scions did many LNLs. These results suggest that RNA silencing was transmitted to non-transgenic tomato and tobacco scions after grafting onto the Sd1 rootstocks and that virus resistance was induced in the scions.     

Do innate killing mechanisms activated by inflammasomes have a role in treating melanoma?

Melanoma, as for many other cancers, undergoes a selection process during progression that limits many innate and adaptive tumor control mechanisms. Immunotherapy with immune checkpoint blockade overcomes one of the escape mechanisms but if the tumor is not eliminated other escape mechanisms evolve that require new approaches for tumor control. Some of the innate mechanisms that have evolved against infections with microorganisms and viruses are proving to be active against cancer cells but require better understanding of how they are activated and what inhibitory mechanisms may need to be targeted. This is particularly so for inflammasomes which have evolved against many different organisms and which recruit a number of cytotoxic mechanisms that remain poorly understood. Equally important is understanding of where these mechanisms will fit into existing treatment strategies and whether existing strategies already involve the innate killing mechanisms.     

Broad-range Glycosidase Activity Profiling

Plants produce hundreds of glycosidases. Despite their importance in cell wall (re)modeling, protein and lipid modification, and metabolite conversion, very little is known of this large class of glycolytic enzymes, partly because of their post-translational regulation and their elusive substrates. Here, we applied activity-based glycosidase profiling using cell-permeable small molecular probes that react covalently with the active site nucleophile of retaining glycosidases in an activity-dependent manner. Using mass spectrometry we detected the active state of dozens of myrosinases, glucosidases, xylosidases, and galactosidases representing seven different retaining glycosidase families. The method is simple and applicable for different organs and different plant species, in living cells and in subproteomes. We display the active state of previously uncharacterized glycosidases, one of which was encoded by a previously declared pseudogene. Interestingly, glycosidase activity profiling also revealed the active state of a diverse range of putative xylosidases, galactosidases, glucanases, and heparanase in the cell wall of Nicotiana benthamiana. Our data illustrate that this powerful approach displays a new and important layer of functional proteomic information on the active state of glycosidases.Carbohydrates are present in all kingdoms of life and are particularly prominent in plants (1). Plants produce carbohydrates as one of their major constituents through their photosynthetic activity. The simplest synthesized forms of carbohydrates are monosaccharide sugars such as glucose, which provides energy for various cellular activities. Carbohydrates also exist in very complex forms. Monosaccharide sugars are attached to one another through covalent glycosidic linkage, which generates di-, oligo-, and polysaccharides. Carbohydrates also attach to non-carbohydrate species (lipids, proteins, hormones) through a glycosidic linkage to form glycoconjugates (2).Glycosidic bonds are hydrolyzed by a group of enzymes termed glycosyl hydrolases (GHs)¹ or glycosidases (3). Because of the tremendous carbohydrate diversity, there are a vast variety of glycosidases, including glucosidases, xylosidases, and galactosidases, that preferentially hydrolyze their respective glycoside substrates. In general, the number of glycosidase-related genes in plants (for instance, Arabidopsis) is relatively high when compared with that in other sequenced organisms (for instance, human) (4). This signifies the unique importance of glycosidases in plants as opposed to other organisms. Based on protein sequence similarities, glycosidases are classified into different GH families. Members of the same GH family share a common mechanism of glycosidic bond cleavage (5).Mechanistically, glycosidases are classified as retaining or inverting enzymes (6). To hydrolyze the glycosidic bond, both retaining and inverting enzymes carry two catalytic glutamate or aspartate residues (or both) (7). Of these two catalytic residues, one acts as a proton donor and the other as a nucleophile/base. The distance between these catalytic residues in the active site of the glycosidases determines the mechanism of hydrolysis. Retaining enzymes have two catalytic residues separated by a distance of ∼5.5 Å, and their hydrolysis mechanism retains the net anomeric configuration of the C1 atom in the sugar molecule. In contrast, inverting enzymes have catalytic residues that are ∼10 Å apart, and these enzymes invert the overall anomeric configuration of the C1 carbon atom in the released sugar (8).Both retaining and inverting glycosidases are present abundantly in plants. The genome of Arabidopsis thaliana encodes for 400 glycosidases, of which 260 are retaining enzymes and 140 are inverting enzymes. Genetic, molecular, and biochemical approaches revealed that glycosidases are localized in different cellular compartments and are important for various biological processes. The majority of plant glycosidases reside in the cell wall, and these enzymes can play major roles in cell wall restructuring (9). Other characterized glycosidases reside in other compartments to regulate glycosylation of proteins and hormones. Despite the importance of GH enzymes, physiological and biochemical functions are assigned to only a few glycosidases (9).Activity-based protein profiling (ABPP) is a powerful tool for monitoring the active state of multiple enzymes without knowledge of their natural substrates (10, 11). ABPP involves chemical probes that react with active site residues in an activity-dependent manner. Thus ABPP displays the availability and reactivity of active site residues in proteins, which are hallmarks for enzyme activity (12). ABPP is particularly attractive because the profiling can be done without purifying the enzymes and can be performed in cell extracts or in living cells. Another key advantage of ABPP is that the activities of large multigene enzyme families can be monitored using broad-range probes. ABPP has had a significant impact on plant science. After the introduction of probes for papain-like cysteine proteases (13, 14), these probes revealed increased protease activities in the tomato and maize apoplasts during immune responses (15, 16) and that these immune proteases are targeted by unrelated inhibitors secreted by fungi, oomycetes, and nematodes (17–24). Likewise, probes for the proteasome displayed unexpected increased proteasome activity during immune responses (25) and revealed that the bacterial effector molecule syringolin A targets the nuclear proteasome (26). We anticipate that more regulatory mechanisms will be discovered through the use of probes introduced for serine hydrolases, metalloproteases, vacuolar processing enzymes, ATP binding proteins, and glutathione transferases (27–32).Cyclophellitol-aziridine-based probes were previously used in animal proteomes to target retaining glucosidases (33). Here we established and applied glycosidase profiling in plants. We discovered that cyclophellitol-aziridine-based probes targeted an unexpectedly broad range of glycosidases representing members of at least seven different GH families. We used these probes to study the active state of glycosidases present in living cells, in different organs and plant species, and in the apoplast of Nicotiana benthamiana.