Recent advances in barley transformation

Barley, an important member of the cereals, has been successfully transformed through various methods such as particle bombardment, Agrobacterium tumefaciens, DNA uptake, and electroporation. Initially, the transformation in barley concentrated on developing protocols using marker genes such as gus, bar, and hpt. Immature embryos and callus derived from immature embryos were targeted for transformation. Subsequently, genes of agronomic and malting importance have been deployed in barley. Particle bombardment appears to be the preferred choice for barley transformation in the majority of the reports, although Agrobacterium-mediated transformation is being used more often. The current review focuses on the challenges encountered in barley transformation such as somaclonal variation, development of transformation systems for commercial cultivars, gene expression, stability and inheritance, and gene flow. Newer markers such as the green fluorescent protein (gfp), firefly luciferase, and phosphomannose isomerase were found to be useful in the selection of transgenic plants. Tissue-specific promoters such as those for B1-hordein and D-hordein genes, and spike-specific promoters, are increasingly used to drive gene expression. The review also describes recent research on gene-tagging through transformation, insertion of disease resistance, and abiotic stress resistance genes, transformation with genes for improved malting quality, nutrient content, feed quality, and the production of feed enzymes and pharmaceutical compounds.     

Carnosic acid: A potent chemopreventive agent against oral carcinogenesis

The present study was aimed to investigate the chemopreventive potential of carnosic acid in 7,12-dimethylbenz(a)anthracene (DMBA)-induced hamster buccal pouch carcinogenesis. The chemopreventive potential was assessed by analyzing the tumor incidence, tumor volume and burden as well as by measuring the status of lipid peroxidation, non-enzymatic and enzymatic antioxidants and phase I and phase II detoxification enzymes. Oral squamous cell carcinoma was developed in the buccal pouch of golden Syrian hamsters by painting with 0.5% DMBA in liquid paraffin three times a week for 14 weeks. In the present study, 100% tumor formation was observed in hamsters treated with DMBA alone. Also, the status of lipid peroxidation, antioxidants and phase I and phase II detoxification enzymes were significantly altered during DMBA-induced oral carcinogenesis. Oral administration of carnosic acid at a dose of 10 mg/kg body weight/day to DMBA-treated animals completely prevented the tumor formation in the hamsters’ buccal pouches. Also, carnosic acid exerted potent anti-lipid peroxidative function and stimulated the detoxification cascade during DMBA-induced hamster buccal pouch carcinogenesis. The results of the present study suggest that the chemopreventive potential of carnosic acid is probably due to its anti-lipid peroxidative potential and modulating effect on carcinogen detoxification enzymes during DMBA-induced oral carcinogenesis.     

LEC-2, a highly variable lectin in the lichen Peltigera membranacea

Lectins are a diverse group of carbohydrate binding proteins often involved in cellular interactions. A lectin gene, lec-2, was identified in the mycobiont of the lichen Peltigera membranacea. Sequencing of lec-2 open reading frames from 21 individual samples showed an unexpectedly high level of polymorphism in the deduced protein (LEC-2), which was sorted into nine haplotypes based on amino acid sequence. Calculations showed that the rates of nonsynonymous versus synonymous nucleotide substitutions deviated significantly from the null hypothesis of neutrality, indicating strong positive selection. Molecular modeling revealed that most amino acid replacements were around the putative carbohydrate-binding pocket, indicating changes in ligand binding. Lectins have been thought to be involved in the recognition of photobiont partners in lichen symbioses, and the hypothesis that positive selection of LEC-2 is driven by variation in the Nostoc photobiont partner was tested by comparing mycobiont LEC-2 haplotypes and photobiont genotypes, as represented by the rbcLX region. It was not possible to pair up the two types of marker sequences without conflicts, suggesting that positive selection of LEC-2 was not due to variation in photobiont partners.     

IQGAP1: a regulator of intracellular spacetime relativity

Activating and inhibiting receptors of lymphocytes collect valuable information about their mikròs kósmos. This information is essential to initiate or to turn off complex signaling pathways. Irrespective of these advances, our knowledge on how these intracellular activation cascades are coordinated in a spatiotemporal manner is far from complete. Among multiple explanations, the scaffolding proteins have emerged as a critical piece of this evolutionary tangram. Among many, IQGAP1 is one of the essential scaffolding proteins that coordinate multiple signaling pathways. IQGAP1 possesses multiple protein interaction motifs to achieve its scaffolding functions. Using these domains, IQGAP1 has been shown to regulate a number of essential cellular events. This includes actin polymerization, tubulin multimerization, microtubule organizing center formation, calcium/calmodulin signaling, Pak/Raf/Mek1/2-mediated Erk1/2 activation, formation of maestrosome, E-cadherin, and CD44-mediated signaling and glycogen synthase kinase-3/adenomatous polyposis coli-mediated β-catenin activation. In this review, we summarize the recent developments and exciting new findings of cellular functions of IQGAP1.     

Genome Sequence of the Plant Growth-Promoting Bacterium Enterobacter cloacae GS1

Here, we present the genome sequence of Enterobacter cloacae GS1. This strain proficiently colonizes rice roots and promotes plant growth by improving plant nutrition. Analyses of the E. cloacae GS1 genome will throw light on the genetic factors involved in root colonization, growth promotion, and ecological success of this rhizobacterium.     

An Orthotopic Bladder Tumor Model and the Evaluation of Intravesical saRNA Treatment

We present a novel method for treating bladder cancer with intravesically delivered small activating RNA (saRNA) in an orthotopic xenograft mouse bladder tumor model. The mouse model is established by urethral catheterization under inhaled general anesthetic. Chemical burn is then introduced to the bladder mucosa using intravesical silver nitrate solution to disrupt the bladder glycosaminoglycan layer and allows cells to attach. Following several washes with sterile water, human bladder cancer KU-7-luc2-GFP cells are instilled through the catheter into the bladder to dwell for 2 hours. Subsequent growth of bladder tumors is confirmed and monitored by in vivo bladder ultrasound and bioluminescent imaging. The tumors are then treated intravesically with saRNA formulated in lipid nanoparticles (LNPs). Tumor growth is monitored with ultrasound and bioluminescence. All steps of this procedure are demonstrated in the accompanying video.     

Mechanistic insights into LDL nanoparticle-mediated siRNA delivery

Although small interfering RNA (siRNA) can silence the expression of disease-related genes, delivery of these highly charged molecules is challenging. Delivery approaches for siRNAs are actively being pursued, and improved strategies are required for nontoxic and efficient delivery for gene knockdown. Low density lipoprotein (LDL) is a natural and endogenous nanoparticle that has a rich history as a delivery vehicle. Here, we examine purified LDL nanoparticles as carriers for siRNAs. When siRNA was covalently conjugated to cholesterol, over 25 chol-siRNA could be incorporated onto each LDL without changing nanoparticle morphology. The resulting LDL-chol-siRNA nanoparticles were selectively taken up into cells via LDL receptor mediated endocytosis, resulting in enhanced gene silencing compared to free chol-siRNA (38% gene knock down versus 0% knock down at 100 nM). However, silencing efficiency was limited by the receptor-mediated entrapment of the LDL-chol-siRNA nanoparticles in endolysosomes. Photochemical internalization demonstrated that endolysosome disruption strategies significantly enhance LDL-mediated gene silencing (78% at 100 nM).     

Studies on nitrosyl hemes in Ni(II)–Fe(II) hybrid hemoglobins

Subunit heterogeneity within a particular subunit in hemoglobin A have been explored with electron paramagnetic resonance spectroscopy using the nitrosyl hemes in Ni-Fe hybrid Hb under various solution conditions. Our previous studies on the crystal structure of NiHb demonstrated the presence of subunit heterogeneity within alpha-subunit. To further cross check this hypothesis, we made a hybrid Hb in which either the alpha- or beta-subunit contains iron, which alone can bind to NO. By this way dynamic exchange between penta- and hexa-coordinated forms within a subunit was confirmed. Upon the addition of inositol hexa phosphate (IHP) to these hybrids, R to T state transition is observed for [alpha(2)(Fe-NO)beta(2)(Ni)] but such a direct transformation is less marked in [alpha(2)(Ni)beta(2)(Fe-NO)]. Hence the bond between N(epsilon) and Fe is fundamental to the structure-function relation in Hb, as the motion of this nitrogen triggers the vast transformation, which occurs in the whole molecule on attachment of NO.     

Membrane degradation, accumulation of Phosphatidic acid, stimulation of catalase activity and nuclear DNA fragmentation during 2,4-d-induced leaf senescence in mustard

We investigated 2,4-D-induced leaf senescence in young mustard seedlings. A set of morphometric, biochemical and molecular parameters were analyzed to characterize senescence markers. In accordance with earlier reports, chloroplast-membrane degradation marked the early phase of leaf senescence based on the analysis of the galactolipid fraction. Degradation of grana occurred earlier to that of the envelope, as revealed by the relative level of their specific galactolipids, namely, monogalactosyl diglyceride and digalactosyl diglyceride. Phospholipids showed extensive degradation resulting in the accumulation of lyso-derivatives of major phospholipids and phosphatidic acid (PA) in senescing leaves. Catalase activity was stimulated by 2,4-D and reflected scavenging of reactive oxygen species. Nuclear DNA degradation, a previously known death signal that represented a point of no return from progression of senescence, occurred late on the 4th day subsequent to 2,4-D supplementation. AgNO₃, an inhibitor of ethylene biosynthesis, inhibited leaf senescence by ca. 54% based on PA content Involvement of 2,4-D, ethylene and abscisic acid in leaf senescence is discussed in relation to hormonal interplay.