Agrobacterium-mediated production of transgenic pigeonpea (Cajanus cajan L.Millsp.) expressing the synthetic bt cry1Ab gene

Conventional breeding methods have not been very successful in producing pest-resistant genotypes of pigeonpea, due to the limited genetic variation in cultivated germplasm. We have developed an efficient method to produce transgenic plants of pigeonpea by incorporating the cry1Ab gene of Bacillus thuringiensis through Agrobacterium tumefaciens-mediated genetic transformation. The novel tissue culture protocol is based on the direct regeneration of adventitious shoot buds in the axillary bud region of in vitro germinating seedlings by suppressing the axillary and primary shoot buds on a medium containing a high concentration of N6-benzyladenine (22.0 µM). The tissue with potential to produce adventitious shoot buds can be explanted and used for co-cultivation with A. tumefaciens carrying the synthetic cry1Ab on a binary vector and driven by a CaMV 35S promoter. Following this protocol, over 75 independently transformed transgenic events of pigeonpea were produced and advanced to T2 generation. Amongst the recovered primary putative transformation events, 60% showed positive gene integration based on initial polymerase chain reaction (PCR) screening. PCR analysis of the progenies from independent transformants followed gene inheritance in a Mendelian ratio and 65% of the transformants showed the presence of single-copy inserts of the introduced genes. Reverse transcription-polymerase chain reaction analysis showed that the transcripts of the introduced genes were normally transcribed and resulted in the expression of Cry1Ab protein in the tested T2 generation plants. Interestingly, the content of Cry1Ab protein as a percent of total soluble protein varied in different tissues of the whole plant, showing the highest expression in flowers (0.1%) and least in the leaves (0.025%) as estimated by enzyme-linked immunosorbent assay. The transgenic plants produced in this study offer immense potential for the improvement of this important legume of the semi-arid tropics for resistance to insect pests

A Method for Isolation and Purification of Peanut Genomic DNA Suitable for Analytical Applications

Numerous methods are available for isolation of plant genomic DNA, but in practice these procedures are empirical due to variability in plant tissue composition. Consistent isolation of quality DNA from peanut (Arachis hypogaea L.) is particularly problematic due to the presence of phenolic compounds and polysaccharides. Inconsistencies in extraction results can be attributed to the age and growth stage of the plant material analyzed. Mature leaves have higher quantities of polyphenols, tannins, and polysaccharides that can contaminate DNA during isolation. We show that four published protocols could not be used to isolate peanut DNA of sufficient quality for PCR amplification or Southern hybridization. We have devised a new protocol that uses DEAE-cellulose purification to isolate peanut DNA useful for downstream applications

Diabetes alters vascular mechanotransduction: pressure-induced regulation of mitogen activated protein kinases in the rat inferior vena cava

BACKGROUND: Diabetes mellitus is an important risk factor for increased vein graft failure after bypass surgery. However, the cellular and molecular mechanism(s) underlying vessel attrition in this population remain largely unexplored. Recent reports have suggested that the pathological remodeling of vein grafts may be mediated by mechanically-induced activation of the mitogen activated protein kinase (MAPK) signaling pathways and the MAPK-related induction of caspase-3 activity. On the basis of these findings, we hypothesized that diabetes may be associated with alterations in how veins "sense" and "respond" to altered mechanical loading. METHODS: Inferior venae cavae (IVC) from the non-diabetic lean (LNZ) and the diabetic obese (OSXZ) Zucker rats were isolated and incubated ex vivo under basal or pressurized conditions (120 mmHg). Protein expression, basal activation and the ability of increased pressure to activate MAPK pathways and apoptosis-related signaling was evaluated by immunoblot analysis. RESULTS: Immunoblot analyses revealed differential expression and activation of extracellular signal-regulated kinase (ERK1/2), p38 and c-Jun NH2-terminal kinase (JNK) MAPKs in the IVCs of diabetic rats as compared to non-diabetic rats. In particular, the expression and basal phosphorylation of p38β- (52.3 ± 11.8%; 45.8 ± 18.2%), JNK 1- (21.5 ± 9.3%; 19.4 ± 11.6%) and JNK3-MAPK (16.8 ± 3.3%; 29.5 ± 17.6%) were significantly higher (P < 0.05) in the diabetic vena cava. An acute increase in IVC intraluminal pressure failed to increase the phosphorylation of ERK1-, JNK-2, or any of the p38-MAPKs in the diabetic obese Zucker rats. Also, IVC loading in the LNZ led to a 276.0 ± 36.0% and 85.8 ± 25.1% (P < 0.05) increase in the cleavage of caspase-3 and caspase-9, respectively, with no effect on these molecules in the OSXZ. No differences were found in the regulation of Bax and Bcl-2 between groups. However, basal expression levels of Akt, phospho-Akt, PTEN, phospho-PTEN and phospho-Bad were higher in the diabetic venae cavae (P < 0.05). CONCLUSION: These data suggest that diabetes is associated with significant alteration in the ability of the vena cava to activate MAPK- and apoptosis-related signaling. Whether these changes are associated with the increased vein graft attrition seen in the diabetic population will require further investigation

Altered Regulation of Contraction-Induced Akt/mTOR/p70S6k Pathway Signaling in Skeletal Muscle of the Obese Zucker Rat

Increased muscle loading results in the phosphorylation of the 70 kDa ribosomal S6 kinase (p70S6k), and this event is strongly correlated with the degree of muscle adaptation following resistance exercise. Whether insulin resistance or the comorbidities associated with this disorder may affect the ability of skeletal muscle to activate p70S6k signaling following an exercise stimulus remains unclear. Here, we compare the contraction-induced activation of p70S6k signaling in the plantaris muscles of lean and insulin resistant obese Zucker rats following a single bout of increased contractile loading. Compared to lean animals, the basal phosphorylation of p70S6k (Thr389; 37.2% and Thr421/Ser424; 101.4%), Akt (Thr308; 25.1%), and mTOR (Ser2448; 63.0%) was higher in obese animals. Contraction increased the phosphorylation of p70S6k (Thr389), Akt (Ser473), and mTOR (Ser2448) in both models however the magnitude and kinetics of activation differed between models. These results suggest that contraction-induced activation of p70S6k signaling is altered in the muscle of the insulin resistant obese Zucker rat

Chronic Paracetamol Treatment Influences Indices of Reactive Oxygen Species Accumulation in the Aging Fischer 344 X Brown Norway Rat Aorta

Previous reports have demonstrated that increased levels of reactive oxygen species (ROS) and alterations in cell signaling characterize aging in the Fischer 344 X Brown Norway (FBN) rat aorta. Other work has suggested that increases in ROS may be related to vascular wall thickening and the development of hypertension. Paracetamol (acetaminophen) is a potent antioxidant that has been found to diminish free radicals in ischemia-reperfusion studies. However, it remains unclear whether chronic paracetamol administration influences signaling or ROS accumulation in the aging aorta. FBN rats (27 months old; n=8) were subjected to 6 months of treatment with a therapeutic dose of paracetamol (30 mg/kg/day) and compared to age-matched untreated FBN rat controls (n=8). Compared to measurements in the aortae of 6-month old animals, tunica media thickness, tissue superoxide levels, and protein oxidation levels were 38 ± 7%, 92 ± 31%, and 7 ± 2% higher in the aortae of 33-month control animals (p ≤0.05). Chronic paracetamol treatment decreased tunica media thickness and the amount of oxidized protein by 13 ± 4% and 30 ± 1%, respectively (p ≤0.05). This finding of diminished aortic thickening was associated with increased phosphorylation (activation) of the mitogen activated protein kinases and diminished levels of the anti-apoptotic protein Bcl-2. Taken together, these data suggest that chronic paracetamol treatment may decrease the deleterious effects of aging in the FBN rat aorta

Crop biofortification through genetic engineering : present status and future directions

Global food system is failing to deliver adequate quantities of healthy, nutritionally balanced food, especially to the resource-poor underprivileged people leading to micronutrient malnutrition. The malnutrition of minerals (Fe, Zn) and vitamin A are major food-related primary health problem among populations of the developing world including India where there is a heavy dependence on cereal-based diets and limited access to meat, fruits and vegetables. Vitamin A deficiency (VAD) alone is significant from the public health point of view resulting in over 330,000 child deaths every year, and about 57% of preschoolers and their mothers having subclinical VAD leading to increased morbidity and risk of mortality. While therapeutic supplementation of vitamin A is currently being addressed through sponsored nutrition programmes, they are not sufficient in covering the affected populations. Biofortification of important crop plants through biotechnological applications is a cost-effective and sustainable solution for alleviating VAD. Genetic engineering is the obvious alternative to enhance the β-carotene levels in crop plants. The development of the ‘golden rice’ proved that, it is possible to redirect a complete biosynthetic pathway of carotenoids by genetic engineering of multiple genes encoding key enzymes of the pathway. Recently, there have been several reports on the development of transgenic crops for enhanced levels provitamin A content in crops like maize, tomato, cassava, potato and mustered. At ICRISAT, transgenic events of groundnut and pigeonpea carrying either a single maize phytoene synthase 1 (psy1) gene or both psy1 and tomato β-lycopene cyclase (β-lyc) have been developed through Agrobacterium-mediated genetic transformation. Preliminary results showed a significant increase in the total carotenoids and β-carotene levels in the transgenic events. Provitamin A enrichment of these crops could have a significant impact on the nourishment and nutrient interactions by playing a major role in the bioavailability and metabolic efficiency in the affected population

Acetaminophen prevents aging-associated hyperglycemia in aged rats: effect of aging-associated hyperactivation of p38-MAPK and ERK1/2

Background Aging-related hyperglycemia is associated with increased oxidative stress and diminished muscle glucose transporter-4 (Glut4) that may be regulated, at least in part, by the mitogen-activated protein kinases (MAPK). Methods To test the possibility that aging-related hyperglycemia can be prevented by pharmacological manipulation of MAPK hyperactivation, aged (27-month old) Fischer 344/NNiaHSD × Brown Norway/BiNia F1 (F344BN) rats were administered acetaminophen (30 mg/kg body weight/day) for 6 months in drinking water. Results Hepatic histopathology, serum aspartate aminotransferase and alanine aminotransferase analyses suggested that chronic acetaminophen did not cause hepatotoxicity. Compared with adult (6-month) and aged (27-month) rats, very aged rats (33-month) had higher levels of blood glucose, phosphorylation of soleus p38-MAPK and extracellular-regulated kinase 1/2 (ERK1/2), superoxide and oxidatively modified proteins (p \u3c 0.05), and these changes were associated with decreased soleus Glut4 protein abundance (p \u3c 0.05). Chronic acetaminophen treatment attenuated age-associated increase in blood glucose by 61.3% (p \u3c 0.05) and increased soleus Glut4 protein by 157.2% (p \u3c 0.05). These changes were accompanied by diminished superoxide levels, decrease in oxidatively modified proteins (−60.8%; p \u3c 0.05) and reduced p38-MAPK and ERK1/2 hyperactivation (−50.4% and − 35.4%, respectively; p \u3c 0.05). Conclusions These results suggest that acetaminophen may be useful for the treatment of age-associated hyperglycemia

Acetaminophen Improves Protein Translational Signaling in Aged Skeletal Muscle

Background: Age-related muscle atrophy is characterized by increased oxidative stress, diminished Akt enzymatic function, and reduced phosphorylation of the mammalian target of rapamycin (mTOR), which can be attenuated by chronic acetaminophen ingestion. Here we hypothesize that age-related impairments in Akt/ mTOR function are associated with reduced protein translational signaling, and that these changes, if present, can be attenuated by acetaminophen treatment. Results: Compared to 6- and 27-month old animals, the expression of the mTOR-complex proteins raptor and GbL and the phosphorylation of tuberin/TSC2 (Thr1462) were reduced in the soleus muscles of very aged rats (33 months old). These changes in Akt/mTOR pathway signaling proteins were in turn associated with decreased phosphorylation of S6 kinase p85S6K (Thr412) and eukaryotic translation initiation factor-4E (eIF4E) binding protein-1 (4EBP1, Thr37/46), reduced phosphorylation of S6 ribosomal protein (Ser235/236), and increased inhibition of eIF4E by binding to 4EBP1. Age-associated alterations in the Akt/mTOR pathway signaling and in the phosphorylation of the stress-responsive eIF2a protein were attenuated by chronic acetaminophen treatment (30 mg/kg body weight per day). Ex vivo incubation of adult muscles with hydrogen peroxide mimicked the age-related decreases seen in eIF4E and 4EBP1 phosphorylation, whereas the inclusion of acetaminophen in the muscle bath attenuated this effect. Conclusion: Aging is associated with impairments in the regulation of proteins thought to be important in controlling mRNA translation, and acetaminophen may be useful for the treatment of age-related muscle atrophy by reducing oxidative stress

Application of Poly(amidoamine) Dendrimers for Use in Bionanomotor Systems

The study and utilization of bionanomotors represents a rapid and progressing field of nanobiotechnology. Here, we demonstrate that poly(amidoamine) (PAMAM) dendrimers are capable of supporting heavy meromyosin dependent actin motility of similar quality to that observed using nitrocellulose, and that microcontact printing of PAMAM dendrimers can be exploited to produce tracks of active myosin motors leading to the restricted motion of actin filaments across a patterned surface. These data suggest that the use of dendrimer surfaces will increase the applicability of using protein biomolecular motors for nanotechnological applications

Diabetes Alters Contraction-Induced Mitogen Activated Protein Kinase Activation in the Rat Soleus and Plantaris

The prescription of anaerobic exercise has recently been advocated for the management of diabetes; however exercise-induced signaling in diabetic muscle remains largely unexplored. Evidence from exercise studies in nondiabetics suggests that the extracellular-signal-regulated kinases (Erk1/2), p38, and c-JUN NH2-terminal kinase (Jnk) mitogen-activated protein kinases (MAPKs) are important regulators of muscle adaptation. Here, we compare the basal and the in situ contraction-induced phosphorylation of Erk1/2- p38- and Jnk-MAPK and their downstream targets (p90rsk and MAPKAP-K2) in the plantaris and soleus muscles of normal and obese (fa/fa) Zucker rats. Compared to lean animals, the time course and magnitude of Erk1/2, p90rsk and p38 phosphorylation to a single bout of contractile stimuli were greater in the plantaris of obese animals. Jnk phosphorylation in response to contractile stimuli was muscle-type dependent with greater increases in the plantaris than the soleus. These results suggest that diabetes alters intramuscular signaling processes in response to a contractile stimulus