Molecular metabolic fingerprinting approach to investigate the effects of borneol on metabolic alterations in the liver of nitric oxide deficient hypertensive rats

Hypertension is one of the major risk factor that underlie a wide range of cardiovascular irregularities which causes functional and metabolic alterations in vascular system and major organs. Nitric oxide is the central regulator of the vascular system and its deficiency leads to increased blood pressure and metabolic alterations in liver. Fourier transform infrared spectroscopy (FTIR) is a vibrational spectroscopic technique that uses infrared radiation to vibrate molecular bonds with in the sample that absorbs it and different samples contain diverse configurations of molecular bonds. Both wavenumber and area of the vibrational spectra can be used to explore the qualitative and quantitative constituent of macromolecules. In this study, we intended to evaluate the protective role of borneol, a natural terpene on liver metabolism in a nitric oxide deficient model of hypertension through interpretation of FTIR spectral information. Results demonstrate that FTIR can successfully indicate the molecular changes that occur in all groups. The over all findings demonstrate that in nitric oxide deficient animal model of hypertension, the liver metabolic program is altered through increasing the structural modification in proteins and triglycerides, and quantitative alteration in proteins, lipids, and glycogen. All the above mentioned modifications were protected by borneol in liver and showed its ability to exert a novel defensive action on hepatic metabolism.     

Expression of the bacteriophage T4 lysozyme gene in tall fescue confers resistance to gray leaf spot and brown patch diseases

Tall fescue (Festuca arundinacea Schreb.) is an important turf and forage grass species worldwide. Fungal diseases present a major limitation in the maintenance of tall fescue lawns, landscapes, and forage fields. Two severe fungal diseases of tall fescue are brown patch, caused by Rhizoctonia solani, and gray leaf spot, caused by Magnaporthe grisea. These diseases are often major problems of other turfgrass species as well. In efforts to obtain tall fescue plants resistant to these diseases, we introduced the bacteriophage T4 lysozyme gene into tall fescue through Agrobacterium-mediated genetic transformation. In replicated experiments under controlled environments conducive to disease development, 6 of 13 transgenic events showed high resistance to inoculation of a mixture of two M. grisea isolates from tall fescue. Three of these six resistant plants also displayed significant resistance to an R. solani isolate from tall fescue. Thus, we have demonstrated that the bacteriophage T4 lysozyme gene confers resistance to both gray leaf spot and brown patch diseases in transgenic tall fescue plants. The gene may have wide applications in engineered fungal disease resistance in various crops.     

A molecular and in silico characterization of Hev b 4, a glycosylated latex allergen

Hev b 4 is a heavily glycosylated latex allergen with seven attached N-glycans, comprising of both oligomannose and complex type structures. Treatment with a mixture of N-glycosidase A and N-glycosidase F resulted in lowering Hev b 4 protein on SDS-gel from 53 to 55kDa to circa 40kDa, this being comparable to the 38.53kDa mass predicted by its cDNA. In Western-immunoblots, the enzymatically deglycosylated Hev b 4 showed negligible binding to IgE from latex allergic patients; the results indicated that IgE essentially binds to Hev b 4 via its N-glycan moiety. Structural modelling of the Hev b 4 was carried out based on the template protein and carbohydrate crystal coordinates of rhamnogalacturonan acetylesterase (PDB ID 1DEO). We managed to link four N-glycan structures on to the Hev b 4 model; the glycans were scattered over the surface of the model. The structural and functional features of Hev b 4 could prove useful to elucidate its exposed epitopes which are important for IgE binding.     

Expression Enhancement of a Rice Polyubiquitin Gene Promoter

An 808 bp promoter from a rice polyubiquitin gene, rubi3, has been isolated. The rubi3 gene contained an open reading frame of 1140 bp encoding a pentameric polyubiquitin arranged as five tandem, head-to-tail repeats of 76 aa. The 1140 bp 5′ UTR intron of the gene enhanced its promoter activity in transient expression assays by 20-fold. Translational fusion of the GUS reporter gene to the coding sequence of the ubiquitin monomer enhanced GUS enzyme activity in transient expression assays by 4.3-fold over the construct containing the original rubi3 promoter (including the 5′ UTR intron) construct. The enhancing effect residing in the ubiquitin monomer coding sequence has been narrowed down to the first 9 nt coding for the first three amino acid residues of the ubiquitin protein. Mutagenesis at the third nucleotide of this 9 nt sequence still maintains the enhancing effect, but leads to translation of the native GUS protein rather than a fusion protein. The resultant 5′ regulatory sequence, consisting of the rubi3 promoter, 5′ UTR exon and intron, and the mutated first 9 nt coding sequence, has an activity nearly 90-fold greater than the rubi3 promoter only (without the 5′ UTR intron), and 2.2-fold greater than the maize Ubi1 gene promoter (including its 5′ UTR intron). The newly created expression vector is expected to enhance transgene expression in monocot plants. Considering the high conservation of the polyubiquitin gene structure in higher plants, the observed enhancement in gene expression may apply to 5′ regulatory sequences of other plant polyubiquitin genes.     

Selective elimination of perennial ryegrass by activation of a pro-herbicide through engineering <Emphasis Type="Italic">E. coli argE</Emphasis> gene

Perennial ryegrass is widely used for overseeding dormant bermudagrass on golf courses and sports fields in Southeastern United States to provide green color and improved playability. Late spring and summer persistence of perennial ryegrass may decrease the quality of the bermudagrass turf and reduce its winter hardiness. To help solve this problem, we developed a strategy to activate a pro-herbicide within the transgenic perennial ryegrass plants and to cause self elimination of the plants. An E. coli argE gene was introduced into perennial ryegrass by the biolistic method, which resulted in four independently transformed green plants. The mRNA of argE gene was detected in three of the plants by RT-PCR. Perennial ryegrass plants expressing the argE transgene were selectively controlled upon application of a pro-herbicide, N-acetyl-l-phosphinothricin (or N-acetyl-PPT), since the N-acetylornithinase encoded by argE gene is able to convert N-acetyl-PPT to the herbicide phosphinothricin (PPT). The non-transgenic bermudagrass plants were unaffected by the treatment. This approach provides a means to selectively remove a group of transgenic plants without affecting other plants growing with them.     

A mutation in the Arabidopsis KT2/KUP2 potassium transporter gene affects shoot cell expansion

Potassium ions (K(+)) are the most abundant cations in plants and are necessary for cell growth. Arabidopsis shy3-1 mutant plants have a short hypocotyl, small leaves, and a short flowering stem, and these defects result from decreased cell expansion. The semidominant shy3-1 mutation changes an amino acid in KT2/KUP2, a K(+) transporter related to the Escherichia coli Kup protein. Second mutations in the KT2/KUP2/SHY3 gene, including presumed null mutations, suppress the shy3-1 phenotypes. Plants with these intragenic suppressor mutations appear similar to wild-type plants, suggesting that KT2/KUP2/SHY3 acts redundantly with other genes. Expression of the shy3-1 mutant version of KT2/KUP2/SHY3 in wild-type plants confers shy3-1-like phenotypes, indicating that shy3-1 probably either causes a gain of function or creates an interfering protein. The shy3-1 mutation does not eliminate the ability of the KT2/KUP2 cDNA to rescue the growth of a potassium transport-deficient E. coli mutant. A P(SHY3)::GUS fusion is expressed in growing portions of the plant. These results suggest that KT2/KUP2/SHY3 mediates K(+)-dependent cell expansion in growing tissues.     

Electron ionization gas chromatography-mass spectrometric determination of residues of thirteen pyrethroid insecticides in whole blood

A new rapid and sensitive electron ionization gas chromatography-mass spectrometry method in selective ion monitoring mode (SIM) was developed for the determination of l3 synthetic pyrethroid insecticide molecules and their stereo isomers in whole blood. The pyrethroid insecticides investigated are allethrin, bifenthrin, cypermethrin, cyphonothrin, cyfluthrin, lambda-cyhalothrin, deltamethrin, fenvalerate, fenpropathrin, imiprothrin, permethrin, prallethrin and transfluthrin. The residues of pyrethroids are extracted from the whole blood using hexane and acetone mixture (80 + 20%) as solvent. All the pyrethroid residues were separated by using a gas chromatography-mass spectrometry operated in electron ionization mode and quantified in selective ion monitoring mode. The method can detect the residues of different pyrethroids down to the level 0.05-2 ng/ml. Recovery experiments conducted in whole blood samples at the fortification level 1-1000 ng/ml showed 91-103% recovery. The applications of the analytical method for the determination of pyrethroid residues in real samples were tested by analyzing 45 human blood samples collected from the population exposed continuously to different pyrethroid based formulations. The results are confirmed by spiking the known quantity of pyrethroids and subsequently their positive detection.     

Protective Role of Quercetin on Polychlorinated Biphenyls (Aroclor‐1254) Induced Oxidative Stress and Apoptosis in Liver of Adult Male Rats

The present study aims to investigate the protective effect of quercetin against Aroclor‐1254–induced hepatotoxicity in rats. Male Wistar rats were grouped into Group I control received vehicle (corn oil; 1 mL/kg bwt); Group II quercetin alone (50 mg/kg bwt/day orally); Group III Aroclor‐1254 (2 mg/kg bwt/day intraperitoneally); Group IV Aroclor‐1254 + quercetin treated for 30 days. The Aroclor‐1254 treatment caused significant alteration in the biochemical parameters (hydrogen peroxide, lipid peroxidation, reduced glutathione levels, and alkaline phosphatase activity). The expressions of apoptotic and antiapoptotic proteins and the liver histology of Aroclor‐1254–exposed rats showed cytoplasmic degeneration along with infiltration of polymorphonuclear cells. Whereas simultaneous treatment with quercetin normalized all the biochemical parameters, consequently it inhibited apoptosis mediated by Aroclor‐1254 by downregulating aryl hydrocarbon receptor, p53 and apoptotic protein (Bax, caspase‐9, caspase‐3) and upregulating the antiapoptotic protein (Bcl‐2) expression patterns; thereby, quercetin reduces alteration in hepatocellular morphology. Thus quercetin exhibited hepatoprotective effect. © 2012 Wiley Periodicals, Inc. J BiochemMol Toxicol 26:522‐532, 2012; View this article online at wileyonlinelibrary.com . DOI 10.1002/jbt.21466