Multicomponent analysis of encapsulated marine oil supplements using high-resolution 1H and 13C NMR techniques

Multicomponent high-resolution 1H and 13C NMR analysis has been employed for the purpose of detecting and quantifying a wide range of fatty acids (as triacylglycerols or otherwise) in encapsulated marine cod liver oil supplements. The 1H NMR technique provided quantitative data regarding the docosahexaenoic acid content of these products, which serves as a valuable index of fish oil quality, and a combination of both 1H and 13C spectroscopies permitted the analysis of many further components therein, including sn-1 monoacylglycerols, sn-1,2 and -1,3 diacylglycerol adducts, together with a range of minor components, such as trans-fatty acids, free glycerol and cholesterol, and added vitamins A and E. The identities of each of the above agents were confirmed by the application of two-dimensional 1H-1H spectroscopies. The NMR techniques employed also uniquely permitted determinations of the content of nonacylglycerol forms of highly unsaturated (or other) fatty acids in these products (i.e., ethyl esters), and therefore served as a means of distinguishing "natural" sources of cod liver oils from those subjected to chemical modification to and/or supplementation with synthetic derivatives such as ethyl docosahexaenoate or eicosopentaenoate. The analytical significance and putative health effects of the results acquired are discussed.     

Binding of bilirubin with albumin-coupled liposomes: implications in the treatment of jaundice

In the present study, we demonstrated the suitability of liposomes as a method of removing plasma bilirubin in hyperbilirubinemic rats. The liposomes have innate tendency to bind with bilirubin through hydrophobic interaction. Among different types of liposomes, the positively charged liposomes were found to have maximum affinity to free bilirubin. However, the entrapment or coupling of serum albumin on the surface of egg phosphatidylcholine liposomes can render a several-fold increase in their bilirubin binding capacity. The proteoliposomes were able to preferentially bind with bilirubin even in the presence of erythrocytes. Interestingly, these liposomes were found to displace bilirubin bound on the surface of erythrocytes as well. The results of the present study further demonstrate that albumin-bearing liposomes were equally effective in removing plasma bilirubin in experimental jaundiced animals. These observations indicate that liposome-mediated selective homing of excess plasma bilirubin to the liver cells (cf. hepatocytes) may help in the development of safer strategy for the treatment of hyperbilirubinemic conditions in the model animals.     

Triterpenoids from the leaves of Psidium guajava

The resin of Commiphora kwo yielded two new octanordammarane triterpenes namely 15 alpha-hydroxymansumbinone and 28-acetoxy-15 alpha-hydroxymansumbinone, along with the four known compounds, mansumbinone, mansumbinol, (16S, 20R)-dihydroxydammar-24-en-3-one and T-cadinol. These structures were elucidated by spectroscopic techniques, including 1D and 2D NMR spectroscopy, and X-ray analysis.     

Cell-free replication of the hepatitis C virus subgenomic replicon

The hepatitis C virus (HCV) contains a plus-strand RNA genome. The 5' noncoding region (NCR) of the viral genome functions as an internal ribosome entry site, and its unique 3' NCR is required for the assembly of the replication complex during initiation of HCV RNA replication. Lohmann et al. (V. Lohmann, F. Korner, J.-O. Koch, U. Herian, L. Theilman, and R. Batenschlager, Science 285:110-113, 1999) developed a subgenomic HCV replicon system, which represents an important tool in studying HCV replication in cultured cells. In this study, we describe a cell-free replication system that utilizes cytoplasmic lysates prepared from Huh-7 cells harboring the HCV subgenomic replicons. These lysates, which contain ribonucleoprotein complexes associated with cellular membranes, were capable of incorporating [alpha(32)P]CTP into newly synthesized RNA from subgenomic replicons in vitro. Replicative forms (RFs) and replicative intermediates (RIs) were synthesized from the endogenous HCV RNA templates. Consistent with previous observations, RFs were found to be resistant to RNase A digestion, whereas RIs were sensitive to RNase treatment. The radiolabeled HCV RF-RI complexes contained both minus and plus strands and were specific to the lysates derived from replicon-expressing cells. The availability of a cell-free replication system offers opportunities to probe the mechanism(s) of HCV replication. It also provides a novel assay for potential therapeutic agents.     

Thermodynamic activation properties of elongation factor 2 (EF-2) proteins from psychrotolerant and thermophilic Archaea

In this study, the thermodynamic activation parameters of cold-adapted proteins from Archaeaa are described for the first time for the irreversible protein unfolding and ribosome-dependent GTPase activity of elongation factor 2 (EF-2) from the psychrotolerant Methanococcoides burtonii and the thermophilic Methanosarcina thermophila. Thermolability of Methanococcoides burtonii EF-2 was demonstrated by a low activation free-energy of unfolding as a result of low activation-enthalpy. Although structural data for EF-2 are presently limited to protein homology modeling, the observed thermodynamic properties are consistent with a low number of noncovvalent bonds or an altered solvent interaction, causing a loss of entropy during the unfolding process. A physiological concentration of potassium aspartate or potassium glutamate was shown to stabilize both proteins against irreversible denaturation by strengthening noncovalent interactions, as indicated by increased activation enthalpies. The transition state of GTPase activity for Methanococcoides burtonii EF-2 was characterized by a lower activation enthalpy than for Methanosarcina thermophila EF-2. The relative entropy changes could be explained by differential displacement of water molecules during catalysis, resulting in similar activation free energies for both proteins. The presence of solutes was shown to facilitate the breaking of enthalpy-driven interactions and structuring of more water molecules during the reaction. By studying the thermodynamic activation parameters of both GTPase activity and unfolding and examining the effects of intracellular solutes and partner proteins (ribosomes), we were able to identify enthalpic and entropic properties that have evolved in the archaeal EF-2 proteins to enable Methanococcoides burtonii and Methanosarcina thermophila to adapt to their respective thermal environments.     

Impact of biocontrol agents Pseudomonas fluorescens CHA0 and its genetically modified derivatives on the diversity of culturable fungi in the rhizosphere of mungbean

AIMS: To assess whether Pseudomonas fluorescens strain CHA0 and its genetically modified derivatives, CHA0/pME3424 (antibiotic over-producer) and CHA89 (antibiotic-deficient) could have an impact on the fungal community structure and composition in the rhizosphere of mungbean. METHODS AND RESULTS: Under glasshouse conditions, mungbean was grown repeatedly in the same soil, which was inoculated with CHA0, CHA0/pME3424, CHA89 or was left untreated. Treatments were applied to soil at the start of each 36-day mungbean growth cycle, and their effects on the diversity of the rhizosphere populations of culturable fungi were assessed at the end of the first, second and third cycles. The effects of CHA0 and CHA0/pME3424 did differ from the controls while CHA89 did not. Whereas all major fungal species were frequently isolated from both bacterized and nonbacterized rhizospheres, certain fungal species were exclusively promoted or specifically suppressed from Pseudomonas-treated soils. In general, fungal diversity and equitability tended to decrease with time while species richness slightly increased. Whilst a total of 29 fungal species were isolated from the mungbean rhizosphere, only eight species colonized the root tissues. CONCLUSIONS: Soil inoculation with Ps. fluorescens CHA0 or CHA0/pME3424 altered fungal community structure in mungbean rhizosphere but strain CHA89 failed to produce such effect. SIGNIFICANCE AND IMPACT OF THE STUDY: Pseudomonas fluorescens-mediated alteration in the composition and structure of fungal communities might have acute or lasting effects on ecosystem functioning. Furthermore, the study provides useful data pertinent to characterization of the fate of genetically modified inoculants (e.g. antibiotic-overproducing Pseudomonas strains) released into the environment.     

Dose-dependent protective effect of selenium in rat model of Parkinson's disease: neurobehavioral and neurochemical evidences

Normal cellular metabolism produces oxidants that are neutralized within cells by antioxidant enzymes and other antioxidants. An imbalance between oxidant and antioxidant has been postulated to lead the degeneration of dopaminergic neurons in Parkinson's disease. In this study, we examined whether selenium, an antioxidant, can prevent or slowdown neuronal injury in a 6-hydroxydopamine (6-OHDA) model of Parkinsonism. Rats were pre-treated with sodium selenite (0.1, 0.2 and 0.3 mg/kg body weight) for 7 days. On day 8, 2 micro L 6-OHDA (12.5 micro g in 0.2% ascorbic acid in normal saline) was infused in the right striatum. Two weeks after 6-OHDA infusion, rats were tested for neurobehavioral activity, and were killed after 3 weeks of 6-OHDA infusion for the estimation of glutathione peroxidase, glutathione-S-transferase, glutathione reductase, glutathione content, lipid peroxidation, and dopamine and its metabolites. Selenium was found to be successful in upregulating the antioxidant status and lowering the dopamine loss, and functional recovery returned close to the baseline dose-dependently. This study revealed that selenium, which is an essential part of our diet, may be helpful in slowing down the progression of neurodegeneration in parkinsonism.     

Non-pathogenic Fusarium solani represses the biosynthesis of nematicidal compounds in vitro and reduces the biocontrol of Meloidogyne javanica by Pseudomonas fluorescens in tomato

AIMS: The aim of the present investigation was to determine the influence of various Fusarium solani strains on the production of nematicidal agent(s) in vitro and biocontrol of Meloidogyne javanica in tomato by Pseudomonas fluorescens strains CHA0 and CHA0/pME3424. METHODS AND RESULTS: Culture filtrates (CF) of P. fluorescens strain CHA0 and its diacetylphloroglucinol-overproducing derivative CHA0/pME3424 caused substantial mortality of M. javanica juveniles in vitro. Bacterial growth medium amended with the growth medium of F. solani repressed the nematicidal activity of the bacteria. Methanol extract of F. solani CF resulting from Czapek's Dox liquid (CDL) medium without zinc amendment repressed the nematicidal activity of the bacteria while the CF obtained from CDL medium amended with zinc did not. Conidial suspension of F. solani strain Fs5 (repressor strain for the biosynthesis of nematicidal compounds in P. fluorescens) reduced biocontrol potential of the bacterial inoculants against M. javanica in tomato while strain Fs3 (non-repressor) did not. CONCLUSIONS: Fusarium solani strains with increased nematicidal activity repress the biosynthesis of nematicidal compounds by P. fluorescens strains in vitro and greatly alter its biocontrol efficacy against root-knot nematode under natural conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: Fusarium solani strains are distributed worldwide and found in almost all the agricultural fields which suggest that some mycotoxin-producing strains will also be found in almost any soil sample taken. Besides the suppressive effect of these metabolite-producing strains on the production of nematicidal compound(s) critical in biocontrol, F. solani strains may also affect the performance of mycotoxin-sensitive biocontrol bacteria effective against plant-parasitic nematodes.     

Angiogenic effects of dual gene transfer of bFGF and PDGF-BB after myocardial infarction

Therapeutic effects of combination of angiogenic growth factors for the treatment of ischemia after myocardial infarction are largely unknown. Plasmids expressing basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF-BB) or their combination with a 1:1 mass ratio were injected into hearts with 7-day-old myocardial infarction. Hearts were harvested after 1 and 4 weeks after gene transfer. The major findings in this chronic myocardial infarction model were that bFGF, PDGF-BB and their combination all had a more pronounced angiogenic effect on the arteriolar than the capillary level. bFGF stimulated both capillary and arteriolar growth while PDGF-BB preferentially stimulated arterioles. The combination increased the amount of both capillaries and arterioles and in addition gave rise to stable capillaries compared to single factor transfer but did not further enhance angiogenesis. No cardiovascular side effects were observed after gene transfer.