Control of Aspergillus niger infection in varieties of Arachis hypogeae L. by supplementation of zinc ions during seed germination

Three varieties of Arachis hypogeae, GG 11, GG 20 and GG 24, were compared for resistance against A. niger. GG 20 showed the least disease severity. Infection with A. niger resulted in a rapid increase in NADPH oxidase, Glutathione reductase (GR) and salicylic acid in all the three varieties, indicating hyper increase of reactive oxygen species (ROS) and activation of phenyl propanoid pathway. Ferric reducing antioxidant power value was found to be decreasing due to infection in all the three varieties, confirming the role of ROS in pathogenesis. Since A. niger was found to cause pathogenesis by oxidative stress, the treatment of zinc was given as an antioxidant and its effect was studied. The application of zinc inhibited NADPH oxidase and GR activity in the control as well as in the infected GG 11 and GG 24 varieties but induced in the tolerant variety GG 20. Because zinc treatment could control the ROS in GG 11 and GG 24 varieties, disease severity was reduced but in GG 20 variety, zinc treatment aggravated ROS levels and also the disease severity. The protein profile of GG 20 in comparison to GG 11 and GG 24 varieties revealed one oligomeric protein of 110 kD as one of the responsible factors for its resistance. Total oil and its iodine value were found little higher in GG 20 variety than in other two varieties. It was found that the control of ROS could control the A. niger infection in Arachis hypogeae.     

Free radical mechanisms in enzyme reactions

Free radicals are formed in prosthetic groups or amino acid residues of certain enzymes. These free radicals are closely related to the activation process in enzyme catalysis, but their formation does not always result in the formation of substrate free radicals as a product of the enzyme reactions. The role of free radicals in enzyme catalysis is discussed.     

Measurement of 7-dehydrocholesterol and cholesterol in hair can be used in the diagnosis of Smith-Lemli-Opitz syndrome

7-dehydrocholesterol (7-DHC) and cholesterol (CHOL) are biomarkers of Smith-Lemli-Opitz Syndrome (SLOS), a congenital autosomal recessive disorder characterized by elevated 7-DHC level in patients. Hair samples have been shown to have great diagnostic and research value, which has long been neglected in the SLOS field. In this study, we sought to investigate the feasibility of using hair for SLOS diagnosis. In the presence of antioxidants (2,6-ditert-butyl-4-methylphenol and triphenylphosphine), hair samples were completely pulverized and extracted by micro-pulverized extraction in alkaline solution or in n-hexane. After microwave-assisted derivatization with N,O-Bis(trimethylsilyl)trifluoroacetamide, the analytes were measured by GC-MS. We found that the limits of determination for 7-DHC and CHOL were 10 ng/mg and 8 ng/mg, respectively. In addition, good linearity was obtained in the range of 50–4000 ng/mg and 30–6000 ng/mg for 7-DHC and CHOL, respectively, which fully meets the requirement for SLOS diagnosis and related research. Finally, by applying the proposed method to real hair samples collected from 14 healthy infants and two suspected SLOS patients, we confirmed the feasibility of hair analysis as a diagnostic tool for SLOS. In conclusion, we present an optimized and validated analytical method for the simultaneous determination of two SLOS biomarkers using human hair.     

Identification of the coding region for the putidaredoxin reductase gene from the plasmid of Pseudomonas putida

The first 12 NH₂-terminal amino acids of the Pseudomonas putida putidaredoxin reductase were shown to be Met-Asn-Ala-Asn-Asp-Asn-Val-Val-Ile-Val-Gly-Thr. Comparison of these data with the DNA sequence of the BamHI-HindIII 197-base fragment derived from the PstI 2.2-kb fragment obtained from the P. putida plasmid showed that the putidaredoxin reductase gene was downstream from the cytochrome P-450 gene and the intergenic region had the 24-nucleotide sequence TAAACACATGGGAGTGCGTGCTAA. The Shine-Dalgarno sequence GGAG was detected in this region. The initiating triplet for the reductase gene was GTG, which normally codes for valine, but in the initiating codon position codes for methionine. From the amino acid sequence and X-ray data comparisons with other flavoproteins, what appears to be the AMP binding region of the FAD can be recognized in the NH₂-terminal portion of the reductase involving residues 5–35.This article was presented during the proceedings of the International Conference on Macromolecular Structure and Function, held at the National Defence Medical College, Tokorozawa, Japan, December 1985.     

Saturation and Utilization of Nitrate Pools in Pea and Sugar Beet Leaves

The critical periods in the saturation of pea and sugar beet leaves with nitrate absorbed by roots were discriminated. In peas, during the first 14 h, all nitrate penetrating leaf cells was concentrated in the cytosol (metabolic pool). During the second period (14–62 h), nitrate began to flow into the vacuole (storage pool), and the filling of the metabolic pool continued. Metabolic pool was saturated by the end of this period (62 h). During the third period (62–110 h), further nitrate accumulation in the cell occurred because of expanding of the storage pool. Its saturation (similarly as total cell saturation) commenced 86 h after the start of nitrate uptake. In sugar beet leaves, both metabolic and storage nitrate pools were saturated by the end of the first period (14 h), and the sizes of these pools did not change during the second period (14–86 h). When pea plants were transferred to the nitrate-free medium, nitrate efflux began from the storage pool until its complete exhausting after 3 days. In sugar beet leaves, nitrate was still present in the storage pool 4 days after plant transfer to the nitrate-free medium. In both crops, nitrate export from the storage pool was aimed at the maintenance of the optimum nitrate concentration in the metabolic pool and, thus, at the maintenance of nitrate reductase activity. A functional diversity of nitrate compartmentation in the cells of various plant species is discussed.     

Occurrence of two structural types of mercury reductases among Gram-positive bacteria

Structural variants of mercury reductase containing the N-terminal domain, which is easily cleaved by trypsin, have been found in Gram-positive bacteria with a low genomic G + C content (Bacillus, Staphylococcus and, possibly, some other genera). Mercury reductases without the N-terminal domain and relatively resistant to limited proteolysis are typical for Gram-positive bacteria with a high genomic G + C content (Arthrobacter, Citreobacterium, Micrococcus, Mycobacterium, Rhodococcus). Both types of mercury reductase genes may be located on plasmids.     

Nitrosating activity in Escherichia coli

Nitrosation activity was measured in Escherichia coli isolates and a range of nitrite reductase (nir) mutants. Activity was only detected in intact cells and could be inhibited by a number of treatments such as sonication and osmotic shock. Aerobically-grown cells had highest nitrosation activity compared to oxygen-limited ones. Inclusion of nitrite in growth media induced high activities of nitrite reductase and for some isolates, nitrosation. Analysis of nir mutants identified two which were unable to nitrosate. This result suggested that NADH-dependent nitrite reductase was implicated either directly or indirectly in nitrosation.     

An integrated network analysis reveals that nitric oxide reductase prevents metabolic cycling of nitric oxide by Pseudomonas aeruginosa

Nitric oxide (NO) is a chemical weapon within the arsenal of immune cells, but is also generated endogenously by different bacteria. Pseudomonas aeruginosa are pathogens that contain an NO-generating nitrite (NO₂⁻) reductase (NirS), and NO has been shown to influence their virulence. Interestingly, P. aeruginosa also contain NO dioxygenase (Fhp) and nitrate (NO₃⁻) reductases, which together with NirS provide the potential for NO to be metabolically cycled (NO→NO₃⁻→NO₂⁻→NO). Deeper understanding of NO metabolism in P. aeruginosa will increase knowledge of its pathogenesis, and computational models have proven to be useful tools for the quantitative dissection of NO biochemical networks. Here we developed such a model for P. aeruginosa and confirmed its predictive accuracy with measurements of NO, O₂, NO₂⁻, and NO₃⁻ in mutant cultures devoid of Fhp or NorCB (NO reductase) activity. Using the model, we assessed whether NO was metabolically cycled in aerobic P. aeruginosa cultures. Calculated fluxes indicated a bottleneck at NO₃⁻, which was relieved upon O₂ depletion. As cell growth depleted dissolved O₂ levels, NO₃⁻ was converted to NO₂⁻ at near-stoichiometric levels, whereas NO₂⁻ consumption did not coincide with NO or NO₃⁻ accumulation. Assimilatory NO₂⁻ reductase (NirBD) or NorCB activity could have prevented NO cycling, and experiments with ΔnirB, ΔnirS, and ΔnorC showed that NorCB was responsible for loss of flux from the cycle. Collectively, this work provides a computational tool to analyze NO metabolism in P. aeruginosa, and establishes that P. aeruginosa use NorCB to prevent metabolic cycling of NO.