3. Technologies for utilizing biological nitrogen fixation in wetland rice: Potentialities,current usage,and limiting factors

Almost all types of N₂-fixing microorganisms are found in lowland rice fields. The resulting N fertility has permitted moderate but constant productivity in fields where no N fertilizer is applied. Current and potential technologies for utilizing biological N₂ fixation in lowland rice production are reviewed in terms of potential, current usage, and limiting factors.Legumes and azolla have been traditionally used as green manure in parts of Asia, permitting yields of 2–4 t/ha. To a limited extent, straw incorporation favors heterotrophic biological N₂ fixation. Recently, inoculation with blue-green algae has been claimed to increase yields by about 10%. Using non-symbiotic N₂-fixing systems is still experimental.Utilization of biological N₂ fixation as an alternative or additional N source for rice is severely limited by technological, environmental, and socioeconomical factors.     

Strategy for the detection of Helicobacter species by amplification of 16S rRNA genes and identification of H. felis in a human gastric biopsy

The aim of the present work was to develop polymerase chain reactions (PCRs) based on the conserved nucleotide sequence of the 16S rRNA gene for detection of bacteria of the Helicobacter genus in human antral biopsy samples. The assay for Helicobacter spp was developed by amplifying a 399-bp 16S rRNA gene sequence specific to the genus Helicobacter. The identity of the amplicon was confirmed by hybridization with an internal probe and by restriction by endonuclease VspI showing two expected fragments of 295 and 104 base pairs. A total of 65 dyspeptic patients from France and New Caledonia were screened for Helicobacter spp infection through the use of the following diagnostic assays on biopsy specimens collected through endoscopy: direct detection of bacteria in histological sections by Giemsa and Warthin Starry staining, urease test and bacterial isolation, PCR for Helicobacter pylori ureC/glmM gene, and PCR targeted to 16S rRNA genes. The 16S rRNA gene PCR assay was able to detect down to 680 bacterial cells, as assessed by agarose gel electrophoresis, and down to 4 bacterial cells by hybridization of amplicon with the internal probe. The 16S rRNA PCR test was 100% specific and sensitive; results obtained with this test were in agreement with the visualization of bacteria by histology. Urease test and culture were 86.4% and 22.7% sensitive, and 96.5 and 100% specific, respectively. The H. pylori ureC/glmM gene-based PCR was 100% specific and only 95.4% sensitive, since one biopsy from a Melanesian patient contained a Helicobacter strain other than H. pylori. For this Melanesian patient, a branch-specific PCR targeting the epsilon branch of Proteobacteria was used to amplify a 967-bp amplicon. This amplicon was sequenced and matched with the H. felis sequence. This was confirmed using an H. felis-specific urease PCR test.     

Characterization of a synthetic 37-residue fragment of a monoclonal antibody against herpes virus by capillary electrophoresis/electrospray (ionspray) mass spectrometry and 252Cf plasma desorption mass spectrometry

A peptide comprising 37 amino acids of the antigen binding site of a monoclonal antibody directed against glycoprotein D of herpes simplex virus was synthesized. The synthetic peptide and the impurities formed in the synthesis were characterized by capillary electrophoresis/ionspray mass spectrometry and by 252Cf plasma desorption-time of flight mass spectrometry. The measured average molecular mass of the synthetic peptide was 4627.16 Da, which was only 0.08 Da higher than the calculated value (4627.08 Da). The plasma desorption mass spectrum of the synthetic peptide showed a protonated molecule at m/z 4624.1, which was 4 Da lower than the calculated one (4628.09 Da). The amino acid sequence of the peptide was confirmed in part by electrospray (ionspray) mass spectrometry using a high nozzle skimmer voltage difference. Five impurities were separated and identified by capillary electrophoresis/mass spectrometry and two of them also appeared in the plasma desorption mass spectrum.     

Consequences of mutations to the phosphorylation site of the alpha-subunit of Na, K-ATPase for ATP binding and E1-E2 conformational equilibrium

Expression of Na, K-ATPase in yeast allowed targeting of alpha beta-units with lethal substitutions at the phosphorylation site alpha 1 (D369N) beta 1 and alpha 1 (D369A) beta 1 at the cell surface at the same concentration of alpha-subunit and [3H] ouabain binding sites as for wild type Na, K-ATPase. Phosphorylation and reaction with vanadate were abolished, and the mutations had no Na, K-ATPase or K-phosphatase activity. Binding of [3H]-ATP at equilibrium revealed an intrinsic high affinity of the D369A mutation for ATP (KD = 2.8 nM) that was 39-fold higher than for wild type Na, K-ATPase (KD = 109 nM). The affinities for ADP were unaffected, indicating that the negative charge at residue 369 determines the contribution of the gamma-phosphate to the free energy of ATP binding. Analysis of the K(+)-ATP antagonism showed that the reduction of charge and hydrophobic substitution at Asp369 of the alpha-subunit caused a large shift in conformational equilibrium toward the E2-form. This was accompanied by a large increase in affinity for [3H] ouabain in Mg2+ medium with KD = 4.9 nM for D369A compared to KD = 51 nM for D369N and KD = 133 nM for wild type, and [3H] ouabain binding (KD = 153 nM) to D369A was detectable even in absence of Mg2+. In addition to its function as receptor of the gamma-phosphate of ATP, Asp369 has important short-range catalytic functions in modulating the affinity for ATP and long-range functions in governing the E1-E2 transitions which are coupled to reorientation of cation sites and changes in affinity for digitalis glycosides.     

Biliary reconstruction for liver transplantation and management of biliary complications: overview and survey of current practices in the United States

Vitamin E (alpha-tocopherol), a well known naturally occurring chain breaking antioxidant and a free radical scavenger was found to exacerbate nickel (Ni) toxicity in mice. Vitamin E (Vit. E) mediated enhancement of nickel toxicity was demonstrated by (i) enhanced mortality in mice treated with Ni and Vit. E (ii) increased hepatic lipid peroxidation, (iii) increased rate of benzoate hydroxylation, and (iv) liposomal membrane damage.     

Investigation of a synthetic peptide as immunogen for a variant epidermal growth factor receptor associated with gliomas

We have previously demonstrated antibody production to a glioma-associated variant form of the human epidermal growth factor receptor in rabbits that had received a synthetic peptide mimicking the unique primary structure of the variant protein as immunogen. We report here the response of mice, rabbits, goats, and macaques immunized by various protocols to this peptide. Titers to both peptide- and cell-elaborated variant receptor were measured, and the capacity to recognize the variant receptor in human tumor samples was determined. Within the range of species and strains investigated, we demonstrated a variable species-associated response to the peptide (rabbits > mice > goats > rats > macaques). Rabbits and a single goat produced specific, high titer antibody activity to the variant receptor protein following immunization with peptide alone. Murine titers to the parent protein were not appreciable following peptide immunization alone; additional immunization with variant receptor as expressed on cell membranes was used to boost this response.