A spin labeling study of the effects of inorganic ions and pH on the conformation of spectrin

The structure of spectrin from human erythrocytes has been investigated by the EPR technique measuring the mobility of the protein spin label, 4-maleimido-2,2,6,6-tetramethylpiperidinooxyl. Conformational changes in the protein induced by variation of the concentrations of NaCl, Na2SO4, KCl, CaCl2 and MgCl2 and of pH have been studied. It could be demonstrated that both Ca2+ and Mg2+ give rise to structural changes by binding to specific sites, whereas the monovalent cations (K+, Na+) seem to act via ionic strength. A model is used to correlate the spin label mobility with the radius of the protein. In the Ca2+- and Mg2+-binding experiments, the decrease in the spin label mobility has been interpreted on the basis of the theory of multiple chemical equilibria. These experiments have been compared with EPR spectra measured at different pH values. The results support the model in that binding of H+, Ca2+ or Mg2+ reduces the charges located on the protein surface: the 'discharging' reduces the repulsive forces on the surface of the molecule and consequently, the protein contracts in discrete steps.     

Prevalence of gene sequences coding for hypervariable regions of Opa (protein II) in Neisseria gonorrhoeae

Opas (protein IIs) are a family of surface-exposed proteins of Neisseria gonorrhoeae. Each strain of N. gonorrhoeae has multiple (10-11) genes encoding for Opas. Identifiable elements in opa genes include the coding repeat within the signal sequence, conserve 5' and 3' regions, and hypervariable regions (HV1 and HV2) located within the structural gene. N. gonorrhoeae strains appear to have many biological properties in common that are either HV-region-mediated or associated with the presence of specific HV regions, suggesting that HV regions could be found in many clinical isolates. Oligonucleotides from three source strains representing three conserved regions of opa, 12 HV1 regions, and 14 HV2 regions were used by dot blot analysis to probe 120 clinical isolates of N. gonorrhoeae. The probe for the coding repeat hybridized to all 120 strains, the 3' conserved-region probe reacted with 98% of the strains, and the 5' conserved-region probe with 90% of the strains. Nine HV1 probes hybridized to 3.3-39.2% of the strains, and 13 of the HV2 probes hybridized to 1.7-25% of the isolates. Analysis of the number of probes that hybridized to each of the isolates showed that 19% did not hybridize with any of the HV1 probes and 25% did not hybridize with any of the HV2 probes. Approximately three-quarters of the isolates hybridized with one, two or three of the HV1 probes or one, two or three of the HV2 probes; 89% of the isolates hybridized to least one HV1 or one HV2 probe. The data indicate that some genes encoding HV regions of N. gonorrhoeae Opa proteins are widely distributed in nature.     

Kinetic properties of Serratia marcescens adenosine 5''-diphosphate glucose pyrophosphorylase.

The regulatory properties of partially purified adenosine 5'-diphosphate-(ADP) glucose pyrophosphorylase from two Serratia marcescens strains (ATCC 274 and ATCC 15365) have been studied. Slight or negligible activation by fructose-P2, pyridoxal-phosphate, or reduced nicotinamide adenine dinucleotide phosphate (NADPH) was observed. These compounds were previously shown to be potent activators of the ADPglucose pyrophosphorylases from the enterics, Salmonella typhimurium, Enterobacter aerogenes, Enterobacter cloacae, Citrobacter freundii, Escherichia aurescens, Shigella dysenteriae, and Escherichia coli. Phosphoenolpyruvate stimulated the rate of ADPglucose synthesis catalyzed by Serratia ADPglucose pyrophosphorylase about 1.5- to 2-fold but did not affect the S0.5 values (concentration of substrate required for 50% maximal stimulation) of the substrates, alpha-glucose-1-phosphate, and adenosine 5'-triphosphate. Adenosine 5'-monophosphate (AMP), a potent inhibitor of the enteric ADPglucose pyrophosphorylase, is an effective inhibitor of the S. marcescens enzyme. ADP also inhibits but is not as effective as AMP. Activators of the enteric enzyme counteract the inhibition caused by AMP. This is in contrast to what is observed for the S. marcescens enzyme. Neither phosphoenolpyruvate, fructose-diphosphate, pyridoxal-phosphate, NADPH, 3-phosphoglycerate, fructose-6-phosphate, nor pyruvate effect the inhibition caused by AMP. The properties of the S. marcescens HY strain and Serratia liquefaciens ADPglucose pyrophosphorylase were found to be similar to the above two S. marcescens enzymes with respect to activation and inhibition. These observations provide another example where the properties of an enzyme found in the genus Serratia have been found to be different from the properties of the same enzyme present in the enteric genera Escherichia, Salmonella, Shigella, Citrobacter, and Enterobacter.     

Diversity and symbiotic effectiveness of beta-rhizobia isolated from sub-tropical legumes of a Brazilian Araucaria Forest

While the occurrence of Betaproteobacteria occupying the nodules of tropical legumes has been shown, little is known about subtropical areas. Araucaria Forest is a subtropical endangered ecosystem, and a better understanding of the legume-rhizobial symbionts may allow their use in land reclamation. The 16S rRNA gene of bacteria isolated from nine leguminous species was sequenced and their nodulation tested in Mimosa scabrella and Phaseolus vulgaris. 196 isolates were identified as eight genotypes: Pantoea, Pseudomonas, Bradyrhizobium sp1-2, Rhizobium, and Burkholderia sp1-3. The majority of the isolates from native plants (87 %) were taxonomically related to β-rhizobia, namely Burkholderia, however the legumes Galactia crassifolia and Collea speciosa were nodulated by both α and β-rhizobia, and Acacia dealbata, an exotic plant, only by α-rhizobia. The nifH genes of some isolates were sequenced and N-fixing potential shown by the acetylene reduction test. Most of the isolates nodulated the test plants, some were effective in M. scabrella, but all presented low efficiency in the exotic promiscuous legume P. vulgaris. Pantoea and Pseudomonas did not nodulate and probably are endophytic bacteria. The presented data shows diversity of α, β and γ-Proteobacteria in nodules of subtropical legumes, and suggests host specificity with β-rhizobia. Potential isolates were found for M. scabrella, indicating that a high N-fixing strain may be further inoculated in plants for use in reforestation.     

Specific expression of the Drosophila midline-jumper retro-transposon in embryonic CNS midline cells

Here we describe of a novel Drosophila LTR-type retrotransposon that is expressed in the embryonic CNS midline glia and in the embryonic germ cells. The element is related to the gypsy and burdock retrotransposons and was termed midline-jumper. In addition to cDNA clones generated from internal retrotransposon sequences, we have identified one cDNA clone that appears to reflect a transposition event, indicating that the midline-jumper retrotransposon is not only transcribed but also able to transpose during Drosophila development.     

Trypanosoma cruzi: inhibition of metacyclogenesis by mannose.

Metacyclogenesis of Trypanosoma cruzi epimastigotes was evaluated in a medium supplemented with Triatoma infestans intestinal homogenate in the presence of sugars and derivates as are mannose, galactose, fucose, N-acetylglucosamine, mannose 6-P, and fructose 1,6-P at a concentration of 25 mM. Only mannose significantly inhibited metacyclogenesis. Sodium metaperiodate and trypsin treatment of the intestinal homogenate also inhibited differentiation. In our opinion there exists a proteinic factor in the intestine of the vector that promotes metacyclogenesis and is incorporated by the parasite. Treatment of the intestinal homogenate with alkaline phosphatase had no effect. Instead, high ionic strength in the medium (0.4 M NaCl) strongly inhibited metacyclogenesis indicating that, in these conditions, the possible binding of the differentiation factor to the parasite surface was inhibited.     

Use of a layered double hydroxide (LDH) to buffer nitrate in soil: long-term nitrate exchange properties under cropping and fallow conditions

The potential use of a layered double hydroxide (LDH) to act as a nitrate buffer system in soil in order to reduce the movement of nitrate was investigated. Long-term plant and soil experiments were carried out under greenhouse conditions with the following objectives: (i) evaluate the nitrate adsorption capacity of the LDH during crop growth, and its influence on N uptake, (ii) study the ability of the LDH to adsorb nitrate mineralized during fallow periods, and its influence on nitrate leaching, (iii) evaluate the reversibility for nitrate exchange of the LDH under cultivation conditions, and (iv) determine the nitrate buffer capacity of the soil after LDH application. The LDH adsorbed nitrate from the soil solution during the growth period without affecting plant N uptake. As a result of the adsorption of nitrate on the LDH, the nitrate-N concentration in the soil solution at harvest was reduced by a factor of ten compared to a soil without LDH. The LDH efficiently adsorbed nitrate that was mineralized in the soil during periods without cultivation, reduced nitrate-N leaching losses by about 80%, and kept this nitrate available for a following crop. The nitrate buffer capacity of the soil after 15months increased from 0.3 (without LDH) to 2.7 with the application of 10g LDH kg^?1 soil. It is concluded that the LDH has a potential to be used as a long-term nitrate exchanger to control the movement of nitrate in soil, and thereby reduce risks of nitrate leaching in crop production in sensible areas.