Growth Temperatures and Temperature Characteristics of Aeromonas

Six of the 13 Aeromonas hydrophila, 1 of 10 A. shigelloides, and none of 10 A. salmonicida were found to be psychrophiles. All of the rest of the strains were mesophiles. The mu values (temperature characteristics) could not be used to distinguish psychrophiles from mesophiles.     

Morphology,nutrition and physiology of Sphaerotilus discophorus

Summary By means of hay infusion-Fe(OH)₃–MnCO₃ enrichment cultures, 11 pure strains of the filamentous, sheathed bacterium, Sphaerotilus discophorus were isolated from streams, rivers and lakes. The morphology of the colonies, filaments and cells are shown in a series of photographs. The strains grew well in dilute organic media but not in many of the common bacteriological media. All strains required thiamin and biotin for growth. Glucose, mannitol, salicin, raffinose, glycerol and other compounds were suitable energy sources and peptone and casein hydrolysate were satisfactory nitrogen sources for growth. The temperature range for growth was 5–35°C and the pH range 6.0–8.6.The sudanophilic granules of S. discophorus are composed of poly--hydroxybutyric acid which may be as much as 40% of the dry weight of the cells. Growth with iron and manganese salts results in extensive deposition of oxides on these metals on the filaments. Chemical analyses of 60 hr cultures showed that the iron content of the filaments ranged from 4.25–7.10% and manganese from 1.12–1.43%, calculated on a dry weight basis. Resting cell suspensions oxidized a variety of sugars, sugar alcohols, amino acids and other compounds and concomitantly assimilated about 90% of these substrates. The properties of S. discophorus are compared with those of S. natans.Based in part on a thesis presented by the senior author in partial fulfillment of the requirements for the degree of Doctor of Philosophy, Washington State University, 1963.     

Aluminum Toxicity: A Case Study on Tobacco (<i>Nicotiana tabacum</i> L.)

Aluminum is an abundant metal in the earth’s crust that turns out to be toxic in acidic environments. Many plants are affected by the presence of aluminum at the whole plant level, at the organ level, and at the cellular level. Tobacco as a cash crop (Nicotiana tabacum L.) is a widely cultivated plant worldwide and is also a good model organism for research. Although there are many articles on Al-phytotoxicity in the literature, reviews on a single species that are economically and scientifically important are limited. In this article, we not only provide the biology associated with tobacco Al-toxicity, but also some essential information regarding the effects of this metal on other plant species (even animals). This review provides information on aluminum localization and uptake process by different staining techniques, as well as the effects of its toxicity at different compartment levels and the physiological consequences derived from them. In addition, molecular studies in recent years have reported specific responses to Al toxicity, such as overexpression of various protective proteins. Besides, this review discusses data on various organelle-based responses, cell death, and other mechanisms, data on tobacco plants and other kingdoms relevant to these studies.     

Preferential recognition of epitopes on peroxynitrite-modified alpha-2-macroglobulin by circulating autoantibodies in rheumatoid arthritis patients

Abstract

Autoimmune responses against post-translationally modified antigens are a hallmark of several autoimmune diseases. In this work, we have studied the changes in alpha-2-macroglobulin (α₂M) upon modification by peroxynitrite. Furthermore, we have evaluated the immunogenicity of modified α₂M in experimental rabbits and rheumatoid arthritis (RA) patients. Peroxynitrite-modified α₂M showed disturbed microenvironment and altered aromatic residues under UV and fluorescence studies. Aggregation, reduction in β-sheet content, production of nitrotyrosine and shift in amide I and II bands were observed in the modified α₂M by polyacrylamide gel electrophoresis besides CD and FTIR spectroscopic analysis. The exposure of hydrophobic clusters and changes in contact positions were observed in ANS and ThT binding assays. Immunological studies using ELISA showed peroxynitrite-modified α₂M as highly immunogenic producing high titre of specific antibodies in immunized rabbits. Cross-reactivity studies revealed the polyspecificity of the elicited antibodies. Direct binding ELISA and competitive inhibition studies confirmed the presence of circulating antibodies in the sera of RA patients having high specificity towards the peroxynitrite-modified α₂M as compared to the native α₂M. Sera from healthy (normal) human subjects showed lower binding with the native and modified protein. This study confirms that peroxynitrite induces structural modifications in α₂M and makes it immunogenic. The presence of neo-antigenic determinants on modified α₂M with enhanced binding for circulating autoantibodies in RA patients could offer new possibilities for diagnosis and etiopathology of the disease.

Communicated by Ramaswamy H. Sarma     

Single versus multiple bioreactor scale-up: economy for high-value products

The economy of scaling-up a bioreactor by increasing the number of units was investigated with respect to an integrated flowsheet. For the production of t-PA from animal cells, a base case flowsheet using a single large bioreactor was compared to a multiple bioreactor case. Simulation of the complete flowsheets for the two cases showed that a multiple bioreactor approach to scale-up increases the return of investment (ROI) of the base process by 122%. This enormous increase in ROI results from the smaller size of the downstream units compared to the base case, since downstream processing accounts for about 80% of the total cost for high value products like t-PA. Proper scheduling of the downstream units allowed sharing of the equipment by the bioreactors. A breakdown of the equipment purchase cost showed that cost related to cell culture equipment increased from 14% for the base case to about 37% for the multiple bioreactor case. The contribution from chromatography columns to the total equipment purchase cost, on the other hand, decreased from 52 to 33%.     

Methionine sulfoxide reductases are essential for virulence of Salmonella typhimurium

Production of reactive oxygen species represents a fundamental innate defense against microbes in a diversity of host organisms. Oxidative stress, amongst others, converts peptidyl and free methionine to a mixture of methionine-S- (Met-S-SO) and methionine-R-sulfoxides (Met-R-SO). To cope with such oxidative damage, methionine sulfoxide reductases MsrA and MsrB are known to reduce MetSOs, the former being specific for the S-form and the latter being specific for the R-form. However, at present the role of methionine sulfoxide reductases in the pathogenesis of intracellular bacterial pathogens has not been fully detailed. Here we show that deletion of msrA in the facultative intracellular pathogen Salmonella (S.) enterica serovar Typhimurium increased susceptibility to exogenous H(2)O(2), and reduced bacterial replication inside activated macrophages, and in mice. In contrast, a ΔmsrB mutant showed the wild type phenotype. Recombinant MsrA was active against free and peptidyl Met-S-SO, whereas recombinant MsrB was only weakly active and specific for peptidyl Met-R-SO. This raised the question of whether an additional Met-R-SO reductase could play a role in the oxidative stress response of S. Typhimurium. MsrC is a methionine sulfoxide reductase previously shown to be specific for free Met-R-SO in Escherichia (E.) coli. We tested a ΔmsrC single mutant and a ΔmsrBΔmsrC double mutant under various stress conditions, and found that MsrC is essential for survival of S. Typhimurium following exposure to H(2)O(2,) as well as for growth in macrophages, and in mice. Hence, this study demonstrates that all three methionine sulfoxide reductases, MsrA, MsrB and MsrC, facilitate growth of a canonical intracellular pathogen during infection. Interestingly MsrC is specific for the repair of free methionine sulfoxide, pointing to an important role of this pathway in the oxidative stress response of Salmonella Typhimurium.     

Mucoadhesive, thermosensitive, prolonged-release vaginal gel for clotrimazole:beta-cyclodextrin complex

The purpose of this study was to achieve a better therapeutic efficacy and patient compliance in the treatment for vaginitis. Clotrimazole (1%) has been formulated in a vaginal gel using the thermosensitive polymer Pluronic F127 (20%) together with mucoadhesive polymers such as Carbopol 934 and hydroxypropylmethylcellulose (0.2% for both). To increase its aqueous solubility, clotrimazole was incorporated as its inclusion complex with 1:1 molar ratio with beta-cyclodextrin. The inclusion complex was thoroughly characterized using various techniques, including 1H NMR spectroscopy, FT IR spectrophotometry, differential scanning calorimetry, scanning electron microscopy, phase solubility studies, and determination of stability constant (k(1:1)). The gelation temperature and rheological behavior of different formulations at varying temperatures were measured. In vitro release profiles of the gels were determined in pH 5.5 citrate buffer. It was observed that complexation with cyclodextrin slowed down the release of clotrimazole considerably. Carbopol 934, on the other hand, was found to interact with beta-cyclodextrin, inducing precipitation. As far as rheological properties are concerned, thermosensitive in situ gelling was obtained with formulations containing drug:cyclodextrin complex rather than with free drug. Thus, the optimum formulation for a controlled-release thermosensitive and mucoadhesive vaginal gel was determined to be clotrimazole:beta-cyclodextrin 1% with 0.2% hydroxypropylmethylcellulose in Pluronic F127 gel (20%) providing continuous and prolonged release of active material above MIC values.     

Thiol Peroxidase Protects Salmonella enterica from Hydrogen Peroxide Stress In Vitro and Facilitates Intracellular Growth

At present, Salmonella is considered to express two peroxiredoxin-type peroxidases, TsaA and AhpC. Here we describe an additional peroxiredoxin, Tpx, in Salmonella enterica and show that a single tpx mutant is susceptible to exogenous hydrogen peroxide (H₂O₂), that it has a reduced capacity to degrade H₂O₂ compared to the ahpCF and tsaA mutants, and that its growth is affected in activated macrophages. These results suggest that Tpx contributes significantly to the sophisticated defense system that the pathogen has evolved to survive oxidative stress.Salmonella is an important human pathogen which causes a variety of diseases, including gastroenteritis, septicemia, and typhoid fever. In the host, salmonellae reside inside phagocytic cells and are exposed to various host defense mechanisms, including oxidative stress (13). The production of superoxide anion (O₂⁻) is crucial, as individuals with chronic granulomatous disease, which is due to a defective phagocyte NADPH oxidase, are more susceptible to infections with Salmonella (10). Likewise, diminished NADPH oxidase activity leads to increased susceptibility to Salmonella in murine macrophages (20-22, 25). Superoxide anion (O₂⁻) is weakly reactive and fails to pass through the bacterial cell wall. After conversion to H₂O₂ by either spontaneous or enzymatic dismutation by superoxide dismutases, it readily diffuses into the bacterial cell and forms reactive hydroxyl radicals (OH) that damage macromolecules such as DNA, proteins, and lipids (12, 17).In principle, Salmonella possesses two classes of enzymes to degrade H₂O₂. Catalases degrade H₂O₂ to water and molecular oxygen independent of an additional reductant. Peroxiredoxin-type peroxidases (peroxiredoxins) reduce organic hydroperoxides to alcohols and hydrogen peroxide to water at the expense of NADH or NADPH. In a recent study by Hébrard et al., three members of the catalase family, KatG, KatE, and KatN, and two members of the peroxiredoxin family, AhpC and TsaA, were characterized in Salmonella (14). Previously it had been shown that single katE, katG, and katN Salmonella mutants did not show increased susceptibility to exogenous H₂O₂ (3, 24). In macrophages a katG katE katN triple mutant had no growth defect, whereas an ahpCF tsaA double mutant showed a reduced growth rate in macrophages (14). These observations point out the multiple routes that have evolved in Salmonella to protect the pathogen against oxidative stress and suggest that peroxiredoxins play a dominant role in the antioxidant defense during infection. In this study we characterized a third peroxiredoxin-type peroxidase, Tpx. Surprisingly, a simple tpx mutant of Salmonella enterica serovar Typhimurium (S. Typhimurium) was more susceptible to exogenous H₂O₂ than the wild type (WT). The mutant grew less well in activated macrophages and showed a reduced peroxidase activity toward H₂O₂.     

Identification of positive yield QTL alleles from exotic soybean germplasm in two backcross populations

Increasing seed yield is an important breeding goal of soybean [Glycine max (L.) Merr.] improvement efforts. Due to the small number of ancestors and subsequent breeding and selection, the genetic base of current soybean cultivars in North America is narrow. The objective of this study was to map quantitative trait loci (QTL) in two backcross populations developed using soybean plant introductions as donor parents. The first population included 116 BC(2)F(3)-derived lines developed using "Elgin" as the recurrent parent and PI 436684 as the donor parent (E population). The second population included 93 BC(3)F(3)-derived lines developed with "Williams 82" as the recurrent parent and PI 90566-1 as the donor parent (W population). The two populations were evaluated with 1,536 SNP markers and during 2?years for seed yield and other agronomic traits. Genotypic and phenotypic data were analyzed using the programs MapQTL and QTLNetwork to identify major QTL and epistatic QTL. In the E population, two yield QTL were identified by both MapQTL and QTLNetwork, and the PI 436684 alleles were associated with yield increases. In the W population, a QTL allele from PI 90566-1 accounted for 30?% of the yield variation; however, the PI region was also associated with later maturity and shorter plant height. No epistasis for seed yield was identified in either population. No yield QTL was previously reported at the regions where these QTL map indicating that exotic germplasm can be a source of new alleles that can improve soybean yield.