GPD1, which encodes glycerol-3-phosphate dehydrogenase, is essential for growth under osmotic stress in Saccharomyces cerevisiae, and its expression is regulated by the high-osmolarity glycerol response pathway.

The yeast Saccharomyces cerevisiae responds to osmotic stress, i.e., an increase in osmolarity of the growth medium, by enhanced production and intracellular accumulation of glycerol as a compatible solute. We have cloned a gene encoding the key enzyme of glycerol synthesis, the NADH-dependent cytosolic glycerol-3-phosphate dehydrogenase, and we named it GPD1. gpd1 delta mutants produced very little glycerol, and they were sensitive to osmotic stress. Thus, glycerol production is indeed essential for the growth of yeast cells during reduced water availability. hog1 delta mutants lacking a protein kinase involved in osmostress-induced signal transduction (the high-osmolarity glycerol response [HOG] pathway) failed to increase glycerol-3-phosphate dehydrogenase activity and mRNA levels when osmotic stress was imposed. Thus, expression of GPD1 is regulated through the HOG pathway. However, there may be Hog1-independent mechanisms mediating osmostress-induced glycerol accumulation, since a hog1 delta strain could still enhance its glycerol content, although less than the wild type. hog1 delta mutants are more sensitive to osmotic stress than isogenic gpd1 delta strains, and gpd1 delta hog1 delta double mutants are even more sensitive than either single mutant. Thus, the HOG pathway most probably has additional targets in the mechanism of adaptation to hypertonic medium.     

Immunodominant Francisella tularensis antigens identified using proteome microarray

Stimulation of protective immune responses against intracellular pathogens is difficult to achieve using non-replicating vaccines. BALB/c mice immunized by intramuscular injection with killed Francisella tularensis (live vaccine strain) adjuvanted with preformed immune stimulating complexes admixed with CpG, were protected when systemically challenged with a highly virulent strain of F. tularensis (Schu S4). Serum from immunized mice was used to probe a whole proteome microarray in order to identify immunodominant antigens. Eleven out of the top 12 immunodominant antigens have been previously described as immunoreactive in F. tularensis. However, 31 previously unreported immunoreactive antigens were revealed using this approach. Twenty four (50%) of the ORFs on the immunodominant hit list belonged to the category of surface or membrane associated proteins compared to only 22% of the entire proteome. There were eight hypothetical protein hits and eight hits from proteins associated with different aspects of metabolism. The chip also allowed us to readily determine the IgG subclass bias, towards individual or multiple antigens, in protected and unprotected animals. These data give insight into the protective immune response and have potentially important implications for the rational design of non-living vaccines for tularemia and other intracellular pathogens.     

Water relations of solute accumulation in Pseudomonas fluorescens

When Pseudomonas fluorescens was grown in a glucose salts medium adjusted with NaCl to a water activity (a_w) value of 0.980, the intracellular glutamic acid concentration increased 23-fold and comprised 90% of the total amino acid pool. This increase was not observed when the a_w of the medium was reduced to 0.980 with sorbitol. Sorbitol was taken up rapidly over a 30 min period and accumulated intracellularly to a level approximately two-fold greater than the concentration in the growth medium. In continuous culture, the specific rate of glutamic acid production and glucose uptake was greater at 0.980 (NaCl) than at 0.997 a_w. The maintenance coefficients for glucose uptake were similar at both a_w values but were 2.4-fold greater for glutamic acid production at 0.980 (NaCl) than at 0.997 a_w.     

Complete genome sequence of the plant pathogen Ralstonia solanacearum strain Po82

Ralstonia solanacearum strain Po82, a phylotype IIB/sequevar 4 strain, was found to be pathogenic to both solanaceous plants and banana. Here, we report the complete genome sequence of Po82 and its comparison with seven published R. solanacearum genomes.     

Directed evolution of the thermostable xylanase from Thermomyces lanuginosus

The thermostability of the endo-beta-1,4-xylanase from Thermomyces lanuginosus (xynA) was improved by directed evolution using error-prone PCR. Transformants expressing the variant xylanases were first selected on 0.4% Remazol Brilliant Blue-xylan and then exposed to 80 degrees C. Whereas the wild type XynA lost 90% activity after 10 min at 80 degrees C, five mutants displayed both higher stabilities and activities than XynA. Four mutants were subjected to further mutagenesis to improve the stability and activity of the xylanase. Subsequent screening revealed three mutants with enhanced thermostability. Mutant 2B7-10 retained 71% of its activity after treatment at 80 degrees C for 60 min and had a half-life of 215 min at 70 degrees C, which is higher than that attained by XynA. Sequence analysis of second generation mutants revealed that mutations were not concentrated in any particular region of the protein and exhibited much variation. The best mutant obtained from this study was variant 2B7-10, which had a single substitution (Y58F) in beta-sheet A of the protein, which is the hydrophilic, solvent-accessible outer surface of the enzyme. Most of the mutants obtained in this study displayed a compromise between stability and activity, the only exception being mutant 2B7-10. This variant showed increased activity and thermostability.     

Synthesis and anti-norovirus activity of pyranobenzopyrone compounds

During the last decade, noroviruses have gained media attention as the cause of large scale outbreaks of gastroenteritis on cruise ships, dormitories, nursing homes, etc. Although noroviruses do not multiply in food or water, they can cause large outbreaks because approximately 10-100 virions are sufficient to cause illness in a healthy adult. Recently, it was shown that the activity of acyl-coenzyme A:cholesterol acyltransferase-1 (ACAT1) enzyme may be important in norovirus infection. In search of anti-noroviral agents based on the inhibition of ACAT1, we synthesized and evaluated the inhibitory activities of a class of pyranobenzopyrone molecules containing amino, pyridine, substituted quinolines, or 7,8-benzoquinoline nucleus. Three of the sixteen evaluated compounds possess ED(50) values in the low micrometer range. 2-Quinolylmethyl derivative 3A and 4-quinolylmethyl derivative 4A showed ED(50) values of 3.4 and 2.4 μM and TD(50) values of >200 and 96.4 μM, respectively. The identified active compounds are suitable for further modification for the development of anti-norovirus agents.     

Heterozygosity for the IVS-I-5 (G-->C) mutation with a G-->A change at codon 18 (Val-->Met; Hb Baden) in cis and a T-->G mutation at codon 126 (Val-->Gly; Hb Dhonburi) in trans resulting in a thalassemia intermedia.

We have analyzed the hemoglobins of a young German patient with beta-thalassemia intermedia and of his immediate family and included in these studies an evaluation of possible nucleotide changes in the beta-globin genes through sequencing of amplified DNA. One chromosome of the propositus and one of his father's carried the GTG-->GGG mutation at codon 126 leading to the synthesis of Hb Dhonburi or alpha 2 beta (2)126(H4)Val-->Gly; this variant is slightly unstable and is associated with mild thalassemic features. His second chromosome and one of his mother's had the common IVS-I-5 (G-->C) mutation that leads to a rather severe beta(+)-thalassemia and the GTG-->ATG mutation at codon 18, resulting in the replacement of a valine residue by a methionine residue. This newly discovered beta-chain variant, named Hb Baden, was present for only 2-3% in both the patient and his mother. This low amount results from a decreased splicing of RNA at the donor splice-site of the first intron that is nearly completely deactivated by the IVS-I-5 (G-->C) thalassemic mutation. The chromosome with the codon 18 (GTG-->ATG) and the IVS-I-5 (G-->C) mutations has thus far been found only in this German family; analysis of 51 chromosomes from patients with the IVS-I-5 (G-->C) mutation living in different countries failed to detect the codon 18 (GTG-->ATG) change.     

Viral coat protein is targeted to, but does not gate, plasmodesmata during cell-to-cell movement of potato virus X

The coat protein (CP) of potato virus X was localized immunocytochemically in infected leaves of susceptible Nicotiana species and shown to be targeted to the central cavity of plasmodesmata in virus-infected cells. A viral deletion mutant, in which the CP gene was replaced with the gene for the green fluorescent protein (GFP), was restricted to single, inoculated cells. However, movement of the mutant virus was rescued on transgenic plants constitutively expressing the CP gene, and the CP was again targeted to plasmodesmata. The CP was not localized to plasmodesmata in uninfected transgenic plants and, in contrast to the plasmodesmata of PVX-infected cells, the plasmodesmata of the transgenic plants did not allow the passage of 10 kDa fluorescent dextrans. We propose that the CP is not involved in plasmodesmal gating per se , but is necessary for transport of the viral RNA to, and possibly through, plasmodesmata.