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A Small Peptide (CEL-1000) Derived from the ß-Chain of the Human Major Histocompatibility Complex Class II Molecule Induces Complete Protection against Malaria in an Antigen-Independent Manner. Yupin Charoenvit, 2004.CEL-1000 (DGQEEKAGVVSTGLIGGG) is a novel potential preventative and therapeutic agent . We report that CEL-1000 confers a high degree of protection against Plasmodium sporozoite challenge in a murine model of malaria, as shown by the total absence of blood stage infection following challenge with 100 sporozoites (100% protection) and by a substantial reduction (400-fold) of liver stage parasite RNA following challenge with 50,000 sporozoites . CEL-1000 protection was demonstrated in A/J (H-2a) and C3H/HeJ (H-2k) mice but not in BALB/c (H-2d) or CAF1 (A/J x BALB/c F1 hybrid) mice . In CEL-1000-treated and protected mice, high levels of gamma interferon (IFN-  ) in serum and elevated frequencies of hepatic and splenic CD4+ IFN--positive T cells were detected 24 h after administration of an additional dose of CEL-1000 . Treatment of A/J mice that received CEL-1000 with antibodies against IFN- just prior to challenge abolished the protection, and a similar treatment with antibodies against CD4+ T cells partially reduced the level of protection, while treatment with control antibodies or antibodies specific for interleukin-12 (IL-12), CD8+ T cells, or NK cells had no effect . Our data establish that the protection induced by CEL-1000 is dependent on IFN- and is partially dependent on CD4+ T cells but is independent of CD8+ T cells, NK cells, and IL-12 at the effector phase and does not induce a detectable antibody response .
Overexpression of Two Different GTPases Rescues a Null Mutation in a Heat-Induced rRNA Methyltransferase. Jacqueline Tan, 2002.The Escherichia coli RrmJ (FtsJ) heat shock protein functions as an rRNA methyltransferase that modifies position U2552 of 23S rRNA in intact 50S ribosomal subunits . An in-frame deletion of the rrmJ (ftsJ) gene leads to severe growth disadvantages under all temperatures tested and causes significant accumulation of ribosomal subunits at the expense of functional 70S ribosomes . To investigate whether overexpression of other E . coli genes can restore the severe growth defect observed in rrmJ null mutants, we constructed an overexpression library from the rrmJ deletion strain and cloned and identified the E . coli genes that were capable of rescuing the rrmJ mutant phenotype . Our intention was to identify other methylases whose specificities overlapped enough with that of RrmJ to allow complementation when overexpressed . To our great surprise, no methylases were found by this method; rather, two small GTPases, Obg (YhbZ) and EngA, when overexpressed in the rrmJ deletion strains, were found to restore the otherwise severely impaired ribosome assembly process and/or stability of 70S ribosomes . 50S ribosomal subunits prepared from these overexpressing strains were shown to still serve as in vitro substrates for purified RrmJ, indicating that the 23S rRNA likely was still lacking the highly conserved Um2552 modification . The apparent lack of this modification, however, no longer caused ribosome defects or a growth disadvantage . Massive overexpression of another related small GTPase, Era, failed to rescue the growth defects of an rrmJ strain . These findings suggest a hitherto unexpected connection between rRNA methylation and GTPase function, specifically that of the two small GTPases Obg and EngA . Experimental Verification of a Sequence-Based Prediction: F1F0-Type ATPase of Vibrio cholerae Transports Protons, Not Na+ Ions. Judith Dzioba, 2003.The membrane energetics of the intestinal pathogen Vibrio cholerae involves both H+ and Na+ as coupling ions . The sequence of the c subunit of V . cholerae F0F1 ATPase suggested that this enzyme is H+ specific, in contrast to the results of previous studies on the Na+-dependent ATP synthesis in closely related Vibrio spp . Measurements of the pH gradient and membrane potential in membrane vesicles isolated from wild-type and  atpE mutant V . cholerae show that the F1F0 ATPase of V . cholerae is an H+, not Na+, pump, confirming the bioinformatics assignments that were based on the Na+-binding model of S . Rahlfs and V . Müller (FEBS Lett . 404:269-271, 1999) . Application of this model to the AtpE sequences from other bacteria and archaea indicates that Na+-specific F1F0 ATPases are present in a number of important bacterial pathogens .
Membrane Topology of PssT, the Transmembrane Protein Component of the Type I Exopolysaccharide Transport System in Rhizobium leguminosarum bv . trifolii Strain TA1. Andrzej Mazur, 2003.The pssT gene was identified as the fourth gene located upstream of the pssNOP gene cluster possibly involved in the biosynthesis, polymerization, and transport of exopolysaccharide (EPS) in Rhizobium leguminosarum bv . trifolii strain TA1 . The hydropathy profile and homology searches indicated that PssT belongs to the polysaccharide-specific transport family of proteins, a component of the type I system of the polysaccharide transport . The predicted membrane topology of the PssT protein was examined with a series of PssT-PhoA fusion proteins and a complementary set of PssT-LacZ fusions . The results generally support a predicted topological model for PssT consisting of 12 transmembrane segments, with amino and carboxyl termini located in the cytoplasm . A mutant lacking the C-terminal part of PssT produced increased amounts of total EPS with an altered distribution of high- and low-molecular-weight forms in comparison to the wild-type RtTA1 strain . The PssT mutant produced an increased number of nitrogen fixing nodules on clover . Absence of Malolactic Activity Is a Characteristic of H+-ATPase-Deficient Mutants of the Lactic Acid Bacterium Oenococcus oeni. Delphine Galland, 2003.The lack of malolactic activity in H+-ATPase-deficient mutants of Oenococcus oeni selected previously was analyzed at the molecular level . Western blot experiments revealed a spot at 60 kDa corresponding to the malolactic enzyme only in the parental strain . Moreover, the mleA transcript encoding the malolactic enzyme was not detected by reverse transcription (RT)-PCR analysis of mutants . These results suggest that the malolactic operon was not transcribed in ATPase-deficient mutants . The mleR gene encoding a LysR-type regulatory protein which should be involved in expression of the malolactic genes was described previously for O . oeni . Results obtained in this study show that the mleR transcript was not detected in the mutants by RT-PCR . No mutation in the nucleotide sequences of the mleR gene and the malolactic operon was found . The effect of a reduction in H+-ATPase activity on L-malate metabolism was then investigated by using other malolactic bacteria . Spontaneous H+-ATPase-deficient mutant strains of Lactococcus lactis and Leuconostoc mesenteroides were isolated by using neomycin resistance . Two mutants were selected . These mutants exhibited ATPase activities that were reduced to 54 and 70% of the activities obtained for the L . lactis and L . mesenteroides parental strains, respectively . These mutants were also acid sensitive . However, in contrast to the ATPase-deficient mutants of O . oeni, activation of L-malate metabolism was observed with the L . lactis and L . mesenteroides mutants under optimal or acidic growth conditions . These data support the suggestion that expression of the genes encoding malolactic enzymes in O . oeni is regulated by the mleR product, as it is in L . lactis . Nevertheless, our results strongly suggest that there is a difference between the regulation of expression of the malolactic locus in O . oeni and the regulation of expression of this locus in less acidophilic lactic acid bacteria . Detection and Quantification of Plectosphaerella cucumerina, a Potential Biological Control Agent of Potato Cyst Nematodes, by Using Conventional PCR, Real-Time PCR, Selective Media, and Baiting. S. D. Atkins, 2003.Potato cyst nematodes (PCN) are serious pests in commercial potato production, causing yield losses valued at approximately $300 million in the European Community . The nematophagous fungus Plectosphaerella cucumerina has demonstrated its potential as a biological control agent against PCN populations by reducing field populations by up to 60% in trials . The use of biological control agents in the field requires the development of specific techniques to monitor the release, population size, spread or decline, and pathogenicity against its host . A range of methods have therefore been developed to monitor P . cucumerina . A species-specific PCR primer set (PcCF1-PcCR1) was designed that was able to detect the presence of P . cucumerina in soil, root, and nematode samples . PCR was combined with a bait method to identify P . cucumerina from infected nematode eggs, confirming the parasitic ability of the fungus . A selective medium was adapted to isolate the fungus from root and soil samples and was used to quantify the fungus from field sites . A second P . cucumerina-specific primer set (PcRTF1-PcRTR1) and a Taqman probe (PcRTP1) were designed for real-time PCR quantification of the fungus and provided a very sensitive means of detecting the fungus from soil . PCR, bait, and culture methods were combined to investigate the presence and abundance of P . cucumerina from two field sites in the United Kingdom where PCN populations were naturally declining . All methods enabled differences in the activity of P . cucumerina to be detected, and the results demonstrated the importance of using a combination of methods to investigate population size and activity of fungi .
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