Impact of intra- and interspecies variation of occludin on its function as coreceptor for authentic hepatitis C virus particles

Hepatitis C virus (HCV) is characterized by a narrow host range and high interindividual variability in the clinical course of infection. Both of these traits are thought to be largely due to genetic variation between species and between individual hosts. The tight junction component occludin (OCLN) is essential for HCV entry into host cells, and the differences between human and murine OCLN are thought to account in part for the inability of HCV to infect mice and hence preclude their use as a convenient small-animal model. This study assesses the impact of genetic variation in OCLN on cell culture-grown HCV (HCVcc) using a newly generated and characterized OCLN(low) subclone of the Huh-7.5 cell line (Huh-7.5 subclone in which endogenous OCLN expression has been downregulated by a short hairpin RNA). We report the frequency of coding nonsynonymous single nucleotide polymorphisms, i.e., polymorphisms resulting in amino acid exchanges, present in the human population and determine their ability to function as HCV (co)receptors. Moreover, we show that murine OCLN can sustain HCVcc entry, albeit with about 5-fold reduced efficiency compared to that of human OCLN. This reduction in efficiency is due solely to two amino acid residues previously identified by others using an HCV pseudoparticle approach. Finally, we use the Huh-7.5/OCLN(low) cell line to show that HCV spread between neighboring cells is strictly dependent on OCLN.     

Glutathione--functions and metabolism in the malarial parasite Plasmodium falciparum

When present as a trophozoite in human erythrocytes, the malarial parasite Plasmodium falciparum exhibits an intense glutathione metabolism. Glutathione plays a role not only in antioxidative defense and in maintaining the reducing environment of the cytosol. Many of the known glutathione-dependent processes are directly related to the specific lifestyle of the parasite. Reduced glutathione (GSH) supports rapid cell growth by providing electrons for deoxyribonucleotide synthesis and it takes part in detoxifying heme, a product of hemoglobin digestion. Free radicals generated in the parasite can be scavenged in reaction sequences involving the thiyl radical GS* as well as the thiolate GS-. As a substrate of glutathione S-transferase, glutathione is conjugated to non-degradable compounds including antimalarial drugs. Furthermore, it is the coenzyme of the glyoxalase system which detoxifies methylglyoxal, a byproduct of the intense glycolysis taking place in the trophozoite. Proteins involved in GSH-dependent processes include glutathione reductase, glutaredoxins, glyoxalase I and II, glutathione S-transferases, and thioredoxins. These proteins, as well as the ATP-dependent enzymes of glutathione synthesis, are studied as factors in the pathophysiology of malaria but also as potential drug targets. Methylene blue, an inhibitor of the structurally known P. falciparum glutathione reductase, appears to be a promising antimalarial medication when given in combination with chloroquine.     

Endogenous and environmental factors influence 35S promoter methylation of a maize A1 gene construct in transgenic petunia and its colour phenotype

Summary 30000 transgenic petunia plants carrying a single copy of the maize A1 gene, encoding a dihydroflavonol reductase, which confers a salmon red flower colour phenotype on the petunia plant, were grown in a field test. During the growing season plants with flowers deviating from this salmon red colour, such as those showing white or variegated phenotypes and plants with flowers exhibiting only weak pigmentation were observed with varying frequencies. While four white flowering plants were shown at the molecular level to be mutants in which part of the A1 gene had been deleted, other white flowering plants, as well as 13 representative plants tested out of a total of 57 variegated individuals were not mutants but rather showed hypermethylation of the 35S promoter directing A1 gene expression. This was in contrast to the homogeneous fully red flowering plants in which no methylation of the 35S promoter was observed. While blossoms on plants flowering early in the season were predominantly red, later flowers on the same plants showed weaker coloration. Once again the reduction of the A1-specific phenotype correlated with the methylation of the 35S promoter. This variation in coloration seems to be dependent not only on exogenous but also on endogenous factors such as the age of the parental plant from which the seed was derived or the time at which crosses were made.     

High Sequence Variability of the ppE18 Gene of Clinical Mycobacterium tuberculosis Complex Strains Potentially Impacts Effectivity of Vaccine Candidate M72/AS01E

The development of an effective vaccine is urgently needed to fight tuberculosis (TB) which is still the leading cause of death from a single infectious agent worldwide. One of the promising vaccine candidates M72/AS01E consists of two proteins subunits PepA and PPE18 coded by Rv0125 and Rv1196. However, preliminary data indicate a high level of sequence variability among clinical Mycobacterium tuberculosis complex (MTBC) strains that might have an impact on the vaccine efficacy. To further investigate this finding, we determined ppE18 sequence variability in a well-characterized reference collection of 71 MTBC strains from 23 phylogenetic lineages representing the global MTBC diversity. In total, 100 sequence variations consisting of 96 single nucleotide polymorphisms (SNPs), three insertions and one deletion were detected resulting in 141 variable positions distributed over the entire gene. The majority of SNPs detected were non-synonymous (n = 68 vs. n = 28 synonymous). Strains from animal adapted lineages, e.g., M. bovis, showed a significant higher diversity than the human pathogens such as M. tuberculosis Haarlem. SNP patterns specific for different lineages as well as for deeper branches in the phylogeny could be identified. The results of our study demonstrate a high variability of the ppE18 gene even in the N-terminal domains that is normally highly conserved in ppe genes. As the N-terminal region interacts with TLR2 receptor inducing a protective anti-inflammatory immune response, genetic heterogeneity has a potential impact on the vaccine efficiency, however, this has to be investigated in future studies.