Expression and characterization of endochitinase C from Serratia marcescens BJL200 and its purification by a one-step general chitinase purification method

In this study we cloned, expressed, purified, and charaterized chitinase C1 from Serratia marcescens strain BJL200. As expected, the BJL200-ChiC1 amino acid sequence of this strain was highly similar to sequences of ChiC1 identified in two other strains of S. marcescens. BJL200-ChiC1 was overproduced in E. coli by the T7 expression system, and purified by a one-step hydrophobic interaction chromatography (HIC) with phenyl-sepharose. BJL200-ChiA and BJL200-ChiB had an approximately 30-fold higher k(cat) and 15 fold-lower K(m) than BJL200-ChiC1 for the oligomeric substrate 4-methylumbelliferyl-beta-D-N-N'-N'-triacetylchitotrioside, while BJL200-ChiC1 was 10-15 times faster than BJL200-ChiB and BJL200-ChiA in degrading the polymeric substrate CM-chitin-RBV. BJL200-ChiC1 degradation of beta-chitin resulted in a range of different chito-oligosaccharides (GlcNAc)(2) (main product), GlcNAc, (GlcNAc)(3), (GlcNAc)(4), and (GlcNAc)(5), indicating endo activity. The purification method used for BJL200-ChiC1 in this study is generally applicable to family 18 chitinases and their mutants, including inactive mutants, some of which tend to bind almost irreversibly to chitin columns. The high specificity of the interaction with the (non-chitinous) column material is mediated by aromatic residues that occur in the substrate-binding clefts and surfaces of the enzymes.     

Genotyping strategy matters when analyzing hypervariable major histocompatibility complex‐Experience from a passerine bird

Genotyping of classical major histocompatibility complex (MHC) genes is challenging when they are hypervariable and occur in multiple copies. In this study, we used several different approaches to genotype the moderately variable MHC class I exon 3 (MHCIe3) and the highly polymorphic MHC class II exon 2 (MHCIIβe2) in the bluethroat (Luscinia svecica). Two family groups (eight individuals) were sequenced in replicates at both markers using Ion Torrent technology with both a single‐ and a dual‐indexed primer structure. Additionally, MHCIIβe2 was sequenced on Illumina MiSeq. Allele calling was conducted by modifications of the pipeline developed by Sommer et al. (BMC Genomics, 14, 2013, 542) and the software AmpliSAS. While the different genotyping strategies gave largely consistent results for MHCIe3, with a maximum of eight alleles per individual, MHCIIβe2 was remarkably complex with a maximum of 56 MHCIIβe2 alleles called for one individual. Each genotyping strategy detected on average 50%–82% of all MHCIIβe2 alleles per individual, but dropouts were largely allele‐specific and consistent within families for each strategy. The discrepancies among approaches indicate PCR biases caused by the platform‐specific primer tails. Further, AmpliSAS called fewer alleles than the modified Sommer pipeline. Our results demonstrate that allelic dropout is a significant problem when genotyping the hypervariable MHCIIβe2. As these genotyping errors are largely nonrandom and method‐specific, we caution against comparing genotypes across different genotyping strategies. Nevertheless, we conclude that high‐throughput approaches provide a major advance in the challenging task of genotyping hypervariable MHC loci, even though they may not reveal the complete allelic repertoire.     

The Mitogen-activated protein kinase p38 links Shiga Toxin-dependent signaling and trafficking

Shiga toxin (Stx) binds to the cell, and it is transported via endosomes and the Golgi apparatus to the endoplasmic reticulum and cytosol, where it exerts its toxic effect. We have recently shown that Stx activates the tyrosine kinase Syk, which in turn induces clathrin phosphorylation and up-regulates Stx uptake. Here, we show that toxin-induced signaling can also regulate another step in intracellular Stx transport. We demonstrate that transport of Stx to the Golgi apparatus is dependent on the mitogen-activated protein kinase p38. Treatment of cells with chemical inhibitors or small interfering RNA targeting p38 inhibited Stx transport to the Golgi and reduced Stx toxicity. This p38 dependence is specific to Stx, because transport of the related toxin ricin was not affected by p38 inhibition. Stx rapidly activated p38, and recruited it to early endosomes in a Ca(2+)-dependent manner. Furthermore, agonist-induced oscillations in cytosolic Ca(2+) levels were inhibited upon Stx stimulation, possibly reflecting Stx-dependent local alterations in cytosolic Ca(2+) levels. Intracellular transport of Stx is Ca(2+) dependent, and we provide evidence that Stx activates a signaling cascade involving cross talk between Ca(2+) and p38, to regulate its trafficking to the Golgi apparatus.     

Epizoochorous seed dispersal in relation to seed availability – an experiment with a red fox dummy

Questions: Is the red fox a potential vector for epizoochorous seed dispersal? Can seed attachment and retention be predicted from plant and seed traits? Location: Grasslands in southern Norway. Methods: Epizoochorous seed attachment on the red fox was studied by walking a dummy fox through the vegetation and comparing seeds found on the dummy with the estimated seed availability in the vegetation. Seed retention, i.e. the ability of different seeds to stay on the fox, was estimated in a separate experiment. Seed attachment and retention were related to plant and seed traits using statistical models that account for heteroscedasticity and zero‐inflated data. Results: The majority of seeds attached to the fox originated from a few species, but also species without specific seed traits that are supposed to enhance epizoochory attached at least some seeds to the fox. The probability of seed attachment was positively related to plant height, bristle and hooked seed appendages, and negatively related to winged appendages, seed mass, and seed sphericity. Seed retention was positively related to the seed traits bristles, hooks and pappus. For several species, the results indicate a high potential for dispersal over long distances. Conclusions: In modern agricultural landscapes, large herbivores are often restricted in their mobility or are found at low densities, and other animal vectors may therefore be important for seed dispersal. In our study, a range of plant species were able to disperse by attaching seeds to, and having their seeds retained in, the fox fur some distance. We suggest that the red fox may be an important vector for epizoochorous seed dispersal in the agricultural landscape.     

Anti-dsDNA Antibodies Promote Initiation,and Acquired Loss of Renal Dnase1 Promotes Progression of Lupus Nephritis in Autoimmune (NZBxNZW)F1 Mice

Background

Lupus nephritis is characterized by deposition of chromatin fragment-IgG complexes in the mesangial matrix and glomerular basement membranes (GBM). The latter defines end-stage disease.

Methodology/Principals

In the present study we determined the impact of antibodies to dsDNA, renal Dnase1 and matrix metalloprotease (MMP) mRNA levels and enzyme activities on early and late events in murine lupus nephritis. The major focus was to analyse if these factors were interrelated, and if changes in their expression explain basic processes accounting for lupus nephritis.

Findings

Early phases of nephritis were associated with chromatin-IgG complex deposition in the mesangial matrix. A striking observation was that this event correlated with appearance of anti-dsDNA antibodies and mild or clinically silent nephritis. These events preceded down-regulation of renal Dnase1. Later, renal Dnase1 mRNA level and enzyme activity were reduced, while MMP2 mRNA level and enzyme activity increased. Reduced levels of renal Dnase1 were associated in time with deficient fragmentation of chromatin from dead cells. Large fragments were retained and accumulated in GBM. Also, since chromatin fragments are prone to stimulate Toll-like receptors in e.g. dendritic cells, this may in fact explain increased expression of MMPs.

Significance

These scenarios may explain the basis for deposition of chromatin-IgG complexes in glomeruli in early and late stages of nephritis, loss of glomerular integrity and finally renal failure.     

The CW domain, a new histone recognition module in chromatin proteins

Post-translational modifications of the N-terminal histone tails, including lysine methylation, have key roles in regulation of chromatin and gene expression. A number of protein modules have been identified that recognize differentially modified histone tails and provide their proteins with the capacity to sense such modifications. Here, we identify the CW domain of plant and animal chromatin-related proteins as a novel module that recognizes different methylated states of lysine 4 on histone H3 (H3K4me). The solution structure of the CW domain of the Arabidopsis ASH1 HOMOLOG2 (ASHH2) histone methyltransferase provides insight into how different CW domains can distinguish different methylated histone tails. We provide evidence that ASHH2 is acting on H3K4me-marked genes, allowing for ASHH2-dependent H3K36 tri-methylation, which contributes to sustained expression of tissue-specific and developmentally regulated genes. This suggests that ASHH2 is a combined 'reader' and 'writer' of the histone code. We propose that different CW domains, dependent on their specificity for different H3K4 methylations, are important for epigenetic memory or participate in switching between permissive and repressive chromatin states.     

Consequences of abnormal CDK activity in S phase

Cyclin Dependent Kinases (CDKs) are important regulators of DNA replication. In this work we have investigated the consequences of increasing or decreasing the CDK activity in S phase. To this end we identified S-phase regulators of the fission yeast CDK, Cdc2, and used appropriate mutants to modulate Cdc2 activity. In fission yeast Mik1 has been thought to be the main regulator of Cdc2 activity in S phase. However, we find that Wee1 has a major function in S phase and thus we used wee1 mutants to investigate the consequences of increased Cdc2 activity. These wee1 mutants display increased replication stress and, particularly in the absence of the S-phase checkpoint, accumulate DNA damage. Notably, more cells incorporate EdU in a wee1^? strain as compared to wildtype, suggesting altered regulation of DNA replication. In addition, a higher number of cells contain chromatin-bound Cdc45, an indicator of active replication forks. In addition, we found that Cdc25 is required to activate Cdc2 in S phase and used a cdc25 mutant to explore a situation where Cdc2 activity is reduced. Interestingly, a cdc25 mutant has a higher tolerance for replication stress than wild-type cells, suggesting that reduced CDK activity in S phase confers resistance to at least some forms of replication stress.     

The Arabidopsis SUVR4 protein is a nucleolar histone methyltransferase with preference for monomethylated H3K9

Proteins containing the evolutionarily conserved SET domain are involved in regulation of eukaryotic gene expression and chromatin structure through their histone lysine methyltransferase (HMTase) activity. The Drosophila SU(VAR)3-9 protein and related proteins of other organisms have been associated with gene repression and heterochromatinization. In Arabidopsis there are 10 SUVH and 5 SUVR genes encoding proteins similar to SU(VAR)3-9, and 4 SUVH proteins have been shown to control heterochromatic silencing by its HMTase activity and by directing DNA methylation. The SUVR proteins differ from the SUVH proteins in their domain structure, and we show that the closely related SUVR1, SUVR2 and SUVR4 proteins contain a novel domain at their N-terminus, and a SUVR specific region preceding the SET domain. Green fluorescent protein (GFP)-fusions of these SUVR proteins preferably localize to the nucleolus, suggesting involvement in regulation of rRNA expression, in contrast to other SET-domain proteins studied so far. A novel HMTase specificity was demonstrated for SUVR4, in that monomethylated histone H3K9 is its preferred substrate in vitro.     

Triage of Women with Minor Cervical Lesions: Data Suggesting a “Test and Treat” Approach for HPV E6/E7 mRNA Testing

Background

Human papillomavirus (HPV) testing is included in the cervical cancer screening program in the triage of women with equivocal (ASC-US) or low-grade (LSIL) cytological lesions. These women have an increased risk for developing high grade dysplasia and cancer (CIN2+) compared to women with normal cytology. However, in order to avoid unnecessary follow-up, as well as overtreatment, a high positive predictive value (PPV) of the triage test is important.

Methodology/Principal Findings

The HPV test PreTect HPV-Proofer, detecting E6/E7 mRNA from the HPV types 16, 18, 31, 33 and 45, is used as triage test together with repeat cytology. PPV data for HPV E6/E7 mRNA testing during the period from January 2006 up to June 2009 are reported. In total, 406 of 2099 women (19.3%) had a positive HPV test result. Of the women with a positive test result and with a histological diagnosis (n = 347), 243 women had histological high-grade dysplasia or cancer (CIN2+), giving a PPV of 70.0% (95% confidence interval [CI], 65.2%–74.8%). For HPV 16 or HPV 33 positive women above 40 years of age, the PPV was 83.7% (95% CI, 73.3%–94.0%) and 84.6% (95% CI, 65.0%–100.0%) respectively. The PPV of test positive women with HSIL cytology was 94.2% (95% CI, 88.7%–99.7%).

Conclusions

When the result in triage is HPV mRNA positive, our data suggest direct treatment for women above 40 years of age or for women with a concurrent cytological HSIL diagnosis, contributing to better clinical safety for these women. In addition, by decreasing the time to treatment, thereby reducing the number of recalls, the patient management algorithm will be considerably improved, in turn reducing follow-up costs as well as unnecessary psychological stress among patients.