Anti-prion activity of protein-bound polysaccharide K in prion-infected cells and animals

Protein-bound polysaccharide K (PSK) is a clinical immunotherapeutic agent that exhibits various biological activities, including anti-tumor and anti-microbial effects. In the present study, we report on the anti-prion activity of PSK. It inhibited the formation of protease-resistant abnormal prion protein in prion-infected cells without any apparent alterations in either the normal prion protein turnover or the autophagic function in the cells. Its anti-prion activity was predominantly composed of the high molecular weight component(s) of the protein portion of PSK. A single subcutaneous dose of PSK slightly but significantly prolonged the survival time of peritoneally prion-infected mice, but PSK-treated mice produced neutralizing antibodies against the anti-prion activity of PSK. These findings suggest that PSK is a new anti-prion substance that may be useful in elucidating the mechanism of prion replication, although the structure of the anti-prion component(s) of PSK requires further evaluation.     

Efficient expansion of mouse primary tenocytes using a novel collagen gel culture method

Development of regenerative therapies for damaged tendons remains a great challenge, largely because of lack of information regarding the mechanisms responsible for differentiation of tenocytes. Mouse tenocytes have not been fully characterized owing to the absence of efficient and reproducible methods for their in vitro expansion without losing phenotypic features. The objective of the study was to establish an improved and reliable method for stable primary culture of mouse tenocytes by using collagen gel. Achilles and tail tendon tissues were harvested and embedded in collagen gel. After 10 days of continuous culture, the gel was digested and cells were passaged on tissue culture-treated plastic dishes. Mouse tenocytes cultured in collagen gel exhibited significantly shorter doubling time and higher numbers of proliferation when maintained on the plastic dishes compared with those cultured without using gel. Transmission electron microscopic analyses showed that cultured tenocytes retained some morphological features of tenocytes in tendon tissues, such as cell–cell junctional complex formation, well-developed rough endoplasmic reticulum, and mitochondria in their cytoplasm. mRNA expression of tenocyte markers (tenomodulin, type I collagen, periostin, and scleraxis) was higher in cells cultured in collagen gel than in those cultured in the absence of gel. Our results show that tenocytes cultured using the collagen gel method express typical lineage markers and exhibit improved growth characteristics, thus providing a stable platform for studying molecular mechanisms that control their differentiation.     

A multi-year assessment of the environmental impact of transgenic Eucalyptus trees harboring a bacterial choline oxidase gene on biomass,precinct vegetation and the microbial community

A 4-year field trial for the salt tolerant Eucalyptus globulus Labill. harboring the choline oxidase (codA) gene derived from the halobacterium Arthrobacter globiformis was conducted to assess the impact of transgenic versus non-transgenic trees on biomass production, the adjacent soil microbial communities and vegetation by monitoring growth parameters, seasonal changes in soil microbes and the allelopathic activity of leaves. Three independently-derived lines of transgenic E. globulus were compared with three independent non-transgenic lines including two elite clones. No significant differences in biomass production were detected between transgenic lines and non-transgenic controls derived from same seed bulk, while differences were seen compared to two elite clones. Significant differences in the number of soil microbes present were also detected at different sampling times but not between transgenic and non-transgenic lines. The allelopathic activity of leaves from both transgenic and non-transgenic lines also varied significantly with sampling time, but the allelopathic activity of leaves from transgenic lines did not differ significantly from those from non-transgenic lines. These results indicate that, for the observed variables, the impact on the environment of codA-transgenic E. globulus did not differ significantly from that of the non-transformed controls on this field trial.     

Biochemical Properties and Atomic Resolution Structure of a Proteolytically Processed β-Mannanase from Cellulolytic Streptomyces sp. SirexAA-E

β-mannanase SACTE_2347 from cellulolytic Streptomyces sp. SirexAA-E is abundantly secreted into the culture medium during growth on cellulosic materials. The enzyme is composed of domains from the glycoside hydrolase family 5 (GH5), fibronectin type-III (Fn3), and carbohydrate binding module family 2 (CBM2). After secretion, the enzyme is proteolyzed into three different, catalytically active variants with masses of 53, 42 and 34 kDa corresponding to the intact protein, loss of the CBM2 domain, or loss of both the Fn3 and CBM2 domains. The three variants had identical N-termini starting with Ala51, and the positions of specific proteolytic reactions in the linker sequences separating the three domains were identified. To conduct biochemical and structural characterizations, the natural proteolytic variants were reproduced by cloning and heterologously expressed in Escherichia coli. Each SACTE_2347 variant hydrolyzed only β-1,4 mannosidic linkages, and also reacted with pure mannans containing partial galactosyl- and/or glucosyl substitutions. Examination of the X-ray crystal structure of the GH5 domain of SACTE_2347 suggests that two loops adjacent to the active site channel, which have differences in position and length relative to other closely related mannanases, play a role in producing the observed substrate selectivity.     

Impaired Clearance of Influenza A Virus in Obese,Leptin Receptor Deficient Mice Is Independent of Leptin Signaling in the Lung Epithelium and Macrophages

Rationale

During the recent H1N1 outbreak, obese patients had worsened lung injury and increased mortality. We used a murine model of influenza A pneumonia to test the hypothesis that leptin receptor deficiency might explain the enhanced mortality in obese patients.

Methods

We infected wild-type, obese mice globally deficient in the leptin receptor (db/db) and non-obese mice with tissue specific deletion of the leptin receptor in the lung epithelium (SPC-Cre/LepR^fl/fl) or macrophages and alveolar type II cells (LysM-Cre/Lepr^fl/fl) with influenza A virus (A/WSN/33 [H1N1]) (500 and 1500 pfu/mouse) and measured mortality, viral clearance and several markers of lung injury severity.

Results

The clearance of influenza A virus from the lungs of mice was impaired in obese mice globally deficient in the leptin receptor (db/db) compared to normal weight wild-type mice. In contrast, non-obese, SP-C-Cre^+/+/LepR^fl/fl and LysM-Cre^+/+/LepR^fl/fl had improved viral clearance after influenza A infection. In obese mice, mortality was increased compared with wild-type mice, while the SP-C-Cre^+/+/LepR^fl/fl and LysM-Cre^+/+/LepR^{fl /fl} mice exhibited improved survival.

Conclusions

Global loss of the leptin receptor results in reduced viral clearance and worse outcomes following influenza A infection. These findings are not the result of the loss of leptin signaling in lung epithelial cells or macrophages. Our results suggest that factors associated with obesity or with leptin signaling in non-myeloid populations such as natural killer and T cells may be associated with worsened outcomes following influenza A infection.     

Absence of Nceh1 augments 25-hydroxycholesterol-induced ER stress and apoptosis in macrophages

An excess of cholesterol and/or oxysterols induces apoptosis in macrophages, contributing to the development of advanced atherosclerotic lesions. In foam cells, these sterols are stored in esterified forms, which are hydrolyzed by two enzymes: neutral cholesterol ester hydrolase 1 (Nceh1) and hormone-sensitive lipase (Lipe). A deficiency in either enzyme leads to accelerated growth of atherosclerotic lesions in mice. However, it is poorly understood how the esterification and hydrolysis of sterols are linked to apoptosis. Remarkably, Nceh1-deficient thioglycollate-elicited peritoneal macrophages (TGEMs), but not Lipe-deficient TGEMs, were more susceptible to apoptosis induced by oxysterols, particularly 25-hydroxycholesterol (25-HC), and incubation with 25-HC caused massive accumulation of 25-HC ester in the endoplasmic reticulum (ER) due to its defective hydrolysis, thereby activating ER stress signaling such as induction of CCAAT/enhancer-binding protein-homologous protein (CHOP). These changes were nearly reversed by inhibition of ACAT1. In conclusion, deficiency of Nceh1 augments 25-HC-induced ER stress and subsequent apoptosis in TGEMs. In addition to reducing the cholesteryl ester content of foam cells, Nceh1 may protect against the pro-apoptotic effect of oxysterols and modulate the development of atherosclerosis.     

Trim41 is required to regulate chromosome axis protein dynamics and meiosis in male mice

Meiosis is a hallmark event in germ cell development that accompanies sequential events executed by numerous molecules. Therefore, characterization of these factors is one of the best strategies to clarify the mechanism of meiosis. Here, we report tripartite motif-containing 41 (TRIM41), a ubiquitin ligase E3, as an essential factor for proper meiotic progression and fertility in male mice. Trim41 knockout (KO) spermatocytes exhibited synaptonemal complex protein 3 (SYCP3) overloading, especially on the X chromosome. Furthermore, mutant mice lacking the RING domain of TRIM41, required for the ubiquitin ligase E3 activity, phenocopied Trim41 KO mice. We then examined the behavior of mutant TRIM41 (ΔRING-TRIM41) and found that ΔRING-TRIM41 accumulated on the chromosome axes with overloaded SYCP3. This result suggested that TRIM41 exerts its function on the chromosome axes. Our study revealed that Trim41 is essential for preventing SYCP3 overloading, suggesting a TRIM41-mediated mechanism for regulating chromosome axis protein dynamics during male meiotic progression.     

The genomic basis of high-elevation adaptation in wild house mice (Mus musculus domesticus) from South America

Understanding the genetic basis of environmental adaptation in natural populations is a central goal in evolutionary biology. The conditions at high elevation, particularly the low oxygen available in the ambient air, impose a significant and chronic environmental challenge to metabolically active animals with lowland ancestry. To understand the process of adaptation to these novel conditions and to assess the repeatability of evolution over short timescales, we examined the signature of selection from complete exome sequences of house mice (Mus musculus domesticus) sampled across two elevational transects in the Andes of South America. Using phylogenetic analysis, we show that house mice colonized high elevations independently in Ecuador and Bolivia. Overall, we found distinct responses to selection in each transect and largely nonoverlapping sets of candidate genes, consistent with the complex nature of traits that underlie adaptation to low oxygen availability (hypoxia) in other species. Nonetheless, we also identified a small subset of the genome that appears to be under parallel selection at the gene and SNP levels. In particular, three genes (Col22a1, Fgf14, and srGAP1) bore strong signatures of selection in both transects. Finally, we observed several patterns that were common to both transects, including an excess of derived alleles at high elevation, and a number of hypoxia-associated genes exhibiting a threshold effect, with a large allele frequency change only at the highest elevations. This threshold effect suggests that selection pressures may increase disproportionately at high elevations in mammals, consistent with observations of some high-elevation diseases in humans.     

Downregulation of p‐COUMAROYL ESTER 3‐HYDROXYLASE in rice leads to altered cell wall structures and improves biomass saccharification

p‐Coumaroyl ester 3‐hydroxylase (C3′H) is a key enzyme involved in the biosynthesis of lignin, a phenylpropanoid polymer that is the major constituent of secondary cell walls in vascular plants. Although the crucial role of C3′H in lignification and its manipulation to upgrade lignocellulose have been investigated in eudicots, limited information is available in monocotyledonous grass species, despite their potential as biomass feedstocks. Here we address the pronounced impacts of C3′H deficiency on the structure and properties of grass cell walls. C3′H‐knockdown lines generated via RNA interference (RNAi)‐mediated gene silencing, with about 0.5% of the residual expression levels, reached maturity and set seeds. In contrast, C3′H‐knockout rice mutants generated via CRISPR/Cas9‐mediated mutagenesis were severely dwarfed and sterile. Cell wall analysis of the mature C3′H‐knockdown RNAi lines revealed that their lignins were largely enriched in p‐hydroxyphenyl (H) units while being substantially reduced in the normally dominant guaiacyl (G) and syringyl (S) units. Interestingly, however, the enrichment of H units was limited to within the non‐acylated lignin units, with grass‐specific γ‐p‐coumaroylated lignin units remaining apparently unchanged. Suppression of C3′H also resulted in relative augmentation in tricin residues in lignin as well as a substantial reduction in wall cross‐linking ferulates. Collectively, our data demonstrate that C3′H expression is an important determinant not only of lignin content and composition but also of the degree of cell wall cross‐linking. We also demonstrated that C3′H‐suppressed rice displays enhanced biomass saccharification.