Genetic Evidence That the Non-Homologous End-Joining Repair Pathway Is Involved in LINE Retrotransposition

Long interspersed elements (LINEs) are transposable elements that proliferate within eukaryotic genomes, having a large impact on eukaryotic genome evolution. LINEs mobilize via a process called retrotransposition. Although the role of the LINE-encoded protein(s) in retrotransposition has been extensively investigated, the participation of host-encoded factors in retrotransposition remains unclear. To address this issue, we examined retrotransposition frequencies of two structurally different LINEs—zebrafish ZfL2-2 and human L1—in knockout chicken DT40 cell lines deficient in genes involved in the non-homologous end-joining (NHEJ) repair of DNA and in human HeLa cells treated with a drug that inhibits NHEJ. Deficiencies of NHEJ proteins decreased retrotransposition frequencies of both LINEs in these cells, suggesting that NHEJ is involved in LINE retrotransposition. More precise characterization of ZfL2-2 insertions in DT40 cells permitted us to consider the possibility of dual roles for NHEJ in LINE retrotransposition, namely to ensure efficient integration of LINEs and to restrict their full-length formation.     

Crystal structure of the human monocyte-activating receptor, "Group 2" leukocyte Ig-like receptor A5 (LILRA5/LIR9/ILT11)

Human leukocyte Ig-like receptor B1 (LILRB1) and B2 (LILRB2) belong to "Group 1" receptors and recognize a broad range of major histocompatibility complex class I molecules (MHCIs). In contrast, "Group 2" receptors show low similarity with LILRB1/B2, and their ligands remain to be identified. To date, the structural and functional characteristics of Group 2 LILRs are poorly understood. Here we report the crystal structure of the extracellular domain of LILRA5, which is an activating Group 2 LILR expressed on monocytes and neutrophils. Unexpectedly, the structure showed large changes in structural conformation and charge distribution in the region corresponding to the MHCI binding site of LILRB1/B2, which are also distinct from killer cell Ig-like receptors and Fc alpha receptors. These changes probably confer the structural hindrance for the MHCI binding, and their key amino acid substitutions are well conserved in Group 2 LILRs. Consistently, the surface plasmon resonance and flow cytometric analyses demonstrated that LILRA5 exhibited no affinities to all tested MHCIs. These results raised the possibility that LILRA5 as well as Group 2 LILRs do not play a role in any MHCI recognition but could possibly bind to non-MHCI ligand(s) on the target cells to provide a novel immune regulation mechanism.     

Release of cytotoxin by macrophages on treatment with human placenta lectin

Human lectin purified from placenta induced release of cytotoxin from a murine macrophage cell line and human peritoneal monocytes. This activity was not due to contamination of the lectin preparation with lipopolysaccharide.     

Identification of Lactobacillus crispatus by polymerase chain reaction targeting S-layer protein gene

AIMS: This study aimed to develop a polymerase chain reaction (PCR) method to identify Lactobacillus crispatus. METHODS AND RESULTS: A primer set (CbsA2F-CbsA2R) for amplifying conserved regions of S-layer genes was designed to identify Lact. crispatus and the specificity of this set was compared with that of another primer set (Cri 16SI-Cri 16SII) which has been reported as a species-specific primer set targeting the 16S rRNA gene. Among species in the Lact. acidophilus A1-A4 groups, when KOD polymerase was used for amplification, the primer set CbsA2F-CbsA2R gave PCR products with Lact. crispatus strains only. However, when Taq polymerase was used, this primer set gave products with one Lact. amylovorus strain as well as with Lact. crispatus strains. The primer set Cri 16SI-Cri 16SII gave PCR products with Lact. crispatus strains and two Lact. acidophilus strains, regardless of whether the polymerase used was KOD or Taq. CONCLUSIONS: A PCR targeting the S-layer gene and amplified with KOD polymerase can identify Lact. crispatus accurately and rapidly. SIGNIFICANCE AND IMPACT OF THE STUDY: To the authors' knowledge, this is the first paper to provide a PCR method for the specific identification of Lact. crispatus.     

The genome-wide screening of yeast deletion mutants to identify the genes required for tolerance to ethanol and other alcohols

A set of homozygous diploid deletion mutants of the yeast Saccharomyces cerevisiae was screened for the genes required for tolerance to aliphatic alcohols. The screen identified 137, 122 and 48 deletion mutants sensitive to ethanol, 1-propanol and 1-pentanol, respectively. A number of the genes required for ethanol tolerance were those also required for tolerance to other alcohols. Numerous mutants with defective genes encoding for vacuolar H+ -ATPase (V-ATPase) were cosensitive to these alcohols. A global screening approach of yeast deletion library mutants was useful in elucidating the mechanisms of alcohol tolerance based on different lipophilicities.     

Pair territoriality in the longnose filefish,oxymonacanthus longirostris

The longnose filefish,Oxymonacanthus longirostris, usually lives in heterosexual pairs, the male and female swimming together and sharing the same territory. Pair territoriality in the species was examined in detail in relation to sexual differences in territorial defense activities. Rigorous pair territoriality was maintained only during the breeding season, although pairs used their home ranges exclusively to a certain extent, during the non-breeding season. The frequency of aggression against other conspecific pairs in the breeding season was higher than in the non-breeding season. Agonistic interactions appear to be over both mates and food resources, the strict pair territoriality in the breeding season possibly being due to mutual mate guarding. In intraspecific aggressive interactions, males usually led their partner females when attacking intruders. The feeding frequency of males was much lower than that of females in the breeding season. Mate removal experiments indicated that females could not defend their original territories solitarily and their feeding frequency decreased. Conversely, males could defend territories solitarily without a decrease in feeding frequency. These results suggest that males contribute most to the defense of the pair territory, with females benefiting from territorial pair-swimming with their partner males.     

Determination of the structural ensemble of the molten globule state of a protein by computer simulations

We have used computer simulations to investigate the structural nature of the molten globule (MG) state of canine milk lysozyme. To sample the conformational space efficiently, we performed replica-exchange umbrella sampling simulations with the radius of gyration as a reaction coordinate. We applied the Weighted Histogram Analysis Method to the trajectory of the simulations to obtain the potential of mean force, from which we identified representative structures corresponding to local minima in the free energy surface. The representative structures obtained in this way are in accord with the characteristics of the MG state reported previously by experimental studies. We conjecture that the MG state comprises a series of partially structured states undergoing relatively fast conformational interchange.     

Endospores of halophilic bacteria of the family Bacillaceae isolated from non-saline Japanese soil may be transported by Kosa event (Asian dust storm)

Background

Natural microbial communities are extremely complex and dynamic systems in terms of their population structure and functions. However, little is known about the in situ functions of the microbial communities.

Results

This study describes the application of proteomic approaches (metaproteomics) to observe expressed protein profiles of natural microbial communities (metaproteomes). The technique was validated using a constructed community and subsequently used to analyze Chesapeake Bay microbial community (0.2 to 3.0 μm) metaproteomes. Chesapeake Bay metaproteomes contained proteins from pI 4–8 with apparent molecular masses between 10–80 kDa. Replicated middle Bay metaproteomes shared ~92% of all detected spots, but only shared 30% and 70% of common protein spots with upper and lower Bay metaproteomes. MALDI-TOF analysis of highly expressed proteins produced no significant matches to known proteins. Three Chesapeake Bay proteins were tentatively identified by LC-MS/MS sequencing coupled with MS-BLAST searching. The proteins identified were of marine microbial origin and correlated with abundant Chesapeake Bay microbial lineages, Bacteroides and α-proteobacteria.

Conclusion

Our results represent the first metaproteomic study of aquatic microbial assemblages and demonstrate the potential of metaproteomic approaches to link metagenomic data, taxonomic diversity, functional diversity and biological processes in natural environments.     

Mucosal Immunogenicity of Genetically Modified Lactobacillus acidophilus Expressing an HIV-1 Epitope within the Surface Layer Protein

Surface layer proteins of probiotic lactobacilli are theoretically efficient epitope-displaying scaffolds for oral vaccine delivery due to their high expression levels and surface localization. In this study, we constructed genetically modified Lactobacillus acidophilus strains expressing the membrane proximal external region (MPER) from human immunodeficiency virus type 1 (HIV-1) within the context of the major S-layer protein, SlpA. Intragastric immunization of mice with the recombinants induced MPER-specific and S-layer protein-specific antibodies in serum and mucosal secretions. Moreover, analysis of systemic SlpA-specific cytokines revealed that the responses appeared to be Th1 and Th17 dominant. These findings demonstrated the potential use of the Lactobacillus S-layer protein for development of oral vaccines targeting specific peptides.