Archaeal aminoacyl-tRNA synthetases interact with the ribosome to recycle tRNAs

Aminoacyl-tRNA synthetases (aaRS) are essential enzymes catalyzing the formation of aminoacyl-tRNAs, the immediate precursors for encoded peptides in ribosomal protein synthesis. Previous studies have suggested a link between tRNA aminoacylation and high-molecular-weight cellular complexes such as the cytoskeleton or ribosomes. However, the structural basis of these interactions and potential mechanistic implications are not well understood. To biochemically characterize these interactions we have used a system of two interacting archaeal aaRSs: an atypical methanogenic-type seryl-tRNA synthetase and an archaeal ArgRS. More specifically, we have shown by thermophoresis and surface plasmon resonance that these two aaRSs bind to the large ribosomal subunit with micromolar affinities. We have identified the L7/L12 stalk and the proteins located near the stalk base as the main sites for aaRS binding. Finally, we have performed a bioinformatics analysis of synonymous codons in the Methanothermobacter thermautotrophicus genome that supports a mechanism in which the deacylated tRNAs may be recharged by aaRSs bound to the ribosome and reused at the next occurrence of a codon encoding the same amino acid. These results suggest a mechanism of tRNA recycling in which aaRSs associate with the L7/L12 stalk region to recapture the tRNAs released from the preceding ribosome in polysomes.     

Plasma membranes from intestinal microvilli and erythrocytes contain cytochromes b5 and P-420

The presence of cytochromes b₅, P-450 and P-420 and activities of NADH- and NADPH-cytochrome c reductases were determined in plasma membranes isolated from microvilli of the chick and rat intestinal epithelium and erythrocyte membranes from chick, rat and man. The results are compared with the amounts of these components found in microsomal fractions from intestinal epithelium and in nuclear membranes from chick erythrocytes. Plasma membranes from intestinal microvilli and from erythrocytes contained significant amounts of NADH-cytochrome c reductase activity and of a pigment spectrophotometrically indistinguishable from rat liver microsomal cytochrome b₅. In addition, cytochrome b₅ fragments were prepared from the membranes by limited trypsin digestion and consisted of two to four components with M_r values in the range 10 000–13 500. In low-temperature difference spectra, the presence of a second cytochrome was noted which was similar to cytochrome P-420. Cytochrome P-450 and NADPH-cytochrome c reductase activities were not detected in plasma membrane fractions in significant concentrations but were present in the corresponding endomembrane fractions. These findings in highly purified, well defined plasma membrane fractions, in which contamination by endomembranes is minimal, strengthen the evidence for the existence of cytochrome-containing redox systems in plasma membranes of various cells and suggest that such redox components are general components of the cell surface. Possible functions and origins of these redox components in plasma membranes are discussed.     

LitDB - Keeping Track of Research Papers From Your Institute Made Simple

Background

In science peer-reviewed publications serve as an important indicator of scientific excellence and productivity. Therefore, every scientist and institution must carefully maintain and update records of their scientific publications. However, in most institutions and universities articles are often managed in a redundant file-based and non-central way. Whereas excellent reference management software packages such as Zotero, Endnote or Mendeley exist to manage bibliographies and references when writing scientific articles, we are not aware of any open source database solution keeping track of publication records from large scientific groups, entire institutions and/or universities.

Results

We here describe LitDB, a novel open source literature database solution for easy maintenance of publication lists assigned to various topics. In the last 2 years more than 50 users have been using LitDB at our research institute. The LitDB system is accessed via a web browser. Publications can be uploaded through direct exports from reference manager libraries or by entering PubMed IDs. Single users or user groups can track their citation counts, h-index and impact factor statistics and gain insights into the publication records of other users. It offers various visualization functions like coauthor networks and provides ways to organize publications from dedicated projects and user groups. The latter is in particular beneficial to manage publication lists of large research groups and research initiatives through a “crowd-sourcing” effort.

Conclusions

Keeping track of papers authored and published by a research group, institute or university is an important and non-trivial task. By using a centralized web-based platform for publication management such as LitDB the compilation of project- and group-related publication lists becomes easily manageable and it is less likely that papers are forgotten along the way.

     

Synthesis,secretion and immunoelectron microscopic demonstration of apolipoprotein B-containing lipoprotein particles in the visceral rat yolk sac

Summary Electron microscopic investigations on the involvement of the fetal membranes of the rat (visceral yolk sac) in the lipid metabolism revealed the occurrence of lipoprotein-sized particles located in cisternal Golgi stacks, Golgi vesicles and secretory vesicles of the cells of the visceral yolk sac epithelium as well as in distended areas of the intercellular space between adjacent epithelial cells. Application of the protein A-gold technique with specific anti-apoB antiserum resulted in a specific location of immunogold both over the different compartments of the lipoprotein pathway (RER, Golgi complex, secretory vesicles) as well as over the distended intercellular spaces, thus confirming these particles to be lipoproteins in nature. Isolated visceral epithelial cells prepared by a tryptic digestion method exhibited some ultrastructural alterations, such as a loss of apical brush border, a change from columnar to spherical cell shape, a decrease in phagolysosomes, but an increase in autophagosomal structures after 6 h incubation at a vitality rate of at least 85%. Within this period the epithelial cells secreted measurable amounts of apoB-containing lipoproteins into the medium floating in the density classes d<1.006 g/ml, d=1.006–1.020 g/ml and d=1.020–1.064 g/ml. The production of the lipoproteins was partly inhibited by cycloheximide indicating the secretion of particles with preformed as well as newly synthesized apoB. Negative staining of the particles revealed an average diameter of 34 nm of VLDL, 31 nm of IDL and 24 nm of LDL. In summary, our studies demonstrate that in the feto-placental unit of the rat the fetal membranes are capable of synthesizing and secreting lipoproteins. The cells of the visceral yolk sac epithelium were shown to be the producers of apoB-containing particles.Abbreviations apo apolipoprotein - ER endoplasmic reticulum - IDL intermediate density-lipoprotein - LDL low density-lipoprotein - VLDL very low density-lipoprotein - PBS phosphate-buffered salt solution - RER rough endoplasmic reticulum - TEM transmission electron microscopy     

Cholera Vaccination Campaign Contributes to Improved Knowledge Regarding Cholera and Improved Practice Relevant to Waterborne Disease in Rural Haiti

Background

Haiti''s cholera epidemic has been devastating partly due to underlying weak infrastructure and limited clean water and sanitation. A comprehensive approach to cholera control is crucial, yet some have argued that oral cholera vaccination (OCV) might result in reduced hygiene practice among recipients. We evaluated the impact of an OCV campaign on knowledge and health practice in rural Haiti.

Methodology/Principal Findings

We administered baseline surveys on knowledge and practice relevant to cholera and waterborne disease to every 10th household during a census in rural Haiti in February 2012 (N = 811). An OCV campaign occurred from May–June 2012 after which we administered identical surveys to 518 households randomly chosen from the same region in September 2012. We compared responses pre- and post-OCV campaign.Post-vaccination, there was improved knowledge with significant increase in percentage of respondents with ≥3 correct responses on cholera transmission mechanisms (odds ratio[OR] 1.91; 95% confidence interval[CI] 1.52–2.40), preventive methods (OR 1.83; 95% CI 1.46–2.30), and water treatment modalities (OR 2.75; 95% CI 2.16–3.50). Relative to pre-vaccination, participants were more likely post-OCV to report always treating water (OR 1.62; 95% CI 1.28–2.05). Respondents were also more likely to report hand washing with soap and water >4 times daily post-vaccine (OR 1.30; 95% CI 1.03–1.64). Knowledge of treating water as a cholera prevention measure was associated with practice of always treating water (OR 1.47; 95% CI 1.14–1.89). Post-vaccination, knowledge was associated with frequent hand washing (OR 2.47; 95% CI 1.35–4.51).

Conclusion

An OCV campaign in rural Haiti was associated with significant improvement in cholera knowledge and practices related to waterborne disease. OCV can be part of comprehensive cholera control and reinforce, not detract from, other control efforts in Haiti.     

Preparation and characterization of the inner coat material associated with fat globule membranes from bovine and human milk

Milk fat globules of many species are characterized by a dense 10–50 nm thick layer sandwiched between the milk fat globule membrane (MFGM) and the outer shell of the fat droplet. This coat material is tightly associated with the membrane and survives isolation and extensive washing of the isolated MFGM. We have prepared these MFGM-associated coat structures from bovine and human milk by removal of membrane and loosely associated material using extractions in low and high salt buffers, non-ionic detergents such as Triton X-100, and/or solutions of lithium diiodosalicylate. Residual fractions obtained after such treatments are devoid of identifiable membrane structures but are enriched in MFGM coat material which appears in the form of densely stained plaques of a finely filamentous texture. MFGM fractions are enriched in some polypeptide bands seen after electrophoresis two of which are especially prominent in both species (band 3, apparent mol. wt 155 000; band 12, apparent mol. wt 67 000). Human and bovine MFGM coat fractions and isolated bovine band 12 polypeptide material separated after dissociation in sodium dodecylsulfate (SDS) by gel filtration, chromatography on hydroxylapatite or preparative electrophoresis in SDS-polyacrylamide gels are intimately associated with small amounts of phospholipids and gangliosides of a pattern different from that of total MFGM, contain carbohydrates (relatively high contents of mannose, glucosamine, galactose, and galactosamine; low levels of fucose and sialic acids) and show similar amino acid compositions. The relationship of band 12 polypeptide to components of MFGM coat preparations from various other species and to components present in other membrane fractions has been examined by immunodiffusion techniques and immunofluorescence microscopy using rabbit, mouse and guinea pig antibodies against purified band 12 polypeptide. Evidence is presented for the occurrence of related polypeptides in MFGM coat preparations from different species. The unusual structure and resistance of the MFGM coat material, especially the occurrence of glycopeptides in association with the cytoplasmic side of a membrane structure, are discussed in relation to the stabilization of the emulsified state of milk fat and the process of milk fat globule budding as well as a general model for local differentiation of membrane character.     

Efficient and fast targeted production of murine models based on ENU mutagenesis

Mice with targeted genetic alterations are the most effective tools for deciphering organismal gene function. We generated an ENU-based parallel C3HeB/FeJ sperm and DNA archive characterized by a high probability to identify allelic variants of target genes as well as high efficiencies in allele retrieval and model revitalization. Our archive size of over 17,000 samples contains approximately 340,000 independent alleles (20 functional mutations per individual sample). Based on an estimated number of approximately 30,000 mouse genes, the parallel sperm/DNA archive should permit the identification and recovery of ten or more alleles per average target gene which translates to a calculated 99% success rate in the discovery of five allelic variants for any given average gene. The low rate of unrelated ENU-induced passenger mutations has no practical impact on the analysis of the allele-specific phenotype at the G3 generation because of dilution and free segregation of such unrelated passenger mutations. To date 39 mouse models representing 33 different genes have been recovered from our archive using in vitro fertilization techniques. The generation time for a murine model heterozygous for a mutation in a gene of interest is less than 2 months, i.e., three to four times faster compared with current embryonic stem-cell–based technologies. We conclude that ENU-based targeted mutagenesis is a powerful tool for the fast and high-throughput production of murine gene-specific models for biomedical research.M. Augustin and R. Sedlmeier contributed equally to this work.M. Nehls and S. Wattler are Co-senior authors.TGCE, temperature gradient capillary electrophoresis; ENU, N-ethyl-N-nitrosurea; SNP, single nucleotide polymorphism; IVF, in vitro fertilization     

CCA paper: a new two-dimensional cyanuric chloride-activated matrix for universal application in molecular biology

A novel two-dimensional cyanuric chloride-activated (CCA) paper has been developed. It is composed of a cellulosic base, covalently bound cyanuric chloride, and microprecipitated complex cyanuric chloride-sodium chloride crystals on its surface. CCA paper covalently binds nucleic acids and proteins. Its binding capacity for nucleic acids is about 400 micrograms/cm2. Sealed into nitrogen-filled bags and stored at -20 degrees C, it retains its binding activity for at least a year and is always ready for use. CCA paper has been successfully used for capillary and electroblotting of DNA, RNA, and proteins (Southern, Northern, and Western blotting) as well as for dot tests. Furthermore, it was applied to colony and plaque hybridization. A unique property of it is that it permits the staining of proteins after blotting and subsequent performance of radioimmunological detection of specific protein components. This has proven advantageous in two-dimensional Western blotting experiments. Of further importance is its ability to bind DNA fragments from one up to several hundred bp from polyacrylamide sequencing gels.     

High frequency of cytokeratin-producing smooth muscle cells in human atherosclerotic plaques

Using immunofluorescence microscopy we show that cells expressing cytokeratins 8 and 18 are frequently enriched in human vascular wall tissue pathologically altered by the appearance of intimal thickenings and atherosclerotic plaques. These cytokeratins occur in cells which also synthesize IFs containing vimentin and/or desmin, and a considerable proportion of the cytokeratin-positive cells has been identified as smooth muscle cells by colocalization of desmin and/or smooth muscle type alpha-actin. The presence of extremely low concentrations of these cytokeratins in such vascular tissues has been confirmed by gel electrophoresis with immunoblotting as well as by Northern blot hybridization using specific cytokeratin cRNA probes. The results are discussed in relation to the recent demonstration that low-level synthesis of cytokeratins 8 and 18 occurs in other muscular tissues and to the specific proliferative activity of these cells.