The part of a long beta hairpin from the scrapie form of the human prion protein is reconstructed in the synthetic CC36 protein

Mechanisms of beta sheet formation by the human prion protein are not clear yet. In this work, we clarified the role of the region containing C‐half of the second helix and N‐half of the third helix of that protein in the process of alpha helix to beta sheet transition. Solid phase automatic synthesis of the original peptide (CC36: Cys179–Cys214) failed because of the beta hairpin formation in the region 206‐MERVVEQMC‐214 with a high beta strand potential. Using Met206Arg and Val210Arg substitutions, we increased the probability of alpha helix formation by that sequence. After that modification, the complete CC36 peptide with disulfide bond has been synthesized. Modified peptide has been studied by circular dichroism (CD) and fluorescence spectrography. According to the CD spectra analysis, the CC36 peptide contains 37% of residues in beta sheet and just 15% in helix. Thermal analysis under the control of CD shows that the secondary structure content of the peptide is stable from 5°C to 80°C. Dissociation of oligomers of the CC36 peptide finishes at 37°C according to the fluorescence analysis. The CC36 peptide is able to bind Mn²⁺ cations, which causes small temperature‐associated structural shifts at concentrations of 2 – 10·10^?6 M. Predicted beta hairpin of the CC36 peptide (two beta strands are: 184‐IKQHTVT‐190 and 197‐TETDVKM‐205) should be the part of a longer beta hairpin from the scrapie form of the prion protein (PrPSc). Analogs of the CC36 peptide may be considered as antigens for the future development of a vaccine against PrPSc. Proteins 2016; 84:1462–1479. © 2016 Wiley Periodicals, Inc.     

Synchronous genetic turnovers across Western Eurasia in Late Pleistocene collared lemmings

Recent palaeogenetic studies indicate a highly dynamic history in collared lemmings (Dicrostonyx spp.), with several demographical changes linked to climatic fluctuations that took place during the last glaciation. At the western range margin of D. torquatus, these changes were characterized by a series of local extinctions and recolonizations. However, it is unclear whether this pattern represents a local phenomenon, possibly driven by ecological edge effects, or a global phenomenon that took place across large geographical scales. To address this, we explored the palaeogenetic history of the collared lemming using a next‐generation sequencing approach for pooled mitochondrial DNA amplicons. Sequences were obtained from over 300 fossil remains sampled across Eurasia and two sites in North America. We identified five mitochondrial lineages of D. torquatus that succeeded each other through time across Europe and western Russia, indicating a history of repeated population extinctions and recolonizations, most likely from eastern Russia, during the last 50 000 years. The observation of repeated extinctions across such a vast geographical range indicates large‐scale changes in the steppe‐tundra environment in western Eurasia during the last glaciation. All Holocene samples, from across the species' entire range, belonged to only one of the five mitochondrial lineages. Thus, extant D. torquatus populations only harbour a small fraction of the total genetic diversity that existed across different stages of the Late Pleistocene. In North American samples, haplotypes belonging to both D. groenlandicus and D. richardsoni were recovered from a Late Pleistocene site in south‐western Canada. This suggests that D. groenlandicus had a more southern and D. richardsoni a more northern glacial distribution than previously thought. This study provides significant insights into the population dynamics of a small mammal at a large geographical scale and reveals a rather complex demographical history, which could have had bottom‐up effects in the Late Pleistocene steppe‐tundra ecosystem.     

Inheritance and genetic mapping of resistance to Alternaria alternata f. sp. lycopersici in Lycopersicon pennellii

The fungal pathogen Alternaria alternata f. sp. lycopersici produces AAL-toxins that function as chemical determinants of the Alternaria stem canker disease in the tomato (Lycopersicon esculentum). In resistant cultivars, the disease is controlled by the Asc locus on chromosome 3. Our aim was to characterize novel sources of resistance to the fungus and of insensitivity to the host-selective AAL-toxins. To that end, the degree of sensitivity of wild tomato species to AAL-toxins was analyzed. Of all members of the genus Lycopersicon, only L. cheesmanii was revealed to be sensitive to AAL-toxins and susceptible to fungal infection. Besides moderately insensitive responses from some species, L. pennellii and L. peruvianum were shown to be highly insensitive to AAL-toxins as well as resistant to the pathogen. Genetic analyses showed that high insensitivity to AAL-toxins from L. pennellii is inherited in tomato as a single complete dominant locus. This is in contrast to the incomplete dominance of insensitivity to AAL-toxins of L. esculentum. Subsequent classical genetics, RFLP mapping and allelic testing indicated that high insensitivity to AAL-toxins from L. pennellii is conferred by a new allele of the Asc locus.     

Improved Procedures for Electron Microscopic Visualization of the Cytoskeleton of Cultured Cells

We have developed an improved electron microscopic procedure appropriate for correlative light and electron microscopy of the cytoskeleton. The procedure is based on detergent extraction, chemical fixation, critical point drying, and platinum/carbon coating of cultured cells and the improvements consist of modifications which are minor individually but collectively of substantial impact. They are: inclusion of polyethylene glycol into the extraction medium; cell lysis at room temperature; fixation by sequential application of glutaraldehyde, tannic acid, and uranyl acetate; horizontal position of specimens during dehydration and drying; and uranyl acetate treatment during dehydration. As a result, we have obtained a greatly improved quality of electron microscopic images together with a high consistency of results. Long and straight actin filaments were clearly seen in stress fibers and newly formed lamellipodia. Their polarity was distinctly revealed by decoration with myosin subfragment 1. Depletion of actin from cytoskeletons by gelsolin treatment allowed for better visualization of myosin, intermediate filaments, and microtubules. Intermediate filaments exposed by this treatment exhibited numerous side projections in a hitherto unreported millipede-like appearance. The suggested procedure was compatible with immunogold labeling as demonstrated with an antibody to tubulin. Correlative light and electron microscopy of cells microinjected with a fluorescent derivative of myosin II was reliable and efficient, producing a close resemblance between the two kinds of images.     

Mouse protection induced by Pseudomonas aeruginosa PAC1R and its defective mutants, Salmonella minnesota Re-mutant and Escherichia coli O14

Abstract Pseudomonas aeruginosa PAC1R and its defective mutants (acetone-killed bacteria), Salmonella minnesota Re mutant (acetone-killed bacteria and Re-LPS) and Escherichia coli O14 (acetone-killed bacteria and enterobacterial common antigen, ECA) were studied in a mouse active protection test. Immunized mice were challenged with wild-type P. aeruginosa strains. It was established that P. aeruginosa LPS-defective mutants induced cross-immunity against different Fisher immunotypes of P. aeruginosa. S. minnesota Re-LPS and ECA gave mice protection against P. aeruginosa .     

Immunological studies of an artificial antigen with specificity of a common polysaccharide antigen of Pseudomonas aeruginosa

Abstract Synthetic d -rhamnan, with the structure of Pseudomonas aeruginosa common polysaccharide antigen (CPA), was conjugated with BSA. The artificial antigen obtained, and the natural antigens, lipopolysaccharides (LPS) of P. aeruginosa and Pseudomonas cerasi with rhamnan chains of the same structure, were studied by ELISA with rabbit antibodies to the d -rhamnan-BSA conjugate and to the P. cerasi O-antigen. Immunological relations between the LPS of P. aeruginosa and P. cerasi determined by CPA as well as between these LPS and d -rhamnan-BSA were revealed by ELISA. O-antiserum to P. cerasi possesses protective activity in the mouse passive protection test when mice are challenged with some P. aeruginosa strains; the antiserum to the d -rhamnan-BSA does not possess protective activity in mice.     

Study of H-2 mutations in mice. IV. A comparison of the mutants M505 and Hz1 by skin grafting and serological techniques

The line B6.M505 is congenic with C57BL/6JY and carries a mutant form of theH-2 ^b haplotype designatedH-2 ^bd . The mutant site 505 was located by the F₁ tests in theK end of theH-2 gene complex. The M505 mice are histoincompatible with the B6.C(Hz1) line (haplotypeH-2 ^ba ) carrying another mutation in theK end ofH-2 ^b . Inability of M505 to complement Hz1 in tests with B6 skin grafting is considered as an evidence that the same gene was altered by both mutations. The gained H antigens of two mutants can cross-react in vivo as revealed by accelerated rejection of Hz1 skin grafts by B6 recipients presensitized with M505 spleen cells. The lost antigenic determinants are not identical as shown by accelerated rejection of B6 skin grafts by Hz1 hosts preimmunized with M505 spleen cells. Absorptions of the antiserum ASY-015, (d×a) anti-i, anti-H-2.33 with M505 spleen cells did not clear forH-2 ⁱ ,H-2 ^b andH-2 ^ba , and absorptions with Hz1 did not clear forH-2 ⁱ ,H-2 ^b , andH-2 ^bd . These results show that changes of histocompatibility determinants may be accompanied by loss of some haptenic determinants in the Hz1 and M505 mutations.     

Repair of uracil residues closely spaced on the opposite strands of plasmid DNA results in double-strand break and deletion formation

Summary The role of closely spaced lesions on both DNA strands in the induction of double-strand breaks and formation of deletions was studied. For this purpose a polylinker sequence flanked by 165 by direct repeats was inserted within the tet gene of pBR327. This plasmid was used to construct DNA containing one or two uracil residues which replaced cytosine residues in the Kpnl restriction site of the polylinker. Incubation of the plasmid DNA construct with Escherichia coli cell-free extracts showed that double-strand breaks occurred as a result of excision repair of the opposing uracil residues by uracil-DNA glycosylase (in extracts from ung ⁺ but not in extracts from ung ^– E. coli strains). Recombination of direct repeats, induced by double-strand breakage of plasmid DNA, can lead to the deletion of the polylinker and of one of the direct repeats, thus restoring the tet ⁺ gene function which can be detected by the appearance of tetracycline-resistant colonies of transformants. Transformation of E. coli cells with single or double uracil-containing DNAs demonstrated that DNA containing two closely spaced uracil residues was tenfold more effective in the induction of deletions than DNA containing only a single uracil residue. The frequency of deletions is increased tenfold in an ung ⁺ E. coli strain in comparison with an ung ^– strain, suggesting that deletions are induced by double-strand breakage of plasmid DNA which occurs in vivo as a result of the excision of opposing uracil residues.     

Cardiolipin synthase A colocalizes with cardiolipin and osmosensing transporter ProP at the poles of Escherichia coli cells

Osmosensing by transporter ProP is modulated by its cardiolipin (CL)‐dependent concentration at the poles of Escherichia coli cells. Other contributors to this phenomenon were sought with the BACterial Two‐Hybrid System (BACTH). The BACTH‐tagged variants T18‐ProP and T25‐ProP retained ProP function and localization. Their interaction confirmed the ProP homo‐dimerization previously established by protein crosslinking. YdhP, YjbJ and ClsA were prominent among the putative ProP interactors identified by the BACTH system. The functions of YdhP and YjbJ are unknown, although YjbJ is an abundant, osmotically induced, soluble protein. ClsA (CL Synthase A) had been shown to determine ProP localization by mediating CL synthesis. Unlike a deletion of clsA, deletion of ydhP or yjbJ had no effect on ProP localization or function. All three proteins were concentrated at the cell poles, but only ClsA localization was CL‐dependent. ClsA was shown to be N‐terminally processed and membrane‐anchored, with dual, cytoplasmic, catalytic domains. Active site amino acid replacements (H224A plus H404A) inactivated ClsA and compromised ProP localization. YdhP and YjbJ may be ClsA effectors, and interactions of YdhP, YjbJ and ClsA with ProP may reflect their colocalization at the cell poles. Targeted CL synthesis may contribute to the polar localization of CL, ClsA and ProP.