Membrane topology of the H+-pyrophosphatase of Streptomyces coelicolor determined by cysteine-scanning mutagenesis

The H+-translocating pyrophosphatase (H+-PPase) is a proton pump that is found in a wide variety of organisms. It consists of a single polypeptide chain that is thought to possess between 14 and 17 transmembrane domains. To determine the topological arrangement of its conserved motifs and transmembrane domains, we carried out a cysteine-scanning analysis by determining the membrane topology of cysteine substitution mutants of Streptomyces coelicolor H+-PPase expressed in Escherichia coli using chemical reagents. First, we prepared a synthetic DNA that encoded the enzyme and constructed a functional cysteine-less mutant by substituting the four cysteine residues. We then introduced cysteine residues individually into 42 sites in its hydrophilic regions and N- and C-terminal segments. Thirty-six of the mutant enzymes retained both pyrophosphatase and H+-translocating activities. Analysis of 29 of these mutant forms using membrane-permeable and -impermeable sulfhydryl reagents revealed that S. coelicolor H+-PPase contains 17 transmembrane domains and that several conserved segments, such as the substrate-binding domains, are exposed to the cytoplasm. Four essential serine residues that were located on the cytoplasmic side were also identified. A marked characteristic of the S. coelicolor enzyme is a long additional sequence that includes a transmembrane domain at the C terminus. We propose that the basic structure of H+-PPases has 16 transmembrane domains with several large cytoplasmic loops containing functional motifs.     

External chirality-triggered helicity control promoted by introducing a beta-Ala residue into the N-terminus of chiral peptides

The noncovalent chiral domino effect (NCDE), defined as chiral interaction upon an N-terminus of a 3(10)-helical peptide, will provide a unique method for structural control of a peptide helix through the use of external chirality. On the other hand, the NCDE has not been considered to be effective for the helicity control of peptides strongly favoring a one-handed screw sense. We here aim to promote the NCDE on peptide helicity using two types of nonapeptides: H-beta-Ala-Delta(Z)Phe-Aib-Delta(Z)Phe-X-(Delta(Z)Phe-Aib)(2)-OCH(3) [Delta(Z)Phe = alpha,beta-didehydrophenylalanine, Aib = alpha-aminoisobutyric acid], where X as the single chirality is L-leucine (1) or L-phenylalanine (2). NMR, IR, and CD spectroscopy as well as energy calculation revealed that both peptides alone form a right-handed 3(10)-helix. The original CD amplitudes or signs in chloroform, irrespective of a strong screw-sense preference in the central chirality, responded sensitively to external chiral information. Namely added Boc-L-amino acid stabilized the original right-handed helix, while the corresponding d-isomer destabilized it or transformed it into a left-handed helix. These peptides were also shown to bind more favorably to an L-isomer from the racemate. Although similar helicity control was observed for analogous nonapeptides bearing an N-terminal Aib residue (Inai, Y.; et al. Biomacromolecules 2003, 4, 122), the present findings demonstrate that the N-terminal replacement by the beta-Ala residue significantly improves the previous NCDE to achieve more effective control of helicity. Semiempirical molecular orbital calculations on complexation of peptide 2 with Boc-(L or D)-Pro-OH reasonably explained the unique conformational change induced by external chirality.     

Detection of p53 gene point mutation using sequence-specific molecularly imprinted PoPD electrode

An amperometric sequence-specific molecularly imprinted single-stranded oligodeoxyribonucleotide (ss-ODN) biosensor was fabricated and characterised in this study. Using ss-ODN as the template and o-phenylenediamine as the functional monomer, the ODN biosensor was fabricated by an electropolymerisation process on an indium-tin oxide (ITO) coated glass substrate. The template ss-ODN was washed out of the ss-ODN/poly(o-phenylenediamine)(PoPD)/ITO electrode using sterilised basic ethanol-water. The resulting ss-ODN imprinted PoPD/ITO electrode was characterised using Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM) and cyclic voltammetry (CV). The amperometric responses, i.e., Δi as a function of the target ss-ODN concentration was studied. The biosensor using ss-ODN imprinted PoPD/ITO as the working electrode showed a linear Δ current response to the target ss-ODN concentration within the range of 0.01-300 fM. The biosensor showed a sensitivity of 0.62 μA/fM, with a response time of 14s. The present novel molecularly imprinted ss-ODN biosensor could greatly benefit in terms of cost-effectiveness, storage stability, ultra sensitivity and selectivity together with the potential for improved commercial genetic sensors.     

Intertypic modular exchanges of genomic segments by homologous recombination at universally conserved segments in human adenovirus species D

Human adenovirus species D (HAdV-D), which is composed of clinically and epidemiologically important pathogens worldwide, contains more taxonomic “types” than any other species of the genus Mastadenovirus, although the mechanisms accounting for the high level of diversity remain to be disclosed. Recent studies of known and new types of HAdV-D have indicated that intertypic recombination between distant types contributes to the increasing diversity of the species. However, such findings raise the question as to how homologous recombination events occur between diversified types since homologous recombination is suppressed as nucleotide sequences diverge. In order to address this question, we investigated the distribution of the recombination boundaries in comparison with the landscape of intergenomic sequence conservation assessed according to the synonymous substitution rate (d_S). The results revealed that specific genomic segments are conserved between even the most distantly related genomes; we call these segments “universally conserved segments” (UCSs). These findings suggest that UCSs facilitate homologous recombination, resulting in intergenomic segmental exchanges of UCS-flanking genomic regions as recombination modules. With the aid of such a mechanism, the haploid genomes of HAdV-Ds may have been reshuffled, resulting in chimeric genomes out of diversified repertoires in the HAdV-D population analogous to the MHC region reshuffled via crossing over in vertebrates. In addition, some HAdVs with chimeric genomes may have had the opportunity to avoid host immune responses thereby causing epidemics.     

Genomic regions involved in yield potential detected by genome-wide association analysis in Japanese high-yielding rice cultivars

Background

High-yielding cultivars of rice (Oryza sativa L.) have been developed in Japan from crosses between overseas indica and domestic japonica cultivars. Recently, next-generation sequencing technology and high-throughput genotyping systems have shown many single-nucleotide polymorphisms (SNPs) that are proving useful for detailed analysis of genome composition. These SNPs can be used in genome-wide association studies to detect candidate genome regions associated with economically important traits. In this study, we used a custom SNP set to identify introgressed chromosomal regions in a set of high-yielding Japanese rice cultivars, and we performed an association study to identify genome regions associated with yield.

Results

An informative set of 1152 SNPs was established by screening 14 high-yielding or primary ancestral cultivars for 5760 validated SNPs. Analysis of the population structure of high-yielding cultivars showed three genome types: japonica-type, indica-type and a mixture of the two. SNP allele frequencies showed several regions derived predominantly from one of the two parental genome types. Distinct regions skewed for the presence of parental alleles were observed on chromosomes 1, 2, 7, 8, 11 and 12 (indica) and on chromosomes 1, 2 and 6 (japonica). A possible relationship between these introgressed regions and six yield traits (blast susceptibility, heading date, length of unhusked seeds, number of panicles, surface area of unhusked seeds and 1000-grain weight) was detected in eight genome regions dominated by alleles of one parental origin. Two of these regions were near Ghd7, a heading date locus, and Pi-ta, a blast resistance locus. The allele types (i.e., japonica or indica) of significant SNPs coincided with those previously reported for candidate genes Ghd7 and Pi-ta.

Conclusions

Introgression breeding is an established strategy for the accumulation of QTLs and genes controlling high yield. Our custom SNP set is an effective tool for the identification of introgressed genome regions from a particular genetic background. This study demonstrates that changes in genome structure occurred during artificial selection for high yield, and provides information on several genomic regions associated with yield performance.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-346) contains supplementary material, which is available to authorized users.     

Crystal structure of the NEMO ubiquitin-binding domain in complex with Lys 63-linked di-ubiquitin

NEMO is essential for activation of the NF-κB signaling pathway, which is regulated by ubiquitination of proteins. The C-terminal leucine zipper of NEMO and its adjacent coiled-coil region (CC2-LZ) reportedly bind to linear ubiquitin chains with 1 μM affinity and to Lys 63-linked chains with 100 μM affinity. Here we report the crystal structure of the CC2-LZ region of mouse NEMO in complex with Lys 63-linked di-ubiquitin (K63-Ub₂) at 2.7 Å resolution. The ubiquitin-binding region consists of a 130 Å-long helix and forms a parallel coiled-coil dimer. The Ile 44-centered hydrophobic patch of ubiquitin is recognized in the middle of the NEMO ubiquitin-binding region. NEMO interacts with each K63-Ub₂via a single ubiquitin-binding site, consistent with low affinity binding with K63-Ub₂.

Structured summary

MINT-7262681: NEMO (uniprotkb:O88522) binds (MI:0407) to Ubiquitin (uniprotkb:P62991) by pull down (MI:0096)MINT-7262667: Ubiquitin (uniprotkb:P62991) and NEMO (uniprotkb:O88522) bind (MI:0407) by X-ray crystallography (MI:0114)     

Retroperitoneal hematoma with bone resorption around the acetabular component after total hip arthroplasty: a case report and review of the literature

ABSTRACT: INTRODUCTION: Vascular complications related to cup-fixating screws penetrating the medial acetabular wall during total hip arthroplasty are not uncommon but rarely are associated with serious adverse events in the late post-operative period. CASE PRESENTATION: We present the case of a 77-year-old Japanese woman who developed progressive extensive bone resorption and large hematoma in the acetabulum 13 years after total hip arthroplasty. On admission to our hospital, she was on oral warfarin (1.5mg/day) for atrial fibrillation. About 5 months after the initiation of anticoagulant therapy, she suffered a major fall followed by massive subcutaneous and pelvic girdle bleeding, predominantly on the medial side of the right thigh, but a fracture or damage of total hip arthroplasty was not evident on an emergency orthopedic evaluation. One year after the accident, a routine follow-up examination showed an asymptomatic osteolytic lesion in the acetabulum on the right pelvis, and 2 years later our patient noticed progressive pain in her right hip during walking. A large osteolytic lesion was noted in the right acetabulum on a plain radiograph. On high-resolution computed tomography and magnetic resonance imaging, a huge granulomatous lesion in the acetabulum was suggestive of chronic hematoma in intrapelvic and extrapelvic gluteal regions. A closer computed tomography examination showed that one of the screws used for fixation of the acetabular component in the total hip arthroplasty had penetrated the acetabular bone and had reached the pelvic cavity. Surgery was performed in a single session by means of two approaches: anterior midline transperitoneal address to resect the low-density mass lesion followed by posterolateral acetabular implant re-settlement. CONCLUSIONS: Though rare, total hip arthroplasty-related late vascular complications could be serious and potentially affect the limb and quality of life.     

Interferon-Inducing Activity of Acidic Polysaccharides

Exocellular phosphomannans produced by four strains of Hansenula yeast were examined as to their interferon-inducing activities in rabbits employing the assay system consisting of vesicular stomatitis virus and primary suckling rabbit kidney cells. The phosphomannan of Hansenula holstii NRRL Y-2155 was shown to be the most potent inducer, and the interferon induced was highly host species-specific, preventing viral cytopathic effects only in the homologous cells. The biological and chemical characteristics of the interferon closely resembled those of endotoxin-induced interferon.