Vascular endothelial growth factor A (VEGF-A) is involved in guidance of VEGF receptor-positive cells to the anterior portion of early embryos

The hemangioblast in the mesoderm gives rise to both angioblasts and hematopoietic stem cells. The movement of hemangioblast precursor cells in the fetal trunk is a critical event in early embryogenesis. Vascular endothelial growth factor (VEGF) signaling is likely involved in this migration given the partial disturbance of VEGF receptor (VEGFR)-positive cell accumulation and migration in VEGFR2 null mice or mice with a truncated VEGFR1. However, it is not clear how the VEGF system regulates this migration or its direction. We show here that the expression of VEGF-A is dominant in the anterior portion of the embryo, whereas VEGFR1 and VEGFR2 are expressed in the posterior portion of the embryo. An inhibitor of VEGFR kinase blocked the migration of VEGFR-positive cells in a whole-embryo culture system. In addition, VEGFR-positive cells migrated toward a VEGFR1- or VEGFR2-specific ligand in vitro. Furthermore, VEGFR-positive cells derived from wild-type or VEGFR2(+/-) mice moved rapidly anteriorly, whereas cells derived from VEGFR2(+/-) mice carrying a truncated VEGFR1 [VEGFR1(TM-TK)(-/-)] migrated little when injected into wild-type mice. These results suggest that the VEGF-A protein concentrated in the anterior region plays an important role in the guidance of VEGFR-positive cells from the posterior portion to the head region by interacting with VEGFR in the mouse embryo.     

Detection of guanine-adenine mismatches by surface plasmon resonance sensor carrying naphthyridine-azaquinolone hybrid on the surface

We have discovered a new molecule naphthyridine–azaquinolone hybrid (Npt–Azq) that strongly stabilized the guanine-adenine (G-A) mismatch in duplex DNA. In the presence of Npt–Azq, the melting temperature (T_m) of 5′-d(CTA ACG GAA TG)-3′/3′-d(GAT TGA CTT AC)-5′ containing a single G-A mismatch increased by 15.4°C, whereas fully matched duplex increased its T_m only by 2.2°C. Npt–Azq was immobilized on the sensor surface for the surface plasmon resonance (SPR) assay to examine SPR detection of duplexes containing a G-A mismatch. Distinct SPR signals were observed when 27mer DNA containing a G-A mismatch was analyzed by the Npt–Azq immobilized sensor surfaces, whereas the signal of the fully matched duplex was ~6-fold weaker in intensity. The SPR signals for the G-A mismatch were proportional to the concentration of DNA in a range up to 1 µM, confirming that the SPR signal is in fact due to the binding of the G-A mismatch to Npt–Azq immobilized on the surface. Examination of all 16 G-A mismatches regarding the flanking sequence revealed that the sensor surface reported here is applicable to eight flanking sequences, covering 50% of all possible G-A mismatches.     

Binding between azurocidin and calreticulin: its involvement in the activation of peripheral monocytes

We found that azurocidin, a secretory protein in neutrophils, binds to calreticulin, a multifunctional chaperone of the endoplasmic reticulum. Azurocidin is known to induce cytokine production in monocytes, but the mechanism of monocyte activation by azurocidin remains unknown. On the other hand, an antibacterial peptide, KLKLLLLLKLK-NH(2) (L5), is known to bind to cell surface calreticulin of human neutrophils, resulting in their activation to produce O(2)(-). Therefore, we examined whether cell surface calreticulin is involved in the activation of human monocytes by azurocidin to produce IL-6. We found that carlreticulin is in fact located on the surface of monocytes and that the IL-6 production stimulated by an azurucidin is inhibited by anti-calreticulin antibody. Possibly, binding between cell surface calreticulin and azurocidin is prerequisite for the activation of monocytes by azurocidin to produce IL-6.     

Iron limitation by transferrin promotes simultaneous cheating of pyoverdine and exoprotease in Pseudomonas aeruginosa

Pseudomonas aeruginosa is a primary bacterial model to study cooperative behaviors because it yields exoproducts such as siderophores and exoproteases that act as public goods and can be exploited by selfish nonproducers behaving as social cheaters. Iron-limited growth medium, mainly casamino acids medium supplemented with transferrin, is typically used to isolate and study nonproducer mutants of the siderophore pyoverdine. However, using a protein as the iron chelator could inadvertently select mutants unable to produce exoproteases, since these enzymes can degrade the transferrin to facilitate iron release. Here we investigated the evolutionary dynamics of pyoverdine and exoprotease production in media in which iron was limited by using either transferrin or a cation chelating resin. We show that concomitant loss of pyoverdine and exoprotease production readily develops in media containing transferrin, whereas only pyoverdine loss emerges in medium treated with the resin. Characterization of exoprotease- and pyoverdine-less mutants revealed loss in motility, different mutations, and large genome deletions (13–33 kb) including Quorum Sensing (lasR, rsal, and lasI) and flagellar genes. Our work shows that using transferrin as an iron chelator imposes simultaneous selective pressure for the loss of pyoverdine and exoprotease production. The unintended effect of transferrin uncovered by our experiments can help to inform the design of similar studies.Subject terms: Bacteriology, Microbial ecology     

Amputation of a C-terminal helix of the γ subunit increases ATP-hydrolysis activity of cyanobacterial F1 ATP synthase

F₁ is a soluble part of F_oF₁-ATP synthase and performs a catalytic process of ATP hydrolysis and synthesis. The γ subunit, which is the rotary shaft of F₁ motor, is composed of N-terminal and C-terminal helices domains, and a protruding Rossman-fold domain located between the two major helices parts. The N-terminal and C-terminal helices domains of γ assemble into an antiparallel coiled-coil structure, and are almost embedded into the stator ring composed of α₃β₃ hexamer of the F₁ molecule. Cyanobacterial and chloroplast γ subunits harbor an inserted sequence of 30 or 39 amino acids length within the Rossman-fold domain in comparison with bacterial or mitochondrial γ. To understand the structure–function relationship of the γ subunit, we prepared a mutant F₁-ATP synthase of a thermophilic cyanobacterium, Thermosynechococcus elongatus BP-1, in which the γ subunit is split into N-terminal α-helix along with the inserted sequence and the remaining C-terminal part. The obtained mutant showed higher ATP-hydrolysis activities than those containing the wild-type γ. Contrary to our expectation, the complexes containing the split γ subunits were mostly devoid of the C-terminal helix. We further investigated the effect of post-assembly cleavage of the γ subunit. We demonstrate that insertion of the nick between two helices of the γ subunit imparts resistance to ADP inhibition, and the C-terminal α-helix is dispensable for ATP-hydrolysis activity and plays a crucial role in the assembly of F₁-ATP synthase.     

Characteristic glycopeptides associated with extreme human longevity identified through plasma glycoproteomics

Background

Glycosylation is highly susceptible to changes of the physiological conditions, and accordingly, is a potential biomarker associated with several diseases and/or longevity. Semi-supercentenarians (SSCs; older than 105?years) are thought to be a model of human longevity. Thus, we performed glycoproteomics using plasma samples of SSCs, and identified proteins and conjugated N-glycans that are characteristic of extreme human longevity.

Heavy ion microbeam irradiation induces ultrastructural changes in isolated single fibers of skeletal muscle

The effects of heavy ion microbeams on muscle fibers isolated from mouse skeletal muscles were examined by electron microscopy. The plasma membranes of heavy ion beam-irradiated areas of muscle fibers showed irregular protrusions and invaginations. In the cytoplasm, an irregular distribution of microfilaments was found near the plasma membrane. Sarcoplasmic reticula in the irradiated regions showed a distended appearance with flocculent material within the lumen. These changes were seen as early as 2 min after irradiation, and persisted until as late as 22 min after irradiation. Many autophagic vacuoles could be seen at 7 min after irradiation. At 22 min, the vacuoles became more prominent and showed more variety. These observations suggest that heavy ion beam irradiation causes disruption of the cellular architecture and the autophagy is involved in removal of this disruption.     

Extracting sequence motifs and the phylogenetic features of SNARE-dependent membrane traffic

The SNARE proteins are required for membrane fusion during intracellular vesicular transport and for its specificity. Only the unique combination of SNARE proteins (cognates) can be bound and can lead to membrane fusion, although the characteristics of the possible specificity of the binding combinations encoded in the SNARE sequences have not yet been determined. We discovered by whole genome sequence analysis that sequence motifs (conserved sequences) in the SNARE motif domains for each protein group correspond to localization sites or transport pathways. We claim that these motifs reflect the specificity of the binding combinations of SNARE motif domains. Using these motifs, we could classify SNARE proteins from 48 organisms into their localization sites or transport pathways. The classification result shows that more than 10 SNARE subgroups are kingdom specific and that the SNARE paralogs involved in the plasma membrane-related transport pathways have developed greater variations in higher animals and higher plants than those involved in the endoplasmic reticulum-related transport pathways throughout eukaryotic evolution.