Synthesis, structure and photocatalytic activity of a remarkably bent, cofacial ethene-linked diiron (III) μ-oxobisporphyrin

Synthesis and characterization of cis and trans 1,2-bis[Chloroiron(III) 5-(2,3,7,8,12,13,17,18-octaethylporphyrinyl)]ethenes are described. Upon treatment with 5% NaOH, cis form of the molecule immediately converts to remarkably bent diiron (III)-μ-oxo bisporphyrin which transforms to cis bisChloroFe(III)porphyrin again by the addition of 5% HCl. This facile transformation is reversible with sharp change in color in which the bisporphyrin platform ‘open’ and ‘close’ its binding pockets with very high vertical flexibility in a single molecular framework. Single crystal X-ray structural characterization reveals cis diiron(III)-μ-oxo bisporphyrin in which Fe-O-Fe unit is remarkable bent with 150.9(2)° angle. Two porphyrin rings in the molecule are not slipped but face-to-face in a fully eclipsed geometry and are placed so close that some of the carbon atoms from each of the macrocycles are driven to be essentially less than the van der Waals contacts (<3.4 Å). Two rings in the oxo-bridged dimer also make the interplanar angle of 27.7° instead of expected angle of 60° due to the bridging alkenic bond. EPR, ¹H NMR and Mössbauer spectral data are indicative of strong anti-ferromagnetic coupling between two high-spin iron(III) centers via bridging oxo group. The complex catalyzes the rapid photoinduced oxygenation of phosphites under mild condition using aerial O₂. Electrochemical data reveals that the diiron(III)-μ-oxo bisporphyrin in dichloromethane undergoes four reversible/quasi-reversible one electron oxidation and one electron reductions. The presence of two porphyrin macrocycles within a short distance in the μ-oxo species makes the porphyrin core highly nonplanar and more electron rich that might responsible for easier oxidations compared to [Fe(OEP)]₂O.     

The Spatial Structure of Variability in a Semi-arid, Fluvial Ecosystem

The arrangement and composition of flowpath types within a given network are thought to govern its functioning. This concept assumes that different flowpath types are functionally distinct. We investigated this assumption in a fluvial ecosystem by comparing the riparian zone, parafluvial zone (in-channel gravel bars), and surface stream. We hypothesized that differences in advection, uptake, and sorption would render material cycles more (a) open and (b) mutable in the surface stream, whereas the converse would occur in the riparian zone, and an intermediate state would be seen in the intervening parafluvial zone. To test our first hypothesis, we predicted that spatial heterogeneity in solute concentrations would be least in the surface stream, greater in the parafluvial zone, and greatest in the riparian zone. Using a null model, we ascertained that this pattern was shown by all solute species we examined (nitrate, ammonium, total dissolved inorganic nitrogen [DIN], dissolved organic N, total dissolved N, soluble reactive phosphorus, dissolved organic carbon, and chloride). To test our second hypothesis, we predicted that temporal change in spatial heterogeneity would be greatest in the surface stream, less in the parafluvial zone, and least in the riparian zone. Nitrate, DIN, and chloride showed this pattern. In particular, surface stream inorganic N was less spatially variable following months of high rainfall. According to an extant hypothesis, these results suggest that inorganic N processing may be a stable function in this ecosystem. Other solute species did not support our second prediction, perhaps because their retention and release dynamics are influenced principally by geochemistry. Generally, our findings indicate that a geomorphic template can generate spatial patterns in ecosystem function, warranting an expansion of the spiraling framework to a variety of flowpath types.     

Asparagales Telomerases which Synthesize the Human Type of Telomeres

The order of monocotyledonous plants Asparagales is attractive for studies of telomere evolution as it includes three phylogenetically distinct groups with telomeres composed of TTTAGGG (Arabidopsis-type), TTAGGG (human-type) and unknown alternative sequences, respectively. To analyze the molecular causes of these switches in telomere sequence (synthesis), genes coding for the catalytic telomerase subunit (TERT) of representative species in the first two groups have been cloned. Multiple alignments of the sequences, together with other TERT sequences in databases, suggested candidate amino acid substitutions grouped in the Asparagales TERT synthesizing the human-type repeat that could have contributed to the changed telomere sequence. Among these, mutations in the C motif are of special interest due to its functional importance in TERT. Furthermore, two different modes of initial elongation of the substrate primer were observed in Asparagales telomerases producing human-like repeats, which could be attributed to interactions between the telomerase RNA subunit (TR) and the substrate. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Molecular imprinted polymer with cloned bacterial protein template enriches authentic target in cell extract

Here we describe a new method for preparing a protein-imprinted polymer with a cloned bacterial protein template, which recognizes/adsorbs authentic target protein present at a relatively low level in cell extract. In this work, cloned pig cyclophilin 18 (pCyP18) was used as a template. The template protein was selectively assembled with memory molecules from their library, which consists of numerous limited length polymer chains with randomly distributed recognition sites and immobilizing sites. These assemblies of protein and memory molecules were adsorbed by porous polymeric beads and immobilized by cross-linking polymerization. After removing the template, binding sites that were complementary to the target protein in size, shape and the position of recognition groups were exposed, and their confirmation was preserved by the cross-linked structure. The synthesized imprinted polymer was used to adsorb authentic pCyP18 from cell extract, and its proportional content was enriched 300 times.     

Template switching within exons 3 and 4 of KV11.1 (HERG) gives rise to a 5' truncated cDNA

K(V)11.1 (HERG) channels contribute to membrane potential in a number of excitable cell types. We cloned a variant of K(V)11.1 from human jejunum containing a 171 bp deletion spanning exons 3 and 4. Expression of a full-length cDNA clone containing this deletion gave rise to protein that trafficked to the cell membrane and generated robust currents. The deletion occurred in a G/C-rich region and identical sequence elements of UGGUGG were located at the deletion boundaries. In recent studies these features have been implicated to cause deletions via template switching during cDNA synthesis. To examine this possibility we compared cDNAs from human brain, heart, and jejunum synthesized at lower (42 degrees C) and higher temperatures (70 degrees C). The 171 bp deletion was absent at the higher temperature. Our results suggest that the sequence and secondary structure of mRNA in the G/C rich region leads to template switching producing a cDNA product with a 171 bp deletion.     

Online immunoaffinity liquid chromatography/tandem mass spectrometry determination of a type II collagen peptide biomarker in rat urine: Investigation of the impact of collision-induced dissociation fluctuation on peptide quantitation

Proteolytic fragments of type II collagen, a major component of joint tissue, have recently been identified as biomarkers for osteoarthritis, a progressive disease associated with cartilage degeneration. A liquid chromatography/tandem mass spectrometry (MS/MS) assay that utilizes online immunoaffinity chromatography and column switching was developed in our laboratory for the neoepitope of type II collagen (NET2C). During method development, peptide collision-induced dissociation (CID) was found to be a significant source of assay variation, which exceeded 10% CV, despite the fact that a stable-isotope-labeled (SIL) internal standard was used to minimize imprecision. This phenomenon was studied in detail using peptides and associated SIL internal standards of varying lengths and amino acid compositions. Variability in peptide CID necessitated the monitoring of multiple MS/MS transitions to obtain acceptable assay precision. The assay was subsequently validated to measure NET2C concentrations in rat urine over the range of 0.1 to 10 ng/mL. The interday accuracy and precision ranged from 3.9 to 13.1 (%CV) and 10.7 to 5.3 (%RE), respectively, across the range of validated concentrations. A specific application of the assay is presented in which the role of estrogen deficiency in the development and progression of osteoarthritis was investigated. In this study, the effect of estrogen on lowering NET2C concentrations in urine in ovariectomized rats was demonstrated.     

Essential role of macrophages in the initiation of allergic rhinitis in mice sensitized intranasally once with cedar pollen: regulation of class switching of immunoglobulin in B cells by controlling interleukin-4 production in T cells of submandibular lymph nodes

The production of allergen-specific IgE antibodies (Abs) in allergen-sensitized patients or animals has a mutual relationship with the immunologic response leading to allergic rhinitis. We recently reported that, after an intranasal injection of cedar pollen into mice, an interleukin-4 (IL-4)-dependent increase in serum nonspecific IgE Abs was a prerequisite for the production of serum allergen-specific IgE Abs. Here, we explored which lymphoid organs were responsive to the intranasally injected allergen and how IL-4 and IgE Abs were produced in the lymphocytes. Time-dependent changes in the total cell numbers and in in vitro IgE Ab production in various lymphoid organs revealed that the submandibular lymph nodes were the main responsible organ. After treatment with allergen (for IgE production) or allergen and complete Freund's adjuvant (for IgG production), we separated submandibular lymph node cells into macrophage-, lymphocyte-, and granulocyte-rich populations by discontinuous Percoll density-gradient centrifugation. Unexpectedly, bulk cells, but not the lymphocyte- or macrophage-rich populations, produced significant amounts of IL-4, IgE, and IgG; whereas production was restored by addition of Mac-1(+) cells from the macrophage-rich to the lymphocyte-rich fraction. Furthermore, a combination of the lymphocyte-rich population (for IgG [or IgE]) production) and the macrophage-rich population (for IgE [or IgG]) production) produced a large amount of IgE (or IgG). These results indicate that, in the initiation of allergic rhinitis, macrophages in the submandibular lymph nodes are essential not only for IL-4 or immunoglobulin production, but also for class switching of immunoglobulin in lymphocytes.     

Fast and accurate modeling of protein-protein interactions by combining template-interface-based docking with flexible refinement

The similarity between folding and binding led us to posit the concept that the number of protein-protein interface motifs in nature is limited, and interacting protein pairs can use similar interface architectures repeatedly, even if their global folds completely vary. Thus, known protein-protein interface architectures can be used to model the complexes between two target proteins on the proteome scale, even if their global structures differ. This powerful concept is combined with a flexible refinement and global energy assessment tool. The accuracy of the method is highly dependent on the structural diversity of the interface architectures in the template dataset. Here, we validate this knowledge-based combinatorial method on the Docking Benchmark and show that it efficiently finds high-quality models for benchmark complexes and their binding regions even in the absence of template interfaces having sequence similarity to the targets. Compared to "classical" docking, it is computationally faster; as the number of target proteins increases, the difference becomes more dramatic. Further, it is able to distinguish binders from nonbinders. These features allow performing large-scale network modeling. The results on an independent target set (proteins in the p53 molecular interaction map) show that current method can be used to predict whether a given protein pair interacts. Overall, while constrained by the diversity of the template set, this approach efficiently produces high-quality models of protein-protein complexes. We expect that with the growing number of known interface architectures, this type of knowledge-based methods will be increasingly used by the broad proteomics community.     

Molecular and expression characterization of a nanos1 homologue in Chinese sturgeon, Acipenser sinensis

The nanos gene family was essential for germ line development in diverse organisms. In the present study, the full-length cDNA of a nanos1 homologue in A. sinensis, Asnanos1, was isolated and characterized. The cDNA sequence of Asnanos1 was 1489 base pairs (bp) in length and encoded a peptide of 228 amino acid residues. Multiple sequence alignment showed that the zinc-finger motifs of Nanos1 were highly conserved in vertebrates. By RT-PCR analysis, Asnanos1 mRNAs were ubiquitously detected in all tissues examined except for the fat, including liver, spleen, heart, ovary, kidney, muscle, intestines, pituitary, hypothalamus, telencephalon, midbrain, cerebellum, and medulla oblongata. Moreover, a specific polyclonal antibody was prepared from the in vitro expressed partial AsNanos1 protein. Western blot analysis revealed that the tissue expression pattern of AsNanos1 was not completely coincided with that of its mRNAs, which was not found in fat, muscle and intestines. Additionally, by immunofluoresence localization, it was observed that AsNanos1 protein was in the cytoplasm of primary oocytes and spermatocytes. The presented results indicated that the expression pattern of Asnanos1 was differential conservation and divergence among diverse species.