Data-Driven Modeling of Intracellular Auxin Fluxes Indicates a Dominant Role of the ER in Controlling Nuclear Auxin Uptake

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Single step recombinant human growth hormone (rhGH) purification from milk by peptide affinity chromatography

Recombinant human growth hormone (rhGH) is used for the treatment of several pathologies, most of them related to growth. Although different expression systems can be used for its production, the milk from transgenic cows is one of the most interesting due to the high rhGH level achieved (5 g/L). We have designed and synthesized short peptides (9 or 10 amino acid long) using Fmoc chemistry and studied their ability to purify rhGH from milk once immobilized on an agarose support. Using spiked milk with the hormone as a sample, rhGH was purified with 88% yield and 92% purity in a single step with a fold purification of 4.5. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:999–1005, 2018     

Universal and group-specific real-time PCR diagnosis of flavescence dorée (16Sr-V), bois noir (16Sr-XII) and apple proliferation (16Sr-X) phytoplasmas from field-collected plant hosts and insect vectors

Three real‐time PCR–based assays for the specific diagnosis of flavescence dorée (FD), bois noir (BN) and apple proliferation (AP) phytoplasmas and a universal one for the detection of phytoplasmas belonging to groups 16Sr‐V, 16Sr‐X and 16Sr‐XII have been developed. Ribosomal‐based primers CYS2Fw/Rv and TaqMan probe CYS2 were used for universal diagnosis in real‐time PCR. For group‐specific detection of FD phytoplasma, ribosomal‐based primers fAY/rEY, specific for 16Sr‐V phytoplasmas, were chosen. For diagnosis of BN and AP phytoplasmas, specific primers were designed on non‐ribosomal and nitroreductase DNA sequences, respectively. SYBR^® Green I detection coupled with melting curve analysis was used in each group‐specific protocol. Field‐collected grapevines infected with FD and BN phytoplasmas and apple trees infected with AP phytoplasma, together with Scaphoideus titanus, Hyalesthes obsoletus and Cacopsylla melanoneura adults, captured in the same vineyards and orchards, were used as templates in real‐time PCR assays. The diagnostic efficiency of each group‐specific protocol was compared with well‐established detection procedures, based on conventional nested PCR. Universal amplification was obtained in real‐time PCR from DNAs of European aster yellows (16Sr‐I), elm yellows (16Sr‐V), stolbur (16Sr‐XII) and AP phytoplasma reference isolates maintained in periwinkles. The same assay detected phytoplasma DNA in all test plants and test insect vectors infected with FD, BN and AP phytoplasmas. Our group‐specific assays detected FD, BN, and AP phytoplasmas with high efficiencies, similar to those obtained with nested PCR and did not amplify phytoplasma DNA of other taxonomic groups. Melting curve analysis was necessary for the correct identification of the specific amplicons generated in the presence of very low target concentrations. Our work shows that real‐time PCR methods can sensitively and rapidly detect phytoplasmas at the universal or group‐specific level. This should be useful in developing defence strategies and for quantitative studies of phytoplasma–plant–vector interactions.     

Rediscovery of the sun bear (Helarctos malayanus)in Yingjiang County,Yunnan Province,China

DEAR EDITOR, The sun bear,Helarctos malayanus (Raffles,1821),is a forestdependent bear species distributed in tropical Southeast Asia.The species was previously reported from scattered localities in southwestern China,which is at the northeastern edge of its global range.Due to the scarcity of reliable recent records,some authorities cast doubt on the continued existence of sun bear in China.Here we present the rediscovery of this species in Yingjiang County,western Yunnan Province,China,near the international border with Myanmar's Kachin State.     

Localization patterns of fibroblast growth factor 1 and its receptors FGFR1 and FGFR2 in postnatal mouse retina

Fibroblast growth factors (FGFs) exert basic functions both during embryonic development and in the adult. The expression of FGFs and their receptors has been reported in mammalian retinas, although information on the organization of the FGF system is still incomplete. Here, we report a detailed double-label immunohistochemical investigation of the localization patterns of FGF1 and its receptors FGFR1 and FGFR2 in adult and early postnatal mouse retinas. In adult retinas, FGF1 is localized to ganglion cells, horizontal cells, and photoreceptor inner and outer segments. FGFR1 is found in ganglion cells and Müller cells, whereas FGFR2 is primarily located in ganglion cells, the nuclei of Müller cells, and glycine-containing amacrine cells. During postnatal development, the patterns of FGF1, FGFR1, and FGFR2 immunostaining are similar to those in the adult, although transient FGF1-expressing cells have been detected in the proximal inner nuclear layer before eye opening. These patterns are consistent with a major involvement of FGF1, FGFR1, and FGFR2 in ganglion cell maturation (during development) and survival (in the adult). Moreover, FGF1 may affect amacrine cell development, whereas Müller cells appear to be regulated via both FGFR1 and FGFR2 throughout postnatal life. In immature retinas, large numbers of amacrine cells, including those containing calbindin and glycine, display both FGF1 and FGFR2 immunoreactivities in their nuclei, suggesting an action of FGF1 on FGFR2 leading to the maturation of these amacrine cells during a restricted period of postnatal development. This work was supported by funding from the Italian Ministry of Education.     

The non-peptide kinin receptor antagonists FR 173657 and SSR 240612: preclinical evidence for the treatment of skin inflammation

Peptide and non-peptide kinin receptor antagonists were evaluated in cutaneous inflammation models in mice. Topical and i.p. application of kinin B(1) and B(2) receptor antagonists caused a significant inhibition of the capsaicin-induced cutaneous neurogenic inflammatory response. The calculated mean ID(50) for Hoe140 and SSR240612 were 23.83 (9.14-62.14) nmol/kg and 0.23 (0.15-0.36) mg/ear, respectively. The I(max) observed for Hoe140, SSR240612, R-715, FR173657, and FR plus SSR were 61+/-5%, 56+/-3%, 65+/-10%, 48+/-8%, and 52+/-4%, respectively. Supporting these results, double B(1) and B(2) kinin receptors knockout mice showed a significant inhibition of capsaicin-induced ear oedema (42+/-7%). However, mice with a single deletion of either B(1) or B(2) receptors exhibited no change in their capsaicin responses. In contrast, all of the examined kinin receptor antagonists were unable to inhibit the oedema induced by TPA and the results from knockout mice confirmed the lack of kinin receptor signaling in this model. These findings show that kinin receptors are present in the skin and that both kinin receptors seem to be important in the neurogenic inflammatory response. Moreover, non-peptide antagonists were very effective in reducing skin inflammation when topically applied, thereby suggesting that they could be useful tools in the treatment of some skin inflammatory diseases.     

Crucial Role for BAFF-BAFF-R Signaling in the Survival and Maintenance of Mature B Cells

Defects in the expression of either BAFF (B cell activating factor) or BAFF-R impairs B cell development beyond the immature, transitional type-1 stage and thus, prevents the formation of follicular and marginal zone B cells, whereas B-1 B cells remain unaffected. The expression of BAFF-R on all mature B cells might suggest a role for BAFF-R signaling also for their in vivo maintenance. Here, we show that, 14 days following a single injection of an anti-BAFF-R mAb that prevents BAFF binding, both follicular and marginal zone B cell numbers are drastically reduced, whereas B-1 cells are not affected. Injection of control, isotype-matched but non-blocking anti-BAFF-R mAbs does not result in B cell depletion. We also show that this depletion is neither due to antibody-dependent cellular cytotoxicity nor to complement-mediated lysis. Moreover, prevention of BAFF binding leads to a decrease in the size of the B cell follicles, an impairment of a T cell dependent humoral immune response and a reduction in the formation of memory B cells. Collectively, these results establish a central role for BAFF-BAFF-R signaling in the in vivo survival and maintenance of both follicular and marginal zone B cell pools.     

Probing the Flexibility of Large Conformational Changes in Protein Structures through Local Perturbations

Protein conformational changes and dynamic behavior are fundamental for such processes as catalysis, regulation, and substrate recognition. Although protein dynamics have been successfully explored in computer simulation, there is an intermediate-scale of motions that has proven difficult to simulate—the motion of individual segments or domains that move independently of the body the protein. Here, we introduce a molecular-dynamics perturbation method, the Rotamerically Induced Perturbation (RIP), which can generate large, coherent motions of structural elements in picoseconds by applying large torsional perturbations to individual sidechains. Despite the large-scale motions, secondary structure elements remain intact without the need for applying backbone positional restraints. Owing to its computational efficiency, RIP can be applied to every residue in a protein, producing a global map of deformability. This map is remarkably sparse, with the dominant sites of deformation generally found on the protein surface. The global map can be used to identify loops and helices that are less tightly bound to the protein and thus are likely sites of dynamic modulation that may have important functional consequences. Additionally, they identify individual residues that have the potential to drive large-scale coherent conformational change. Applying RIP to two well-studied proteins, Dihdydrofolate Reductase and Triosephosphate Isomerase, which possess functionally-relevant mobile loops that fluctuate on the microsecond/millisecond timescale, the RIP deformation map identifies and recapitulates the flexibility of these elements. In contrast, the RIP deformation map of α-lytic protease, a kinetically stable protein, results in a map with no significant deformations. In the N-terminal domain of HSP90, the RIP deformation map clearly identifies the ligand-binding lid as a highly flexible region capable of large conformational changes. In the Estrogen Receptor ligand-binding domain, the RIP deformation map is quite sparse except for one large conformational change involving Helix-12, which is the structural element that allosterically links ligand binding to receptor activation. RIP analysis has the potential to discover sites of functional conformational changes and the linchpin residues critical in determining these conformational states.