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The aim of this study was to evaluate the cytotoxicity of the prepared Gold Nanoparticles from Triphala Plant extract and of the indigenous mouthwash prepared from these Triphala based gold nanoparticles (AuNP’s) using Artemia salina test. Its objective was to assess whether the prepared mouthwash was safe and biocompatible to use as an alternative to commercial mouthwashes in orthodontic patients to reduce the plaque load around brackets and bands as an adjuvant to tooth brushing. 15 g of brine shrimps was hatched in salt solution after incubating them for 24 – h. Prepared AuNP’s at concentrations 0µL (microliter), 5µL, 10µL, 20µL, 30µL, 50µL were added using a micropipette in one of the microtiter plates and mouthwash prepared from these AuNP’s were added at concentrations 0µL, 10µL, 20µL, 30µL, 40µL, 50µL in another microtiter plate. 10 Hatched nauplii (brine shrimps) were added in each of these microtiter plates and incubated for 24 – h. All nauplii were alive at the end of 24 – h in the microtiter plate containing AuNP’s. At concentrations 0µL, 10µL, 20µL, 30µL nauplii were alive and at 40µL, 50µL concentrations no nauplii were alive at the end of 24 – h in the microtiter plate containing mouthwash. Triphala mediated AuNP’s showed no toxic effect on the brine shrimps. AuNP’s based mouthwash showed excellent compatibility at lower concentrations. Mild toxic effects were elicited at higher concentrations. Hence, AuNP’s could be used to prepare mouthwashes at 20- 30 µL concentrations.

A Mn(ii)-catalyzed efficient C-H alkylation of imidazoheterocycles with methyl ketones has been developed via dehydrogenative C(sp3)-C(sp2) coupling which can serve as a novel approach toward hydrocarboxylated imidazolopyridines. The merit of this strategy is illustrated by excellent functional group tolerance and the use of cheap and readily available starting materials.

Wet reclamation of waste sodium silicate-bonded sand produces much alkaline sewage and causes pollution. Recycling water glass from wet reclamation sewage of the waste sodium silicate-bonded sand can solve pollution issues and generate economic benefits. In this work, the wet reclamation sewage was filtered, and the filtrate was causticized with a quicklime powder to produce a lye. The effects of causticization temperature, causticization time, and the amount of quicklime powder on the causticization rate were studied. The lye was used to dissolve the silica in the filtration residue to prepare a sodium silicate solution. The effects of the mass of filtration residue, dissolution temperature, and dissolution time on sodium silicate modulus were studied. Finally, the recycled water glass was obtained by concentrating the sodium silicate solution, and the bonding strength of the recycled water glass was tested. The results showed that the causticization rate could be improved by increasing the amount of quicklime powder, causticization temperature, and causticization time, and the highest causticization rate was above 92%. Amorphous silica in the filtration residue dissolved in the lye. Increasing the amount of the filtration residue, dissolution temperature, and dissolution time could improve the sodium silicate modulus. The bonding strength of the recycled water glass was close to that of commercial water glass. The recycled water glass could be used as a substitute for the commercial water glass.

DNA nanomaterials have attracted ever-increasing attention over the past decades due to their incomparable programmability and multifunctionality. In particular, DNA dendrimer nanostructures, as a major research focus, have been applied in the fields of biosensing, therapeutics, and protein engineering, benefiting from their highly branched configuration. With the aid of specific recognition probes and inherent signal amplification, DNA dendrimers can achieve ultrasensitive detection of nucleic acids, proteins, cells, and other substances, such as lipopolysaccharides (LPS), adenosine triphosphate (ATP), and exosomes. By virtue of their void-containing structures and biocompatibility, DNA dendrimers can deliver drugs or functional nucleic acids into target cells in chemotherapy, immunotherapy, and gene therapy. Furthermore, DNA dendrimers are being applied in protein engineering for efficient directed evolution of proteins. This review summarizes the main research progress of DNA dendrimers, concerning their assembly methods and biomedical applications as well as the emerging challenges and perspectives for future research.

Transposable elements exist widely throughout plant genomes and play important roles in plant evolution. Auxin is an important regulator that is traditionally associated with root development and drought stress adaptation. The DEEPER ROOTING 1 (DRO1) gene is a key component of rice drought avoidance. Here, we identified a transposon that acts as an autonomous auxin-responsive promoter and its presence at specific genome positions conveys physiological adaptations related to drought avoidance. Rice varieties with high and auxin-mediated transcription of DRO1 in the root tip show deeper and longer root phenotypes and are thus better adapted to drought. The INDITTO2 transposon contains an auxin response element and displays auxin-responsive promoter activity; it is thus able to convey auxin regulation of transcription to genes in its proximity. In the rice Acuce, which displays DRO1-mediated drought adaptation, the INDITTO2 transposon was found to be inserted at the promoter region of the DRO1 locus. Transgenesis-based insertion of the INDITTO2 transposon into the DRO1 promoter of the non-adapted rice variety Nipponbare was sufficient to promote its drought avoidance. Our data identify an example of how transposons can act as promoters and convey hormonal regulation to nearby loci, improving plant fitness in response to different abiotic stresses. This article is protected by copyright. All rights reserved.

Hypertrophic scar (HS) has been considered as a great concern for patients and a challenging problem for clinicians as it can cause functional debility, cosmetic disfigurement and psychological trauma. Although many methods have been developed to prevent and treat HS, the scarless healing is still a worldwide medical problem. In this study, palmatine-loaded poly( ε-caprolactone)/gelatin nanofibrous scaffolds (PCL/GE/PALs) were fabricated by electrospinning, and their effects on wound healing and HS formation were investigated. These nanofiber mats exhibit good antibacterial and antioxidant activities. In vitro studies indicate PCL/GE/PAL scaffolds can facilitate the adhesion, spreading and proliferation of L929 fibroblasts. In vivo tests demonstrate the full-thickness wounds treated with PCL/GE/PAL scaffolds heal about 3.5 days earlier than those in the control group. Scar elevation index measurements and histological analyses reveal PCL/GE/PAL scaffolds significantly inhibit HS formation, with the decrease in the thickness of dermis and epidermis, the number of fibroblasts, as well as the density of collagen and microvascular. Accelerating wound healing and inhibiting HS formation of these scaffolds are contributed to the sustained release of palmatine. The present work validates the potential use of palmatine-loaded electrospun nanofibrous scaffold PCL/GE/PALs as a functional wound dressing for healing wounds and preventing HS formation.

In this paper, an ultrasound method was applied in the wet reclamation process, and the feasibility and parameters of the ultrasound-assisted wet reclamation were studied, and the mechanism of the ...

Escherichia coli has been considered as a promising host for the production of N-glycosylated therapeutic proteins and glycoconjugate vaccines. In this study, we developed a simple and efficient strategy for improving the production of N-glycosylated recombinant proteins by combining auto-induction with the use of a leaky E. coli strain. A leaky E. coli strain, designated as CLM37-Δlpp, was engineered by deleting the Braun’s lipoprotein (lpp) gene of E. coli strain CLM37. Three distinct acceptor model N-glycosylated proteins, glyco-tagged human tenth fibronectin type III domain (FN3-Gly), enhanced green fluorescent protein (eGFP-Gly), and scFv of vascular endothelial growth factor receptor 3 (scFv-VEGFR3-Gly) were then expressed in CLM37-Δlpp, which carried an N-glycosylation machinery from Campylobacter jejuni for the investigation of glycoprotein production. As much as 75%, 65%, and 60% of the glycosylated FN3-Gly, eGFP-Gly, and scFv-VEGFR3-Gly, respectively, were found in the culture medium. The yields of glycosylated FN3-Gly, eGFP-Gly, and scFv-VEGFR3-Gly were 106 ± 7.4 mg/L, 65 ± 2.5 mg/L, and 62 ± 4.3 mg/L, respectively, which were more than three folds the corresponding yields obtained when these proteins were expressed in CLM37, the unmodified strain. The results suggested that this simplified approach could improve the production of N-glycosylated proteins with E. coli to facilitate large-scale production.

To improve the production yield of N-glycosylated anti-VEGFR2 (vascular endothelial growth factor receptor 2) monobody (FN3VEGFR2-Gly) in lpp knockout Escherichia coli cells harboring Campylobacter jejuni N-glycosylation pathway. The leaky CLM37-Δlpp strain efficiently secreted FN3VEGFR2-Gly into culture medium. The extracellular levels of glycosylated FN3VEGFR2-Gly in CLM37-Δlpp culture medium were approximately 11 and 15 times higher compared to those in CLM37 cells via IPTG and auto-induction, respectively. In addition, the highest level of total glycosylated FN3VEGFR2-Gly (70 ± 3.4 mg/L) was found in culture medium via auto-induction. Furthermore, glycosylated FN3VEGFR2-Gly was more stable than unglycosylated FN3VEGFR2-Gly in this expression system, but their bioactivities were relatively similar. Lpp knockout leaky E. coli strain combined with auto-induction method can enhance the extracellular production of homogenous N-glycosylated FN3VEGFR2-Gly, and facilitate the downstream protein purification. The findings of this study may provide practical implications for the large-scale production and cost-effective harvesting of N-glycosylation proteins.

Metabolic bone diseases are global public health concerns and are primarily caused by uncontrolled osteoclast (OC) formation and activation. During OC differentiation, intracellular reactive oxygen species (ROS) stimulated by receptor activator of nuclear factor kappa-B ligand (RANKL) can serve as the signaling molecules to promote osteoclastic genes expression. Nuclear factor erythroid-2 related factor 2 (NRF2), a master mediator of cellular antioxidant response, also plays a critical role in OC differentiation through the regulation of redox homeostasis. In this study, we investigated the effects of three NRF2 inducers on osteoclastogenesis, including Bardoxolone methyl (CDDO-Me), Sulforaphane (SFN), and tert-butylhydroquinone (tBHQ). By treating RAW cells with three compounds, we found that NRF2 was activated and its downstream antioxidant genes were upregulated, and the RANKL-induced intracellular ROS production and osteoclastogenesis were impaired. Additionally, the expression of nuclear factor of activated T cells c1 (NFATC1), C-FOS and tumor necrosis factor alpha (TNFα) were inhibited after acute exposures (6 h) to the three compounds. Furthermore, suppressed the expression of osteoclast differentiation-associated genes, tartrate-resistant acid phosphatase (TRAP), cathepsin K (CTSK), matrix metalloproteinase-9 (MMP-9) and dendritic cell-specific transmembrane protein (DC-STAMP) were observed after prolonged exposures (5 days) to the compounds. Taken together, these results suggest that CDDO-Me, SFN and tBHQ attenuate RANKL-induced osteoclastogenesis via activation of NRF2-mediated antioxidant response. Among these compounds, relatively low concentrations of CDDO-Me showed stronger active and inhibitory effects on antioxidant response and osteoclastogenesis, respectively.

How the nervous system regulates bone remodeling is an exciting area of emerging research in bone biology. Accumulating evidence suggest that neurotransmitter-mediated inputs from neurons may act directly on osteoclasts. Dopamine is a neurotransmitter that can be released by hypothalamic neurons to regulate bone metabolism through the hypothalamic-pituitary-gonadal axis. Dopamine is also present in sympathetic nerves that penetrate skeletal structures throughout the body. It has been shown that dopamine suppresses osteoclast differentiation via a D2-like receptors (D2R)-dependent manner, but the intracellular secondary signaling pathway has not been elucidated. In this study, we found that cAMP-response element binding protein (CREB) activity responds to dopamine treatment during osteoclastogenesis. Considering the critical role of CREB in osteoclastogenesis, we hypothesize that CREB may be a critical target in dopamine’s regulation of osteoclast differentiation. We confirmed that D2R is also present in RAW cells and activated by dopamine. Binding of dopamine to D2R inhibits the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) signaling pathway which ultimately decreases CREB phosphorylation during osteoclastogenesis. This was also associated with diminished expression of osteoclast markers that are downstream of CREB. Pharmacological activation of adenylate cyclase (to increase cAMP production) and PKA reverses the effect of dopamine on CREB activity and osteoclastogenesis. Therefore, we have identified D2R/cAMP/PKA/CREB as a candidate pathway that mediates dopamine’s inhibition of osteoclast differentiation. These findings will contribute to our understanding of how the nervous and skeletal systems interact to regulate bone remodeling. This will enable future work toward elucidating the role of the nervous system in bone development, repair, aging, and degenerative disease.

Purpose Radiation therapy for head and neck cancer commonly leads to radiation sialadenitis. Emerging evidence has indicated that phenylephrine pretreatment reduces radiosensitivity in the salivary gland; however, the underlying cytoprotective mechanism remains unclear. Nicotinamide phosphoribosyltransferase ( NAMPT ) is not only a key enzyme for the nicotinamide adenine dinucleotide salvage pathway, but also a cytokine participating in cell survival, metabolism, and longevity, with a broad effect on cellular functions in physiology and pathology. However, the regulatory events of NAMPT in response to the irradiated salivary gland are unknown. Methods and Materials The cell viability of primary cultured submandibular gland cells was determined using the PrestoBlue assay. NAMPT expression was measured using reverse transcriptase polymerase chain reaction and Western blotting in vitro and in vivo. Silent information regulator 1 ( SIRT1 ) and phosphorylated Akt protein levels were examined by Western blotting. The cellular locations of NAMPT and SIRT1 were detected by immunohistochemistry. NAMPT promoter activity was assessed using the luciferase reporter gene assay. Results NAMPT was mainly distributed in the cytoplasm of granular convoluted tubule cells and ductal cells in normal submandibular glands. mRNA and protein expression of NAMPT was downregulated after radiation but upregulated with phenylephrine pretreatment both in vivo and in vitro. Moreover, the protein expression of phosphorylated Akt and SIRT1 was decreased in irradiated glands, and phenylephrine pretreatment restored the expression of both. SIRT1 was mainly located in the cell nucleus and cytoplasm in the normal submandibular gland. Phenylephrine dramatically enhanced the expression of SIRT1 , which was significantly reduced by radiation. Furthermore, phenylephrine induced a marked increase of NAMPT promoter activity. Conclusions These findings reveal the regulatory mechanisms of NAMPT expression, which help to understand the mechanism of the cytoprotective role of phenylephrine on irradiated tissues.

Escherichia coli cells have been considered as promising hosts for producing N-glycosylated proteins since the successful production of N-glycosylated protein in E. coli with the pgl (N-linked protein glycosylation) locus from Campylobacter jejuni. However, one hurdle in producing N-glycosylated proteins in large scale using E. coli is inefficient glycan glycosylation. In this study, we developed a strategy for the production of N-glycosylated proteins with high efficiency via an optimized auto-induction method. The 10th human fibronectin type III domain (FN3) was engineered with native glycosylation sequon DFNRSK and optimized DQNAT sequon in C-terminus with flexible linker as acceptor protein models. The resulting glycosylation efficiencies were confirmed by Western blots with anti-FLAG M1 antibody. Increased efficiency of glycosylation was obtained by changing the conventional IPTG induction to auto-induction method, which increased the glycosylation efficiencies from 60% and 75% up to 90% and 100% respectively. Moreover, in the condition of inserting the glycosylation sequon in the loop of FN3 (the acceptor sequon with local structural conformation), the glycosylation efficiency was increased from 35% to 80% by our optimized auto-induction procedures. To justify the potential for general application of the optimized auto-induction method, the reconstituted lsg locus from Haemophilus influenzae and PglB from C. jejuni were utilized, and this led to 100% glycosylation efficiency. Our studies provided quantitative evidence that the optimized auto-induction method will facilitate the large-scale production of pure exogenous N-glycosylation proteins in E. coli cells.

Waterflood, or other forms of immiscible displacement, have been the most widely used mechanism for secondary recovery and the determination of individual phase's relative permeability has been crucial for many reservoir engineering applications. Traditionally there are two ways of determining the relative permeability from laboratory experiments: the steady state experiment and the unsteady state experiment. The steady state experiment has the disadvantages of being time-consuming, obtaining few points on the relative permeability curves, as well as not being identical to the true reservoir displcacement process. On the other hand, the Johnson-Bossler-Naumann (JBN) method, based on Buckley-Leverett (B-L) immiscible displacement theory, is the standard procedure used to determine relative permeability from unsteady state experiments. However, the neglect of capillary pressure effect in the Buckley-Leverett theory has placed limitations on the method, such as requiring high flow rates. In order to remove the limitations of the displacement experiments and give more flexibility to the experiment configuration, an analytic model with capillary corrections is presented in the context of viscous dominated flow to study the impact caused by capillarity. The solution shows a fixed water-oil saturation bank at the outlet whose magnitude depends upon the wettability of the system. The analytic model is able to predict the saturation profile, considering capillarity, under various rock and fluid properties and experimental configurations. Therefore, the model could be used to calculate accurate relative permeability by matching experimental pressure drop and production reponses, correcting for capillarity. Also, through this method, a dimensionless parameter could be provided to quantify the effect caused by capillary end effect based on the length scale affected. It reflects the influence from a variety of aspects, not only from the experimental settings.

Anabaena Sensory Rhodopsin (ASR) is a unique microbial rhodopsin that displays photocromism, interacts with soluble transducer, and may be involved in gene regulation. Here we report nearly complete spectroscopic 13C and 15N assignments of ASR reconstituted in lipids, obtained using two- and three-dimensional magic angle spinning solid state NMR spectroscopy on alternately 13C labeled samples. The obtained chemical shifts are used to characterize the protein backbone conformation. They suggest that lipid-reconstituted ASR has a fold generally similar to that seen in earlier X-ray studies, but with a number of important differences. SSNMR detects double conformations for a number of residues on the cytoplasmic side.

Overexpression of isotope-labeled multi-spanning eukaryotic membrane proteins for structural NMR studies is often challenging. On the one hand, difficulties with achieving proper folding, membrane insertion, and native-like post-translational modifications frequently disqualify bacterial expression systems. On the other hand, eukaryotic cell cultures can be prohibitively expensive. One of the viable alternatives, successfully used for producing proteins for solution NMR studies, is yeast expression systems, particularly Pichia pastoris. We report on successful implementation and optimization of isotope labeling protocols, previously used for soluble secreted proteins, to produce homogeneous samples of a eukaryotic seven-transmembrane helical protein, rhodopsin from Leptosphaeria maculans. Even in shake-flask cultures, yields exceeded 5 mg of purified uniformly (13)C,(15)N-labeled protein per liter of culture. The protein was stable (at least several weeks at 5°C) and functionally active upon reconstitution into lipid membranes at high protein-to-lipid ratio required for solid-state NMR. The samples gave high-resolution (13)C and (15)N solid-state magic angle spinning NMR spectra, amenable to a detailed structural analysis. We believe that similar protocols can be adopted for challenging mammalian targets, which often resist characterization by other structural methods.

In this work, we demonstrate that Homonuclear Rotary Resonance Recoupling (HORROR) can be used to reintroduce carbonyl–carbonyl interresidue dipolar interactions and to achieve efficient polarization transfer between carbonyl atoms in uniformly 13C,15N-labeled peptides and proteins. We show that the HORROR condition is anisotropically broadened and overall shifted to higher radio frequency intensities because of the CSA effects. These effects are analyzed theoretically using Average Hamiltonian Theory. At spinning frequencies used in this study, 22 kHz, this broadening is experimentally found to be on the order of a kilohertz at a proton field of 600 MHz. To match HORROR condition over all powder orientations, variable amplitude radio frequency (RF) fields are required, and efficient direct transfers on the order of 20–30% can be straightforwardly established. Two- and three-dimensional chemical shift correlation experiments establishing long-range interresidue connectivities (e.g., (N[i]–CO[i − 2])) are demonstrated on the model peptide N-acetyl-valine-leucine, and on the third immunoglobulin binding domain of protein G. Possible future developments are discussed.

Proteorhodopsins are typical retinal-binding light-driven proton pumps of heptahelical architecture widely distributed in marine and freshwater bacteria. Recently, we have shown that green proteorhodopsin (GPR) can be prepared in a lipid-bound state that gives well-resolved magic angle spinning (MAS) NMR spectra in samples with different patterns of reverse labelling. Here, we present 3D and 4D sequential chemical shift assignments identified through experiments conducted on a uniformly (13)C,(15)N-labelled sample. These experiments provided the assignments for 153 residues, with a particularly high density in the transmembrane regions ( approximately 74% of residues). The extent of assignments permitted a detailed examination of the secondary structure and dynamics in GPR. In particular, we present experimental evidence of mobility of the protein's termini and of the A-B, C-D, and F-G loops, the latter being possibly coupled to the GPR ion-transporting function.

The genome of thylakoidless cyanobacterium Gloeobacter violaceus encodes a fast-cycling rhodopsin capable of light-driven proton transport. We characterize the dark state, the photocycle, and the proton translocation pathway of GR spectroscopically. The dark state of GR contains predominantly all-trans-retinal and, similar to proteorhodopsin, does not show the light/dark adaptation. We found an unusually strong coupling between the conformation of the retinal and the site of Glu132, the homolog of Asp96 of BR. Although the photocycle of GR is similar to that of proteorhodopsin in general, it differs in accumulating two intermediates typical for BR, the L-like and the N-like states. The latter state has a deprotonated cytoplasmic proton donor and is spectrally distinct from the strongly red-shifted N intermediate known for proteorhodopsin. The proton uptake precedes the release and occurs during the transition to the O intermediate. The proton translocation pathway of GR is similar to those of other proton-pumping rhodopsins, involving homologs of BR Schiff base proton acceptor and donor Asp85 and Asp96 (Asp121 and Glu132). We assigned a pair of FTIR bands (positive at 1749 cm−1 and negative at 1734 cm−1) to the protonation and deprotonation, respectively, of these carboxylic acids.

The 18.5 kDa isoform of myelin basic protein (MBP) is a peripheral membrane protein that maintains the structural integrity of the myelin sheath of the central nervous system by conjoining the cytoplasmic leaflets of oligodendrocytes and by linking the myelin membrane to the underlying cytoskeleton whose assembly it strongly promotes. It is a multifunctional, intrinsically disordered protein that behaves primarily as a structural stabilizer, but with elements of a transient or induced secondary structure that represent binding sites for calmodulin or SH3-domain-containing proteins, inter alia. In this study we used solid-state NMR (SSNMR) and Fourier transform infrared (FTIR) spectroscopy to study the conformation of 18.5 kDa MBP in association with actin microfilaments and bundles. FTIR spectroscopy of fully (13)C,(15)N-labeled MBP complexed with unlabeled F-actin showed induced folding of both protein partners, viz., some increase in beta-sheet content in actin, and increases in both alpha-helix and beta-sheet content in MBP, albeit with considerable extended structure remaining. Solid-state NMR spectroscopy revealed that MBP in MBP-actin assemblies is structurally heterogeneous but gains ordered secondary structure elements (both alpha-helical and beta-sheet), particularly in the terminal fragments and in a central immunodominant epitope. The overall conformational polymorphism of MBP is consistent with its in vivo roles as both a linker (membranes and cytoskeleton) and a putative signaling hub.

We describe a simple protocol to achieve homonuclear J-decoupling in the indirect dimensions of multidimensional experiments, and to enhance spectral resolution of the backbone Calpha carbons in the 3D NCACX experiment. In the proposed protocol, the refocusing of the Calpha-CO homonuclear J-couplings is achieved by applying an off-resonance selective pi pulse to the CO spectral region in the middle of Calpha chemical shift evolution. As is commonly used in solution NMR, a compensatory echo period is used to refocus the unwanted chemical shift evolution of Calpha spins, which takes place during the off-resonance selective pulse. The experiments were carried out on the beta1 immunoglobulin binding domain of protein G (GB1). In GB1, such implementation results in significantly reduced line widths, and leads to an overall sensitivity enhancement.

Numerous fungal genomes encode homologs of bacteriorhodopsin (BR), but only two fungal rhodopsins were overexpressed and characterized spectroscopically. Neurospora rhodopsin (NR) is a slow-cycling sensory rhodopsin-like protein, while Leptosphaeria rhodopsin (LR) is a BR-like proton pump. Recently, we found that a conservative replacement of the cytoplasmic proton donor Asp150 by Glu converts LR into an NR-like protein. In this work, we search for structural reasons for the dramatic differences in their photochemistry by mutating the hydrogen-bonding partner of Asp150 (Thr87) and three additional residues (Thr233, Asp248, and Gly271) selected by comparison of the primary structures of NR and LR. We conclude that while these residues may contribute to the differences between LR and NR, they are not crucial for the optimization of the Schiff base reprotonation by Asp150, and that the dramatic effect of the D150E mutation is not a simple result of the introduction of a bulkier glutamate sidechain.

Radiotherapy for malignant tumors of the head and neck commonly leads to radiation-induced sialadenitis as a result of radiation-induced salivary gland dysfunction. We demonstrated previously that phenylephrine could protect the irradiated submandibular gland against apoptosis, although the mechanism is unclear. In this study, we investigated the influence of phenylephrine pretreatment on the expressions of aquaporin 5 (AQP5) and c-Jun N-terminal kinase (JNK) that were presumed to have a role in radiation-induced salivary gland dysfunction. Rats pretreated with phenylephrine (5 mg/kg) were locally irradiated (20 Gy) in the head and neck region. The submandibular glands were removed on day 7 after irradiation. The expression of AQP5 and activation of JNK were measured by immunohistochemistry and Western blot. The localization of AQP5 at the apical and lateral plasma membrane of acinar cells was significantly reduced by irradiation, but markedly enhanced with phenylephrine pretreatment. The protein expression of AQP5 was decreased by 84.91% in irradiated glands, whereas it was fully recovered to the control level in phenylephrine-pretreated glands. Moreover, many acinar, ductal and granular convoluted tubular cells in the irradiated glands exhibited intense immunoreactivity for p-JNK, while in the phenylephrine-pretreated irradiated glands, only a few acinar cells exhibited very faint immunoreactivity for p-JNK. The protein expression level of p-JNK was increased by 41.65% in the irradiated alone glands, but was significantly decreased in the phenylephrine-pretreated irradiated glands. These results suggest that the protective mechanism of phenylephrine might be related to the improved expression of AQP5 and decreased activation of JNK. Pretreatment with phenylephrine in patients undergoing radiotherapy may provide a helpful strategy for suppression of radiation-induced sialadenitis.

It was found recently that Anabaena sensory rhodopsin (ASR), which possibly serves as a photoreceptor for chromatic adaptation, interacts with a soluble cytoplasmic transducer. The X-ray structure of the transducer-free protein revealed an extensive hydrogen-bonded network of amino acid residues and water molecules in the cytoplasmic half of ASR, in high contrast to its haloarchaeal counterparts. Using time-resolved spectroscopy of the wild-type and mutant ASR in the visible and infrared ranges, we tried to determine whether this hydrogen-bonded network is used to translocate protons and whether those proton transfers are important for interaction with the transducer. We found that the retinal Schiff base deprotonation, which occurs in the M intermediate of the photocycle of all-trans-ASR, results in protonation of Asp217 on the cytoplasmic side of the protein. The deprotonation of the Schiff base induces a conformational change of ASR observed through the perturbation of associated lipids. We suggest that the cytoplasmic shuttling of protons in the photocycle of all-trans-ASR and the ensuing conformational changes might activate the transducer. Consequently, the M intermediate may be the signaling state of ASR.

A homologue of type I rhodopsin was found in the unicellular Gloeobacter violaceus PCC7421, which is believed to be primitive because of the lack of thylakoids and peculiar morphology of phycobilisomes. The Gloeobacter rhodopsin (GR) gene encodes a polypeptide of 298 amino acids. This gene is localized alone in the genome unlike cyanobacterium Anabaena opsin, which is clustered together with 14 kDa transducer gene. Amino acid sequence comparison of GR with other type I rhodopsin shows several conserved residues important for retinal binding and H+ pumping. In this study, the gene was expressed in Escherichia coli and bound all-trans retinal to form a pigment (lambda max = 544 nm at pH 7). The pK(a) of proton acceptor (Asp121) for the Schiff base, is approximately 5.9, so GR can translocate H+ under physiological conditions (pH 7.4). In order to prove the functional activity in the cell, pumping activity was measured in the sphaeroplast membranes of E. coli and one of Gloeobacter whole cell. The efficient proton pumping and rapid photocycle of GR strongly suggests that Gloeobacter rhodopsin functions as a proton pumping in its natural environment, probably compensating the shortage of energy generated by chlorophyll-based photosynthesis without thylakoids.

The HslU-HslV complex functions as a bacterial proteasome, degrading substrate polypeptides to preserve cellular homeostasis. Here, we use methyl-Transverse Relaxation-Optimized Spectroscopy (TROSY) and highly deuterated, methyl-protonated samples to study the 230 kDa dodecameric HslV protease component that is structurally homologous to the stacked pair of β7-rings of the proteasome. Chemical shift assignments for over 95% of the methyl groups are reported. From the pH dependence of methyl chemical shifts, a pKa of 7.7 is measured for the amine group of the catalytic residue T1, confirming that it can act as a proton acceptor during the initial step in substrate proteolysis. Analyses involving a series of single site mutants in HslV, localized to HslU binding sites or regions undergoing significant changes on HslU binding, have identified hot spots whose perturbation leads to an allosteric pathway of propagated changes in structure and ultimately, substrate proteolysis efficiency. HslV plasticity is explored through methyl-TROSY (13)C relaxation dispersion experiments that are sensitive to millisecond timescale dynamics. The data support a dynamic coupling between residues involved in both HslU and substrate binding and residues localized to the active sites of HslV that facilitate the allostery between these distal sites. An important role for dynamics has also been observed in the archaeal proteasome, suggesting a more generally conserved role of motion in the function of these barrel-like protease structures.

The oxidation kinetics of the Ti3Al based alloys with Nb (0 to 20 at%) or Si (0 to 15 at%) addition was carefully measured at 800 and 900°C, respectively. The oxide scale was characterised by high temperature in-situ XRD, SEM, EPMA and conventional XRD. The weight gains of the alloys were greatly reduced by Nb or Si addition. The improvement of the oxidation resistance in the Ti3Al alloys by Nb addition is attributed to the formation of a compact scale. The formation of a compact scale is favoured not only by doping effect but also by the TiN layer which barriers the outward Al diffusion into the scale to form porous intermixed titania/alumina. Si addition promotes the formation of a compact TiO2 layer in the oxide scale and the formation of SiO2, thus the oxidation resistance of the Ti3Al based alloys is improved. The oxidation behaviour of the alloys with Si addition is a combinative function of the α2-Ti3Al matrix and the Ti5Si3 silicide phase.

The frequency-dependent moment (FDM) method proposed by Iwata [Chem. Phys. Lett. 102, 544 (1983)] has been applied to the calculation of the second- and third-order frequency-dependent polarizabilities. It is demonstrated that the FDM ab initio calculations on the HF molecule give the same numerical results as the sum-over-state (SOS) calculations. It is important that the FDM requires only the ground-state eigensolution of the CI matrix while the SOS requires all the eigensolutions. Calculations were also made on H2O and CH4. The FDM method becomes very effective when the CI matrix is so large that its direct diagonalization becomes formidable.

Determination of structure of integral membrane proteins, especially in their native environment, is a formidable challenge in structural biology. Here we demonstrate that magic angle spinning solid-state NMR spectroscopy can be used to determine structures of membrane proteins reconstituted in synthetic lipids, an environment similar to the natural membrane. We combined a large number of experimentally determined interatomic distances and local torsional restraints to solve the structure of an oligomeric membrane protein of common seven-helical fold, Anabaena sensory rhodopsin (ASR). We determined the atomic resolution detail of the oligomerization interface of the ASR trimer, and the arrangement of helices, side chains and the retinal cofactor in the monomer.

We used high-resolution proton-detected multidimensional NMR to study the solvent-exposed parts of a seven-helical integral membrane proton pump, proteorhodopsin (PR). PR samples were prepared by growing the apoprotein on fully deuterated medium and reintroducing protons to solvent-accessible sites through exchange with protonated buffer. This preparation leads to NMR spectra with proton resolution down to ca. 0.2 ppm at fast spinning (28 kHz) in a protein back-exchanged at a level of 40%. Novel three-dimensional proton-detected chemical shift correlation spectroscopy allowed for the identification and resonance assignment of the solvent-exposed parts of the protein. Most of the observed residues are located at the membrane interface, but there are notable exceptions, particularly in helix G, where most of the residues are susceptible to H/D exchange. This helix contains Schiff base-forming Lys231, and many conserved polar residues in the extracellular half, such as Asn220, Tyr223, Asn224, Asp227, and Asn230. We proposed earlier that high mobility of the F-G loop may transiently expose a hydrophilic cavity in the extracellular half of the protein, similar to the one found in xanthorhodopsin. Solvent accessibility of helix G is in line with this hypothesis, implying that such a cavity may be a part of the proton-conducting pathway lined by this helix.

Proteorhodopsin (PR) is a recently discovered ubiquitous eubacterial retinal-binding light-driven proton pump. Almost 1000 PR variants are widely distributed in species of marine and freshwater bacteria, suggesting PR's important photobiological role. PR is a typical seven-transmembrane alpha-helical membrane protein and as such poses a significant challenge to structural studies. Attempts to crystallize PR have not been successful, and its three-dimensional structure remains unknown. We show that PR reconstituted in lipids gives well-resolved magic-angle spinning NMR spectra of high signal-to-noise ratio. We report sequential assignment of 13C and 15N backbone and side-chain chemical shifts for 103 of 238 residues in PR, achieved by three-dimensional chemical shift correlation experiments performed on two samples with different patterns of reverse labeling. The chemical shift analysis gives a number of important structural insights not available from other studies: we have established protonation states of several carboxylic acids, identified the boundaries and distortions of transmembrane alpha-helices, and detected secondary structure elements in the loops. We confirmed that internal Asp227, which was proposed to form part of the Schiff base counterion, is ionized, while Glu142, which is located close to the extracellular surface, is neutral, in agreement with earlier predictions. We infer that, similar to bacteriorhodopsin's structure, PR has a proline kink in helix C, a non-proline kink in helix G, a short beta-turn in the B-C loop, and a short alpha-helical segment in the E-F loop.

Many fungal rhodopsins, eukaryotic structural homologues of the archaeal light-driven proton pump bacteriorhodopsin, have been discovered in the course of genome sequencing projects. Recently, two fungal rhodopsins were characterized in vitro and exhibited very different photochemical behavior. Neurospora rhodopsin possesses a slow photocycle and shows no ion transport, reminiscent of sensory rhodopsins, while Leptosphaeria rhodopsin has a fast bacteriorhodopsin-like photocycle and pumps protons light-dependently. Such a dramatic difference is surprising considering the very high degree of sequence homology of the two proteins. In this paper, we investigate whether the chemical structure of a cytoplasmic carboxylic acid, the homologue of Asp-96 of bacteriorhodopsin serving as a proton donor for the retinal Schiff base, can define the photochemical properties of fungal rhodopsins. We studied mutants of Leptosphaeria rhodopsin in which this aspartic acid was replaced with Glu or Asn using spectroscopy in the infrared and visible ranges. We show that Glu at this position is inefficient as a proton donor similar to a nonprotonatable Asn. Moreover, this replacement induces long-range structural perturbations of the retinal environment, as evidenced by changes in the vibrational bands of retinal (especially, hydrogen-out-of-plane modes) and neighboring aspartic acids and water molecules. The conformational coupling of the mutation site to the retinal may be mediated by helical rearrangements as suggested by the changes in amide and proline vibrational bands. We conclude that the difference in the photochemical behavior of fungal rhodopsins from Leptosphaeria and Neurospora may be ascribed, to some extent, to the replacement of the cytoplasmic proton donor Asp with Glu.

The structures, magnetic properties, and magnetic‐entropy‐changes of La1−zRz(Fe1−x−yCoxAly)13 (R=Ce, Pr, Nd, Sm, Gd, and Dy) were studied. It was found that the crystal structures of La(Fe0.73−xCoxAl0.27)13 maintain the LaCo13‐type cubic structure. The relationship between saturation magnetic moments per 3d atom and the number of 3d electrons in the compounds of La(Fe0.73−xCoxAl0.27)13 system can be explained by the rigid‐band model at Co‐rich side and disagrees with the model at Fe‐rich side. When 0.02≤x≤1.0, the compounds have a nearby room temperature Curie temperature, a high saturation magnetization and a high magnetic‐entropy‐change. When x=0.02 and the external magnetic field is 1.11 MA/m, the maximum magnetic‐entropy‐change of the La(Fe0.71Co0.02Al0.27)13 compound is 13 kJ/m3⋅K which is about half of that of metal Gd at the same external magnetic field. The saturation magnetization of the compound increases and the maximum magnetic‐entropy‐change decreases when La is substituted by Ce. The compoun...

An enzymatic method for synthesis of labelled thyroxine glucuronide (T4G) and triiodothyronine glucuronide (T3G) from labelled thyroxine (T4) and triiodothyronine (T3) is presented. The synthetic glucuronides are completely digested by beta-glucuronidase, with recovery of the parent T4 or T3. They have distinctive elution patterns on HPLC and on Sephadex G25 chromatography, and can be clearly separated from T4 and T3 as well as from synthetic T4 sulfate (T4S) and T3 sulfate (T3S). On LH 20 chromatography, elution of T4G and T3G is intermediate between that of T4 and T3 and that of T4S and T3S. T3G can be well separated from other thyronines by HPLC alone, but T4G coelutes with rT3 on HPLC; these are then separated by adding a Sephadex G25 chromatography step. Biosynthetic 131I-T3G and 125I-T4G from the bile of a cat given 131I-T3 and 125I-T4 had similar HPLC chromatographic patterns to those of synthetic T3G and T4G. That the identified peaks from analysis of the bile were indeed T3G and T4G was confirmed by recovery of the parent T3 and T4 after beta-glucuronidase digestion.

Solid-state NMR spectroscopy is an efficient tool for following conformational dynamics of membrane proteins at atomic resolution. We used this technique for the site-specific detection of light-induced hydrogen-deuterium exchange in the lipid-embedded heptahelical transmembrane photosensor Anabaena sensory rhodopsin to pinpoint the location of its conformational changes upon activation. We show that the light-induced conformational changes result in a dramatic, but localized, increase in the exchange in the transmembrane regions. Most notably, the cytoplasmic half of helix G and the cytoplasmic ends of helices B and C exchange more extensively, probably as a result of their relative displacement in the activated state, allowing water to penetrate into the core of the protein. These light-induced rearrangements must provide the structural basis for the photosensory function of Anabaena sensory rhodopsin.

Binding of thyroxine (T4), triiodothyronine (T3) and their glucuronide and sulfate conjugates, T4G, T3G, T4S and T3S, was assessed by equilibrium dialysis in plasma from 6 euthyroid humans and 5 euthyroid cats. In humans, the dialyzable percentages of T4, T4G and T4S were 0.029 +/- 0.003 (mean +/- S.D.), 0.14 +/- 0.04 and 0.08 +/- 0.02 respectively. For T3, T3G and T3S they were 0.31 +/- 0.04, 0.33 +/- 0.07 and 0.22 +/- 0.02. In cats, the corresponding values for T4, T4G and T4S were 0.057 +/- 0.009, 0.20 +/- 0.10 and 0.53 +/- 0.04; for T3, T3G and T3S they were 0.47 +/- 0.08, 0.51 +/- 0.11, and 0.53 +/- 0.11. We conclude that glucuronide conjugation has little or no inhibiting effect on the plasma binding of T3, and that T3S is actually more tightly bound than T3 in human (but not feline) plasma. On the other hand, conjugation to either the glucuronide or the sulfate markedly reduced T4 binding in plasma from both species.