Structural and functional effects of manipulating the degree of methylesterification in a model homogalacturonan with a pseudo-random fungal pectin methylesterase followed by a processive methylesterase

Kim, Yang,Cameron, Randall G.,Williams, Martin A.K.,Luzio, Gary A. Elsevier 2018 Food hydrocolloids Vol.77 No.-

<P><B>Abstract</B></P> <P>We explored the possibility of controlling charge distribution in the homogalacturonan regions of pectin to produce a population of demethylesterified molecules with desirable functional properties by utilizing consecutive treatments with pectin methylesterases (PME) having different modes of action. A fungal PME from <I>Aspergillus aculeatus</I> (<I>Aa</I>-PME), with a pseudo-random mode of action, was used to demethylesterify a extremely high methylesterified HG (DM 94%, average degree of polymerization 246) by reducing the degree of methylesterification (DM) from 94% to either 70% or 80%. A second demethylesterification step, to 50% DM, was performed using a processive PME from <I>Carica papaya</I> (<I>CpL</I>-PME). Introduced demethylesterified blocks were released either by exhaustive or limited endo polygalacturonase (EPG) digestion. Degree of blockiness (DB), absolute degree of blockiness (DB<SUB>abs</SUB>), average demethylesterified block size ( B S ¯ ) and number of average sized demethylesterified blocks per molecule ( B N ¯ ) were estimated. B S ¯ and B N ¯ as well as DB/DB<SUB>abs</SUB> differed depending on the initial DM reduction by <I>Aa</I>PME, the number of activity units of <I>CpL</I>PME used and the reaction pH (<I>P</I> < 0.05). Consecutive demethylesterification of HG by <I>AaPME</I> to 80% DM and then by <I>CpL</I>PME to 50% DM at pH 4.5 produced significantly longer oligomer blocks compared to <I>Aa-PME</I> demethylesterification to 70% DM followed by <I>CpL</I>-PME to 50% DM at pH 7.0. Limited EPG digestion released nearly intact demethylesterified blocks and the released oligomers were coupled with <I>in silico</I> modeling. Resulting oligomer distribution corresponded to the <I>in silico</I> mode of action representing contiguous demethylesterification of 10 GalA residues rather than that of random or complete block-wise demethylesterification. Calcium-mediated gels of the modified HGs displayed G′ higher than G″ values and both moduli differed significantly according to the amount of <I>CpL</I>PME applied even though their final DMs were identical. These results suggest the possibility of controlling B S ¯ and engineering a population of demethylesterified pectin molecules with specified demethylesterified B S ¯ and functional properties.</P> <P><B>Highlights</B></P> <P> <UL> <LI> A fungal and a plant pectin methylesterase were sequentially applied to a model homogalacturonan. </LI> <LI> Average demethylesterified block size and number differed based on the treatment. </LI> <LI> A processive multiple attack mode of action best explained block distributions. </LI> <LI> Significant correlations between B S ¯ , B N ¯ , degree of blockiness and G′ were observed. </LI> <LI> Functional properties could be customized using PMEs of different mode of actions. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>

Production of a Monoclonal Antibody by Ascites, Hollow Fiber System, and Transgenic Plants for Vaccine Production Using CB.Hep-1 mAb as a Study Case

Rodolfo Valdés,Andrés Tamayo,Marcos González,Sigifredo Padilla,Déborah Geada,William Ferro,Lorely Milá,Leonardo Gómez,Rosario Alemán,Alberto Leyva,Cristina García,Otto Mendoza,Tatiana Alvarez,Lamay Do 한국생물공학회 2012 Biotechnology and Bioprocess Engineering Vol.17 No.1

Monoclonal antibody (mAb) production methods (ascites, in vitro technologies, transgenic animals, and dicot or monocot transgenic plants; moss, algae) have been improved since they were first developed in 1975. In this study, we illustrate a summary of a study case in which mice, a hollow fiber system, and tobacco transgenic plants were assessed for the production of mAb for vaccine manufacturing and vaccine production. Monoclonal antibody (mAb) production methods (ascites, in vitro technologies, transgenic animals, and dicot or monocot transgenic plants; moss, algae) have been improved since they were first developed in 1975. In this study, we illustrate a summary of a study case in which mice, a hollow fiber system, and tobacco transgenic plants were assessed for the production of mAb for vaccine manufacturing and vaccine production.

β-Arrestin/AP-2 Interaction in G Protein-coupled Receptor Internalization : IDENTIFICATION OF A β-ARRESTIN BINDING SITE IN β_2-ADAPTIN

Laporte, Ste´phane A.,Miller, William E.,Kim, Kyeong-Man,Caron, Marc G. 전남대학교 약품개발연구소 2002 약품개발연구지 Vol.11 No.-

β-Arrestins, proteins involved in the turn-off of G protein-coupled receptor (GPCR) activation, bind to the β_2-adaptin subunit of the clathrin adaptor AP-2. The interaction of β_2-adaptin with β-Arrestin involves critical arginine residues in the C-terminal domain of β-Arrestin and plays an important role in initiating clathrin-mediated endocytosis of the β_2-adrenergic receptor(β_2AR) (Laporte, S. A., Oakley, R. H., Holt, J. A., Barak, L. S., and Caron, M. G. (2000) J. Biol. Chem. 276, 23120-23126). However, the β-arrestin-binding site in β_2-adaptin has not been identified, and little is known about the role of β-arrestin/AP-2 interaction in the endocytosis of other GPCRs. Using in vitro binding assays, we have identified two glutamate residues (Glu-849 and Glu-902) in β_2-adaptin that are important in β-arrestin binding. These residues are located in the platform subdomain of the C terminus β_2-adaptin, where accessory/adapter endocytic proteins for other classes of receptors interact, distinct from the main site where clathrin interact. The functional significance of the β-arrestin/AP-2/clathrin complex in the endocytosis of GPCRs such as the β_2AR and vasopressin type Ⅱ receptor was evaluated using mutant constructs of the β_2-adaptin C terminus containing either the clathrin and the β-arrestin binding domains or the β-arrestin-binding domain alone. When expressed in human embryonic kidney 293 cells, both constructs acted as dominant negatives inhibiting the agonist-induced internalization of the β_2AR and the vasopressin type Ⅱ receptor. In addition, although the β_2-adaptin construct containing both the clathrin and β-arrestin binding domains was able to block the endocytosis of transferrin receptors, a β_2-adaptin construct capable of assoociating with β-arrestin but lacking its high affinity clathrin interaction did not interfere with transferrin receptor endocytosis. These results suggest that the interaction of β-arrestin with β_2-adaptin represents a selective endocytic trigger for several members of the GPCR family.

Background Level of Atmospheric Radon-222 Concentrations at Gosan Station, Jeju Island, Korea in 2011

Won-Hyung Kim,Hee-Jung Ko,Chul-Goo Hu,Haeyoung Lee,Chulkyu Lee,S. Chambers,A. G. Williams,Chang-Hee Kang 대한화학회 2014 Bulletin of the Korean Chemical Society Vol.35 No.4

Real-time monitoring of hourly atmospheric radon (Rn-222) concentration was performed throughout 2011 at Gosan station, Jeju Island, one of the least polluted regions in Korea, in order to characterize the background levels, and temporal variations on diurnal to seasonal time-scales. The annual mean radon concentration for 2011 was 2527 ± 1356 mBq m−3, and the seasonal cycle was characterized by a broad winter maximum, and narrow summer minimum. Mean monthly radon concentrations, in descending order of magnitude, were Oct > Sep > Feb > Nov > Jan > Dec > Mar > Aug > Apr > Jun > May > Jul. The maximum monthly mean value (3595 mBq m−3, October), exceeded the minimum value (1243 mBq m−3, July), by almost a factor of three. Diurnal composite hourly concentrations increased throughout the night to reach their maximum (2956 mBq m−3) at around 7 a.m., after which they decreased to their minimum value (2259 mBq m−3) at around 3 p.m. Back trajectory analyses indicated that the highest radon events typically exhibited long-term continental fetch over Asia before arriving at Jeju. In contrast, low radon events were generally correlated with air mass fetch over the North Pacific Ocean. Radon concentrations typical of predominantly continental, and predominantly oceanic fetch, differed by a factor of 3.8.

Background Level of Atmospheric Radon-222 Concentrations at Gosan Station, Jeju Island, Korea in 2011

Kim, Won-Hyung,Ko, Hee-Jung,Hu, Chul-Goo,Lee, Haeyoung,Lee, Chulkyu,Chambers, S.,Williams, A.G.,Kang, Chang-Hee Korean Chemical Society 2014 Bulletin of the Korean Chemical Society Vol.35 No.4

Real-time monitoring of hourly atmospheric radon (Rn-222) concentration was performed throughout 2011 at Gosan station, Jeju Island, one of the least polluted regions in Korea, in order to characterize the background levels, and temporal variations on diurnal to seasonal time-scales. The annual mean radon concentration for 2011 was $2527{\pm}1356$ mBq $m^{-3}$, and the seasonal cycle was characterized by a broad winter maximum, and narrow summer minimum. Mean monthly radon concentrations, in descending order of magnitude, were Oct > Sep > Feb > Nov > Jan > Dec > Mar > Aug > Apr > Jun > May > Jul. The maximum monthly mean value (3595 mBq $m^{-3}$, October), exceeded the minimum value (1243 mBq $m^{-3}$, July), by almost a factor of three. Diurnal composite hourly concentrations increased throughout the night to reach their maximum (2956 mBq $m^{-3}$) at around 7 a.m., after which they decreased to their minimum value (2259 mBq $m^{-3}$) at around 3 p.m. Back trajectory analyses indicated that the highest radon events typically exhibited long-term continental fetch over Asia before arriving at Jeju. In contrast, low radon events were generally correlated with air mass fetch over the North Pacific Ocean. Radon concentrations typical of predominantly continental, and predominantly oceanic fetch, differed by a factor of 3.8.

A computational model of ureteral peristalsis and an investigation into ureteral reflux

G. Hosseini,C. Ji,D. Xu,M. A. Rezaienia,E. Avital,A. Munjiza,J. J. R. Williams,J. S. A. Green 대한의용생체공학회 2018 Biomedical Engineering Letters (BMEL) Vol.8 No.1

The aim of this study is to create a computationalmodel of the human ureteral system that accuratelyreplicates the peristaltic movement of the ureter for avariety of physiological and pathological functions. Theobjectives of this research are met using our in-house fluidstructuraldynamics code (CgLes–Y code). A realisticperistaltic motion of the ureter is modelled using a novelpiecewise linear force model. The urodynamic responsesare investigated under two conditions of a healthy and adepressed contraction force. A ureteral pressure during thecontraction shows a very good agreement with correspondingclinical data. The results also show a dependencyof the wall shear stresses on the contraction velocity and itconfirms the presence of a high shear stress at the proximalpart of the ureter. Additionally, it is shown that an inefficientlumen contraction can increase the possibility of acontinuous reflux during the propagation of peristalsis.

A human mitochondrial poly(A) polymerase mutation reveals the complexities of post-transcriptional mitochondrial gene expression

Wilson, William C.,Hornig-Do, Hue-Tran,Bruni, Francesco,Chang, Jeong Ho,Jourdain, Alexis A.,Martinou, Jean-Claude,Falkenberg, Maria,Spå,hr, Henrik,Larsson, Nils-Gö,ran,Lewis, Richard J.,Hewit Oxford University Press 2014 Human Molecular Genetics Vol.23 No.23

<P>The p.N478D missense mutation in human mitochondrial poly(A) polymerase (mtPAP) has previously been implicated in a form of spastic ataxia with optic atrophy. In this study, we have investigated fibroblast cell lines established from family members. The homozygous mutation resulted in the loss of polyadenylation of all mitochondrial transcripts assessed; however, oligoadenylation was retained. Interestingly, this had differential effects on transcript stability that were dependent on the particular species of transcript. These changes were accompanied by a severe loss of oxidative phosphorylation complexes I and IV, and perturbation of <I>de novo</I> mitochondrial protein synthesis. Decreases in transcript polyadenylation and in respiratory chain complexes were effectively rescued by overexpression of wild-type mtPAP. Both mutated and wild-type mtPAP localized to the mitochondrial RNA-processing granules thereby eliminating mislocalization as a cause of defective polyadenylation. <I>In vitro</I> polyadenylation assays revealed severely compromised activity by the mutated protein, which generated only short oligo(A) extensions on RNA substrates, irrespective of RNA secondary structure. The addition of LRPPRC/SLIRP, a mitochondrial RNA-binding complex, enhanced activity of the wild-type mtPAP resulting in increased overall tail length. The LRPPRC/SLIRP effect although present was less marked with mutated mtPAP, independent of RNA secondary structure. We conclude that (i) the polymerase activity of mtPAP can be modulated by the presence of LRPPRC/SLIRP, (ii) N478D mtPAP mutation decreases polymerase activity and (iii) the alteration in poly(A) length is sufficient to cause dysregulation of post-transcriptional expression and the pathogenic lack of respiratory chain complexes.</P>

Spatial resolution of a glass-ceramic X-ray storage phosphor

A. Edgar,G.V.M. Williams,S. Schweizer,J.-M. Spaeth 한국물리학회 2006 Current Applied Physics Vol.6 No.3

The spatial resolution of a glass-ceramic X-ray storage phosphor imaging plate has been measured using a scanned and focussed laserbeam. The glass ceramic comprises orthorhombic phase barium chloride crystals containing europium ions embedded in a uorozirco-nate glass matrix. The modulation transfer function extends to signicantly higher frequencies than for a commercial imaging plate basedmercial plate. The modulation transfer function has a bimodal character which we ascribe to component responses from the focussedlaser beam volume and from the surrounding scattering halo. A limitation on performance of this particular glass ceramic is the occur-rence of bubbles and other defects which scatter the read-out light beam and reduce the uniformity of the response.

Optical scattering in glass ceramics

A. Edgar,G.V.M. Williams,J. Hamelin 한국물리학회 2006 Current Applied Physics Vol.6 No.3

We analyse measurements of the optical extinction coefficient for fluorozirconate glasses containing a volume fraction of 0.2 of barium chloride nano-particles. We find that the glass ceramics are about a factor of six more transparent than predicted by Rayleigh scattering theory, based on estimates of particle sizes from X-ray diffraction and electron microscopy measurements. A numerical simulation of scattering using the discrete-dipole approximation has been made in order to investigate whether this particular discrepancy, and the ‘‘ultra-transparency’’ observed in other glass ceramics in general, can be explained by particle proximity and interaction effects as has been proposed in the literature. The simulated scattering system behaves essentially as a collection of independent scatterers up to a volume fraction of at least 25%, and so these effects are not responsible for the high transparency in this system.

Lithium borate glass ceramics as thermal neutron imaging plates

G.A. Appleby,C.M. Bartle,G.V.M. Williams,A. Edgar 한국물리학회 2006 Current Applied Physics Vol.6 No.3

A new glass ceramic composed of a lithium borate glass matrix containing Eu-doped BaCl2 crystals has been developed for thermalneutron radiography, and has been tested for sensitivity toc-radiation. It is shown to exhibit photostimulated luminescence (PSL) uponX- and c-irradiation. The PSL arises from orthorhombic BaCl2 nanocrystallites in the glass ceramics, which also contain a PSL-inactivephase of cubic BaCl2. The relative PSL eciency of the glass ceramics has a strong dependence on gamma energy, which correlates withrelative half-thickness. The eect of uoride doping in the glass ceramics has been found to substantially increase the PSL, which rangesfrom. 0.6% without uoride doping to. 8% with 0.2 F:Cl concentration, all relative to Fujilm’s commercial neutron imaging plateBAS-ND.