Phylogenetic and experimental characterization of an acyl-ACP thioesterase family reveals significant diversity in enzymatic specificity and activity.

PubMed

Jing, Fuyuan; Cantu, David C; Tvaruzkova, Jarmila; Chipman, Jay P; Nikolau, Basil J; Yandeau-Nelson, Marna D; Reilly, Peter J

2011-08-10

Acyl-acyl carrier protein thioesterases (acyl-ACP TEs) catalyze the hydrolysis of the thioester bond that links the acyl chain to the sulfhydryl group of the phosphopantetheine prosthetic group of ACP. This reaction terminates acyl chain elongation of fatty acid biosynthesis, and in plant seeds it is the biochemical determinant of the fatty acid compositions of storage lipids. To explore acyl-ACP TE diversity and to identify novel acyl ACP-TEs, 31 acyl-ACP TEs from wide-ranging phylogenetic sources were characterized to ascertain their in vivo activities and substrate specificities. These acyl-ACP TEs were chosen by two different approaches: 1) 24 TEs were selected from public databases on the basis of phylogenetic analysis and fatty acid profile knowledge of their source organisms; and 2) seven TEs were molecularly cloned from oil palm (Elaeis guineensis), coconut (Cocos nucifera) and Cuphea viscosissima, organisms that produce medium-chain and short-chain fatty acids in their seeds. The in vivo substrate specificities of the acyl-ACP TEs were determined in E. coli. Based on their specificities, these enzymes were clustered into three classes: 1) Class I acyl-ACP TEs act primarily on 14- and 16-carbon acyl-ACP substrates; 2) Class II acyl-ACP TEs have broad substrate specificities, with major activities toward 8- and 14-carbon acyl-ACP substrates; and 3) Class III acyl-ACP TEs act predominantly on 8-carbon acyl-ACPs. Several novel acyl-ACP TEs act on short-chain and unsaturated acyl-ACP or 3-ketoacyl-ACP substrates, indicating the diversity of enzymatic specificity in this enzyme family. These acyl-ACP TEs can potentially be used to diversify the fatty acid biosynthesis pathway to produce novel fatty acids.

Phylogenetic and experimental characterization of an acyl-ACP thioesterase family reveals significant diversity in enzymatic specificity and activity

PubMed Central

2011-01-01

Background Acyl-acyl carrier protein thioesterases (acyl-ACP TEs) catalyze the hydrolysis of the thioester bond that links the acyl chain to the sulfhydryl group of the phosphopantetheine prosthetic group of ACP. This reaction terminates acyl chain elongation of fatty acid biosynthesis, and in plant seeds it is the biochemical determinant of the fatty acid compositions of storage lipids. Results To explore acyl-ACP TE diversity and to identify novel acyl ACP-TEs, 31 acyl-ACP TEs from wide-ranging phylogenetic sources were characterized to ascertain their in vivo activities and substrate specificities. These acyl-ACP TEs were chosen by two different approaches: 1) 24 TEs were selected from public databases on the basis of phylogenetic analysis and fatty acid profile knowledge of their source organisms; and 2) seven TEs were molecularly cloned from oil palm (Elaeis guineensis), coconut (Cocos nucifera) and Cuphea viscosissima, organisms that produce medium-chain and short-chain fatty acids in their seeds. The in vivo substrate specificities of the acyl-ACP TEs were determined in E. coli. Based on their specificities, these enzymes were clustered into three classes: 1) Class I acyl-ACP TEs act primarily on 14- and 16-carbon acyl-ACP substrates; 2) Class II acyl-ACP TEs have broad substrate specificities, with major activities toward 8- and 14-carbon acyl-ACP substrates; and 3) Class III acyl-ACP TEs act predominantly on 8-carbon acyl-ACPs. Several novel acyl-ACP TEs act on short-chain and unsaturated acyl-ACP or 3-ketoacyl-ACP substrates, indicating the diversity of enzymatic specificity in this enzyme family. Conclusion These acyl-ACP TEs can potentially be used to diversify the fatty acid biosynthesis pathway to produce novel fatty acids. PMID:21831316

Measurements of gluconeogenesis and glycogenolysis: A methodological review

USDA-ARS?s Scientific Manuscript database

Gluconeogenesis is a complex metabolic process that involves multiple enzymatic steps regulated by myriad factors, including substrate concentrations, the redox state, activation and inhibition of specific enzyme steps, and hormonal modulation. At present, the most widely accepted technique to deter...

Cloning and characterization of a new broadspecific β-glucosidase from Lactococcus sp. FSJ4.

PubMed

Fang, Shujun; Chang, Jie; Lee, Yong Seok; Guo, Weiliang; Choi, Yong Lark; Zhou, Yongcan

2014-01-01

A β-glucosidase gene bglX was cloned from Lactococcus sp. FSJ4 by the method of shotgun. The bglX open reading frame consisted of 1,437 bp, encoding 478 amino acids. SDS-PAGE showed a recombinant bglX monomer of 54 kDa. Substrate specificity study revealed that the enzyme exhibited multifunctional catalysis activity against pNPG, pNPX and pNPGal. This enzyme shows higher activity against aryl glycosides of xylose than those of glucose or galactose. The enzyme exhibited the maximal activity at 40 °C, and the optimal pH was 6.0 with pNPG and 6.5 with pNPX as the substrates. Molecular modeling and substrate docking showed that there should be one active center responsible for the mutifuntional activity in this enzyme, since the active site pocket was substantially wide to allow the entry of pNPG, pNPX and pNPGal, which elucidated the structure-function relationship in substrate specificities. Substrate docking results indicated that Glu180 and Glu377 were the essential catalytic residues of the enzyme. The CDOCKER_ENERGY values obtained by substrate docking indicated that the enzyme has higher activity against pNPX than those of pNPG and pNPGal. These observations are in conformity with the results obtained from experimental investigation. Therefore, such substrate specificity makes this β-glucosidase of great interest for further study on physiological and catalytic reaction processes.

Molecular mechanisms of substrate recognition and specificity of botulinum neurotoxin serotype F.

PubMed

Chen, Sheng; Wan, Hoi Ying

2011-01-15

BoNTs (botulinum neurotoxins) are both deadly neurotoxins and natural toxins that are widely used in protein therapies to treat numerous neurological disorders of dystonia and spinal spasticity. Understanding the mechanism of action and substrate specificity of BoNTs is a prerequisite to develop antitoxin and novel BoNT-derived protein therapy. To date, there is a lack of detailed information with regard to how BoNTs recognize and hydrolyse the substrate VAMP-2 (vesicle-associated membrane protein 2), even though it is known to be cleaved by four of the seven BoNT serotypes, B, D, F, G and TeNT (tetanus neurotoxin). In the present study we dissected the molecular mechanisms of VAMP-2 recognition by BoNT serotype F for the first time. The initial substrate recognition was mediated through sequential binding of VAMP-2 to the B1, B2 and B3 pockets in LC/F (light chain of BoNT serotype F), which directed VAMP-2 to the active site of LC/F and stabilized the active site substrate recognition, where the P2, P1' and P2' sites of VAMP-2 were specifically recognized by the S2, S1' and S2' pockets of LC/F to promote substrate hydrolysis. The understanding of the molecular mechanisms of LC/F substrate recognition provides insights into the development of antitoxins and engineering novel BoNTs to optimize current therapy and extend therapeutic interventions.

A Modern Understanding of the Traditional and Nontraditional Biological Functions of Angiotensin-Converting Enzyme

PubMed Central

Ong, Frank S.; Blackwell, Wendell-Lamar B.; Shah, Kandarp H.; Giani, Jorge F.; Gonzalez-Villalobos, Romer A.; Shen, Xiao Z.; Fuchs, Sebastien

2013-01-01

Angiotensin-converting enzyme (ACE) is a zinc-dependent peptidase responsible for converting angiotensin I into the vasoconstrictor angiotensin II. However, ACE is a relatively nonspecific peptidase that is capable of cleaving a wide range of substrates. Because of this, ACE and its peptide substrates and products affect many physiologic processes, including blood pressure control, hematopoiesis, reproduction, renal development, renal function, and the immune response. The defining feature of ACE is that it is composed of two homologous and independently catalytic domains, the result of an ancient gene duplication, and ACE-like genes are widely distributed in nature. The two ACE catalytic domains contribute to the wide substrate diversity of ACE and, by extension, the physiologic impact of the enzyme. Several studies suggest that the two catalytic domains have different biologic functions. Recently, the X-ray crystal structure of ACE has elucidated some of the structural differences between the two ACE domains. This is important now that ACE domain-specific inhibitors have been synthesized and characterized. Once widely available, these reagents will undoubtedly be powerful tools for probing the physiologic actions of each ACE domain. In turn, this knowledge should allow clinicians to envision new therapies for diseases not currently treated with ACE inhibitors. PMID:23257181

A Measure of the Broad Substrate Specificity of Enzymes Based on ‘Duplicate’ Catalytic Residues

PubMed Central

Chakraborty, Sandeep; Ásgeirsson, Bjarni; Rao, Basuthkar J.

2012-01-01

The ability of an enzyme to select and act upon a specific class of compounds with unerring precision and efficiency is an essential feature of life. Simultaneously, these enzymes often catalyze the reaction of a range of similar substrates of the same class, and also have promiscuous activities on unrelated substrates. Previously, we have established a methodology to quantify promiscuous activities in a wide range of proteins. In the current work, we quantitatively characterize the active site for the ability to catalyze distinct, yet related, substrates (BRASS). A protein with known structure and active site residues provides the framework for computing ‘duplicate’ residues, each of which results in slightly modified replicas of the active site scaffold. Such spatial congruence is supplemented by Finite difference Poisson Boltzmann analysis which filters out electrostatically unfavorable configurations. The congruent configurations are used to compute an index (BrassIndex), which reflects the broad substrate profile of the active site. We identify an acetylhydrolase and a methyltransferase as having the lowest and highest BrassIndex, respectively, from a set of non-homologous proteins extracted from the Catalytic Site Atlas. The acetylhydrolase, a regulatory enzyme, is known to be highly specific for platelet-activating factor. In the methyltransferase (PDB: 1QAM), various combinations of glycine (Gly38/40/42), asparagine (Asn101/11) and glutamic acid (Glu59/36) residues having similar spatial and electrostatic profiles with the specified scaffold (Gly38, Asn101 and Glu59) exemplifies the broad substrate profile such an active site may provide. ‘Duplicate’ residues identified by relaxing the spatial and/or electrostatic constraints can be the target of directed evolution methodologies, like saturation mutagenesis, for modulating the substrate specificity of proteins. PMID:23166637

Structural basis of RND-type multidrug exporters

PubMed Central

Yamaguchi, Akihito; Nakashima, Ryosuke; Sakurai, Keisuke

2015-01-01

Bacterial multidrug exporters are intrinsic membrane transporters that act as cellular self-defense mechanism. The most notable characteristics of multidrug exporters is that they export a wide range of drugs and toxic compounds. The overexpression of these exporters causes multidrug resistance. Multidrug-resistant pathogens have become a serious problem in modern chemotherapy. Over the past decade, investigations into the structure of bacterial multidrug exporters have revealed the multidrug recognition and export mechanisms. In this review, we primarily discuss RND-type multidrug exporters particularly AcrAB-TolC, major drug exporter in Gram-negative bacteria. RND-type drug exporters are tripartite complexes comprising a cell membrane transporter, an outer membrane channel and an adaptor protein. Cell membrane transporters and outer membrane channels are homo-trimers; however, there is no consensus on the number of adaptor proteins in these tripartite complexes. The three monomers of a cell membrane transporter have varying conformations (access, binding, and extrusion) during transport. Drugs are exported following an ordered conformational change in these three monomers, through a functional rotation mechanism coupled with the proton relay cycle in ion pairs, which is driven by proton translocation. Multidrug recognition is based on a multisite drug-binding mechanism, in which two voluminous multidrug-binding pockets in cell membrane exporters recognize a wide range of substrates as a result of permutations at numerous binding sites that are specific for the partial structures of substrate molecules. The voluminous multidrug-binding pocket may have numerous binding sites even for a single substrate, suggesting that substrates may move between binding sites during transport, an idea named as multisite-drug-oscillation hypothesis. This hypothesis is consistent with the apparently broad substrate specificity of cell membrane exporters and their highly efficient ejection of drugs from the cell. Substrates are transported through dual multidrug-binding pockets via the peristaltic motion of the substrate translocation channel. Although there are no clinically available inhibitors of bacterial multidrug exporters, efforts to develop inhibitors based on structural information are underway. PMID:25941524

Structural basis of RND-type multidrug exporters.

PubMed

Yamaguchi, Akihito; Nakashima, Ryosuke; Sakurai, Keisuke

2015-01-01

Bacterial multidrug exporters are intrinsic membrane transporters that act as cellular self-defense mechanism. The most notable characteristics of multidrug exporters is that they export a wide range of drugs and toxic compounds. The overexpression of these exporters causes multidrug resistance. Multidrug-resistant pathogens have become a serious problem in modern chemotherapy. Over the past decade, investigations into the structure of bacterial multidrug exporters have revealed the multidrug recognition and export mechanisms. In this review, we primarily discuss RND-type multidrug exporters particularly AcrAB-TolC, major drug exporter in Gram-negative bacteria. RND-type drug exporters are tripartite complexes comprising a cell membrane transporter, an outer membrane channel and an adaptor protein. Cell membrane transporters and outer membrane channels are homo-trimers; however, there is no consensus on the number of adaptor proteins in these tripartite complexes. The three monomers of a cell membrane transporter have varying conformations (access, binding, and extrusion) during transport. Drugs are exported following an ordered conformational change in these three monomers, through a functional rotation mechanism coupled with the proton relay cycle in ion pairs, which is driven by proton translocation. Multidrug recognition is based on a multisite drug-binding mechanism, in which two voluminous multidrug-binding pockets in cell membrane exporters recognize a wide range of substrates as a result of permutations at numerous binding sites that are specific for the partial structures of substrate molecules. The voluminous multidrug-binding pocket may have numerous binding sites even for a single substrate, suggesting that substrates may move between binding sites during transport, an idea named as multisite-drug-oscillation hypothesis. This hypothesis is consistent with the apparently broad substrate specificity of cell membrane exporters and their highly efficient ejection of drugs from the cell. Substrates are transported through dual multidrug-binding pockets via the peristaltic motion of the substrate translocation channel. Although there are no clinically available inhibitors of bacterial multidrug exporters, efforts to develop inhibitors based on structural information are underway.

Structure of human POFUT2: insights into thrombospondin type 1 repeat fold and O-fucosylation

PubMed Central

Chen, Chun-I; Keusch, Jeremy J; Klein, Dominique; Hess, Daniel; Hofsteenge, Jan; Gut, Heinz

2012-01-01

Protein O-fucosylation is a post-translational modification found on serine/threonine residues of thrombospondin type 1 repeats (TSR). The fucose transfer is catalysed by the enzyme protein O-fucosyltransferase 2 (POFUT2) and >40 human proteins contain the TSR consensus sequence for POFUT2-dependent fucosylation. To better understand O-fucosylation on TSR, we carried out a structural and functional analysis of human POFUT2 and its TSR substrate. Crystal structures of POFUT2 reveal a variation of the classical GT-B fold and identify sugar donor and TSR acceptor binding sites. Structural findings are correlated with steady-state kinetic measurements of wild-type and mutant POFUT2 and TSR and give insight into the catalytic mechanism and substrate specificity. By using an artificial mini-TSR substrate, we show that specificity is not primarily encoded in the TSR protein sequence but rather in the unusual 3D structure of a small part of the TSR. Our findings uncover that recognition of distinct conserved 3D fold motifs can be used as a mechanism to achieve substrate specificity by enzymes modifying completely folded proteins of very wide sequence diversity and biological function. PMID:22588082

Tuning Fluorescence Direction with Plasmonic Metal–Dielectric– Metal Substrates

PubMed Central

Choudhury, Sharmistha Dutta; Badugu, Ramachandram; Nowaczyk, Kazimierz; Ray, Krishanu; Lakowicz, Joseph R.

2013-01-01

Controlling the emission properties of fluorophores is essential for improving the performance of fluorescence-based techniques in modern biochemical research, medical diagnosis, and sensing. Fluorescence emission is isotropic in nature, which makes it difficult to capture more than a small fraction of the total emission. Metal– dielectric–metal (MDM) substrates, discussed in this Letter, convert isotropic fluorescence into beaming emission normal to the substrate. This improves fluorescence collection efficiency and also opens up new avenues for a wide range of fluorescence-based applications. We suggest that MDM substrates can be readily adapted for multiple uses, such as in microarray formats, for directional fluorescence studies of multiple probes or for molecule-specific sensing with a high degree of spatial control over the fluorescence emission. SECTION: Physical Processes in Nanomaterials and Nanostructures PMID:24013521

Nonpeptide-Based Small-Molecule Probe for Fluorogenic and Chromogenic Detection of Chymotrypsin.

PubMed

Wu, Lei; Yang, Shu-Hou; Xiong, Hao; Yang, Jia-Qian; Guo, Jun; Yang, Wen-Chao; Yang, Guang-Fu

2017-03-21

We report herein a nonpeptide-based small-molecule probe for fluorogenic and chromogenic detection of chymotrypsin, as well as the primary application for this probe. This probe was rationally designed by mimicking the peptide substrate and optimized by adjusting the recognition group. The refined probe 2 exhibits good specificity toward chymotrypsin, producing about 25-fold higher enhancement in both the fluorescence intensity and absorbance upon the catalysis by chymotrypsin. Compared with the most widely used peptide substrate (AMC-FPAA-Suc) of chymotrypsin, probe 2 shows about 5-fold higher binding affinity and comparable catalytical efficiency against chymotrypsin. Furthermore, it was successfully applied for the inhibitor characterization. To the best of our knowledge, probe 2 is the first nonpeptide-based small-molecule probe for chymotrypsin, with the advantages of simple structure and high sensitivity compared to the widely used peptide-based substrates. This small-molecule probe is expected to be a useful molecular tool for drug discovery and chymotrypsin-related disease diagnosis.

Functional characterization of the Mycobacterium tuberculosis zinc metallopeptidase Zmp1 and identification of potential substrates.

PubMed

Petrera, Agnese; Amstutz, Beat; Gioia, Magda; Hähnlein, Janine; Baici, Antonio; Selchow, Petra; Ferraris, Davide M; Rizzi, Menico; Sbardella, Diego; Marini, Stefano; Coletta, Massimo; Sander, Peter

2012-07-01

Zinc metallopeptidases of bacterial pathogens are widely distributed virulence factors and represent promising pharmacological targets. In this work, we have characterized Zmp1, a zinc metallopeptidase identified as a virulence factor of Mycobacterium tuberculosis and belonging to the neprilysin (NEP; M13) family, whose X-ray structure has been recently solved. Interestingly, this enzyme shows an optimum activity toward a fluorogenic substrate at moderately acidic pH values (i.e., 6.3), which corresponds to those reported for the Mtb phagosome where this enzyme should exert its pathological activity. Substrate specificity of Zmp1 was investigated by screening a peptide library. Several sequences derived from biologically relevant proteins were identified as possible substrates, including the neuropeptides bradykinin, neurotensin, and neuropeptide FF. Further, subsequences of other small bioactive peptides were found among most frequently cleaved sites, e.g., apelin-13 and substance P. We determined the specific cleavage site within neuropeptides by mass spectrometry, observing that hydrophobic amino acids, mainly phenylalanine and isoleucine, are overrepresented at position P1'. In addition, the enzymatic mechanism of Zmp1 toward these neuropeptides has been characterized, displaying some differences with respect to the synthetic fluorogenic substrate and indicating that the enzyme adapts its enzymatic action to different substrates.

The fungal cultivar of leaf-cutter ants produces specific enzymes in response to different plant substrates

DOE Office of Scientific and Technical Information (OSTI.GOV)

Khadempour, Lily; Burnum-Johnson, Kristin E.; Baker, Erin S.

Herbivores use symbiotic microbes to help gain access to energy and nutrients from plant material. Leaf-cutter ants are a paradigmatic example, having tremendous impact on their ecosystems as dominant generalist herbivores through cultivation of a fungus, Leucoagaricus gongylophorous. Here we examine how this mutualism could facilitate the flexible substrate incorporation of the ants by providing leaf-cutter ant subcolonies four substrate types: leaves, flowers, oats, and a mixture of all three. Through metaproteomic analysis of the fungus gardens, we were able to identify and quantify 1766 different fungal proteins, including 161 biomass-degrading enzymes. This analysis revealed that fungal protein profiles weremore » significantly different between subcolonies fed different substrates with the highest abundance of cellulolytic enzymes observed in the leaf and flower treatments. When the fungus garden is provided with leaves and flowers, which contain the majority of their energy in recalcitrant material, it increases its production of proteins that break down cellulose: endoglucanases, exoglucanase and β-glucosidase. Further, the complete metaproteomes for the leaves and flowers treatments were very similar, the mixed treatment closely resembled the treatment with oats alone. This suggests that when provided a mixture of substrates, the fungus garden preferentially produces enzymes necessary for breakdown of simpler, more digestible substrates. This flexible, substrate-specific response of the fungal cultivar allows the leaf-cutter ants to derive energy from a wide range of substrates, which may contribute to their ability to be dominant generalist herbivores.« less

Functional characterization of a novel β-fructofuranosidase from Bifidobacterium longum subsp. infantis ATCC 15697 on structurally diverse fructans.

PubMed

Ávila-Fernández, Á; Cuevas-Juárez, E; Rodríguez-Alegría, M E; Olvera, C; López-Munguía, A

2016-07-01

In this study, we describe the isolation of a gene encoding a novel β-fructofuranosidase from Bifidobacterium longum subsp. infantis ATCC 15697, and the characterization of the enzyme, the second one found in this strain, significantly different in primary sequence to the already reported bifidobacterial β-fructofuranosidases. The gene, found through genome-mining was expressed in Escherichia coli C41(DE3). The recombinant enzyme (B.longum_l1) has a molecular weight of 75 kDa, with optimal activity at 50°C, pH 6·0-6·5, and a remarkable stability with a half-life of 75·5 h at 50°C. B.longum_l1 has a wide specificity for fructans, hydrolysing all substrates through an exo-type mechanism, including Oligofructose P95 (β2-1 fructooligosaccharides (FOS), DP 2-8), Raftilose Synergy 1(β2-1 FOS & inulin, DP 2-60), Raftiline HP (inulin, DP 2-60), bacterial inulin (3000 kDa) and levan (8·3 & 3500 kDa), Agave fructans (mixed fructans, DP 3-29) and levan-type FOS (β2-6 FOS, DP 2-8), with the highest relative activity and turnover number found for levan-type FOS. The apparent affinity of the enzyme for levan-type FOS and Oligofructose P95 was found to be 9·2 and 4·6 mmol l(-1) (Km ) with a specific activity of 908 and 725 μmol min(-1)  mg(-1) of protein (k2 ), respectively, more than twice the activity for sucrose. B.longum_l1 is a wide substrate specificity enzyme, which may contribute to the competitiveness and persistence of this strain in the colon. The bifidobacterial β-fructofuranosidase activity was evaluated with a wide variety of substrates including noncommercial fructans, such as levan-type and mixed agave fructans. Its activity on these substrates certainly strengthens their commercial prebiotic character and contributes to the understanding of bifidobacteria stimulation by structurally diverse fructans. © 2016 The Society for Applied Microbiology.

Complementary Proteomic and Biochemical Analysis of Peptidases in Lobster Gastric Juice Uncovers the Functional Role of Individual Enzymes in Food Digestion.

PubMed

Bibo-Verdugo, Betsaida; O'Donoghue, Anthony J; Rojo-Arreola, Liliana; Craik, Charles S; García-Carreño, Fernando

2016-04-01

Crustaceans are a diverse group, distributed in widely variable environmental conditions for which they show an equally extensive range of biochemical adaptations. Some digestive enzymes have been studied by purification/characterization approaches. However, global analysis is crucial to understand how digestive enzymes interplay. Here, we present the first proteomic analysis of the digestive fluid from a crustacean (Homarus americanus) and identify glycosidases and peptidases as the most abundant classes of hydrolytic enzymes. The digestion pathway of complex carbohydrates was predicted by comparing the lobster enzymes to similar enzymes from other crustaceans. A novel and unbiased substrate profiling approach was used to uncover the global proteolytic specificity of gastric juice and determine the contribution of cysteine and aspartic acid peptidases. These enzymes were separated by gel electrophoresis and their individual substrate specificities uncovered from the resulting gel bands. This new technique is called zymoMSP. Each cysteine peptidase cleaves a set of unique peptide bonds and the S2 pocket determines their substrate specificity. Finally, affinity chromatography was used to enrich for a digestive cathepsin D1 to compare its substrate specificity and cold-adapted enzymatic properties to mammalian enzymes. We conclude that the H. americanus digestive peptidases may have useful therapeutic applications, due to their cold-adaptation properties and ability to hydrolyze collagen.

Identification and binding mechanism of phage displayed peptides with specific affinity to acid-alkali treated titanium.

PubMed

Sun, Yuhua; Tan, Jing; Wu, Baohua; Wang, Jianxin; Qu, Shuxin; Weng, Jie; Feng, Bo

2016-10-01

Acid-alkali treatment is one of means widely used for preparing bioactive titanium surfaces. Peptides with specific affinity to titanium surface modified by acid-alkali two-steps treatment were obtained via phage display technology. Out of the eight new unique peptides, titanium-binding peptide 54 displayed by monoclonal M13 phage at its pIII coat protein (TBP54-M13 phage) was proved to have higher binding affinity to the substrate. The binding interaction occurred at the domain from phenylalanine at position 1 to arginine at position 6 in the sequences of TBP54 (FAETHRGFHFSF) mainly via the reaction of these residues with the Ti surface. Together the coordination and electrostatic interactions controlled the specific binding of the phage to the substrate. The binding affinity was dependent on the surface basic hydroxyl group content. In addition, the phage showed a different interaction way with the Ti surface without acid-alkali treatment along with an impaired affinity. This study could provide more understanding of the interaction mechanism between the selected peptide and its specific substrate, and develop a promising method for the biofunctionalization of titanium. Copyright © 2016 Elsevier B.V. All rights reserved.

Wide-Bandgap CIAS Thin-film Photovoltaics with Transparent Back Contacts for Next-Generation Single and Multijunction Devices

NASA Technical Reports Server (NTRS)

Woods, Lawrence M.; Kalla, Ajay; Gonzalez, Damian; Ribelin, Rosine

2005-01-01

Future spacecraft and high-altitude airship (HAA) technologies will require high array specific power (W/kg), which can be met using thin-film photovoltaics (PV) on lightweight and flexible substrates. It has been calculated that the thin-film array technology, including the array support structure, begins to exceed the specific power of crystalline multi-junction arrays when the thin-film device efficiencies begin to exceed 12%. Thin-film PV devices have other advantages in that they are more easily integrated into HAA s, and are projected to be much less costly than their crystalline PV counterparts. Furthermore, it is likely that only thin-film array technology will be able to meet device specific power requirements exceeding 1 kW/kg (photovoltaic and integrated substrate/blanket mass only). Of the various thin-film technologies, single junction and radiation resistant CuInSe2 (CIS) and associated alloys with gallium, aluminum and sulfur have achieved the highest levels of thin-film device performance, with the best efficiency, reaching 19.2% under AM1.5 illumination conditions and on thick glass substrates.(3) Thus, it is anticipated that single- and tandem-junction devices with flexible substrates and based on CIS and related alloys could achieve the highest levels of thin-film space and HAA solar array performance.

Comprehensive Evaluation for Substrate Selectivity of Cynomolgus Monkey Cytochrome P450 2C9, a New Efavirenz Oxidase.

PubMed

Hosaka, Shinya; Murayama, Norie; Satsukawa, Masahiro; Uehara, Shotaro; Shimizu, Makiko; Iwasaki, Kazuhide; Iwano, Shunsuke; Uno, Yasuhiro; Yamazaki, Hiroshi

2015-07-01

Cynomolgus monkeys are widely used as primate models in preclinical studies, because of their evolutionary closeness to humans. In humans, the cytochrome P450 (P450) 2C enzymes are important drug-metabolizing enzymes and highly expressed in livers. The CYP2C enzymes, including CYP2C9, are also expressed abundantly in cynomolgus monkey liver and metabolize some endogenous and exogenous substances like testosterone, S-mephenytoin, and diclofenac. However, comprehensive evaluation regarding substrate specificity of monkey CYP2C9 has not been conducted. In the present study, 89 commercially available drugs were examined to find potential monkey CYP2C9 substrates. Among the compounds screened, 20 drugs were metabolized by monkey CYP2C9 at a relatively high rates. Seventeen of these compounds were substrates or inhibitors of human CYP2C9 or CYP2C19, whereas three drugs were not, indicating that substrate specificity of monkey CYP2C9 resembled those of human CYP2C9 or CYP2C19, with some differences in substrate specificities. Although efavirenz is known as a marker substrate for human CYP2B6, efavirenz was not oxidized by CYP2B6 but by CYP2C9 in monkeys. Liquid chromatography-mass spectrometry analysis revealed that monkey CYP2C9 and human CYP2B6 formed the same mono- and di-oxidized metabolites of efavirenz at 8 and 14 positions. These results suggest that the efavirenz 8-oxidation could be one of the selective markers for cynomolgus monkey CYP2C9 among the major three CYP2C enzymes tested. Therefore, monkey CYP2C9 has the possibility of contributing to limited specific differences in drug oxidative metabolism between cynomolgus monkeys and humans. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

Identification of Carboxypeptidase Substrates by C-Terminal COFRADIC.

PubMed

Tanco, Sebastian; Aviles, Francesc Xavier; Gevaert, Kris; Lorenzo, Julia; Van Damme, Petra

2017-01-01

We here present a detailed procedure for studying protein C-termini and their posttranslational modifications by C-terminal COFRADIC. In fact, this procedure can enrich for both C-terminal and N-terminal peptides through a combination of a strong cation exchange fractionation step at low pH, which removes the majority of nonterminal peptides in whole-proteome digests, while the actual COFRADIC step segregates C-terminal peptides from N-terminal peptides. When used in a differential mode, C-terminal COFRADIC allows for the identification of neo-C-termini generated by the action of proteases, which in turn leads to the identification of protease substrates. More specifically, this technology can be applied to determine the natural substrate repertoire of carboxypeptidases on a proteome-wide scale.

Rad-Release

ScienceCinema

None

2017-12-09

The R&D 100 Award winning Rad-Release Chemical Decontamination Technology is a highly effective (up to 99% removal rate), affordable, patented chemical-foam-clay decontamination process tailored to specific radiological and metal contaminants, which is applicable to a wide variety of substrates. For more information about this project, visit http://www.inl.gov/rd100/2011/rad-release/

Substrates of the Arabidopsis thaliana protein isoaspartyl methyltransferasel identified using phage display and biopanning

USDA-ARS?s Scientific Manuscript database

The role of PROTEIN ISOASPARTYL-METHYLTRANSFERASE (PIMT) in repairing a wide assortment of damaged proteins in a host of organisms has been inferred from the affinity of the enzyme for isoaspartyl residues in a plethora of amino acid contexts. The identification of specific PIMT target proteins in p...

The fungal cultivar of leaf-cutter ants produces specific enzymes in response to different plant substrates.

PubMed

Khadempour, Lily; Burnum-Johnson, Kristin E; Baker, Erin S; Nicora, Carrie D; Webb-Robertson, Bobbie-Jo M; White, Richard A; Monroe, Matthew E; Huang, Eric L; Smith, Richard D; Currie, Cameron R

2016-11-01

Herbivores use symbiotic microbes to help derive energy and nutrients from plant material. Leaf-cutter ants are a paradigmatic example, cultivating their mutualistic fungus Leucoagaricus gongylophorus on plant biomass that workers forage from a diverse collection of plant species. Here, we investigate the metabolic flexibility of the ants' fungal cultivar for utilizing different plant biomass. Using feeding experiments and a novel approach in metaproteomics, we examine the enzymatic response of L. gongylophorus to leaves, flowers, oats or a mixture of all three. Across all treatments, our analysis identified and quantified 1766 different fungal proteins, including 161 putative biomass-degrading enzymes. We found significant differences in the protein profiles in the fungus gardens of subcolonies fed different plant substrates. When provided with leaves or flowers, which contain the majority of their energy as recalcitrant plant polymers, the fungus gardens produced more proteins predicted to break down cellulose: endoglucanase, exoglucanase and β-glucosidase. Further, the complete metaproteomes for the leaves and flowers treatments were very similar, while the mixed substrate treatment closely resembled the treatment with oats alone. This indicates that when provided a mixture of plant substrates, fungus gardens preferentially break down the simpler, more digestible substrates. This flexible, substrate-specific enzymatic response of the fungal cultivar allows leaf-cutter ants to derive energy from a wide range of substrates, which likely contributes to their ability to be dominant generalist herbivores. © 2016 John Wiley & Sons Ltd.

The fungal cultivar of leaf-cutter ants produces specific enzymes in response to different plant substrates

PubMed Central

Khadempour, Lily; Burnum-Johnson, Kristin E.; Baker, Erin S.; Nicora, Carrie D.; Webb-Robertson, Bobbie-Jo M.; White, Richard A.; Monroe, Matthew E.; Huang, Eric L.; Smith, Richard D.; Currie, Cameron R.

2016-01-01

Herbivores use symbiotic microbes to help derive energy and nutrients from plant material. Leaf-cutter ants are a paradigmatic example, cultivating their mutualistic fungus Leucoagaricus gongylophorus on plant biomass that workers forage from a diverse collection of plant species. Here, we investigate the metabolic flexibility of the ants’ fungal cultivar for utilizing different plant biomass. Using feeding experiments and a novel approach in metaproteomics, we examine the enzymatic response of L. gongylophorus to leaves, flowers, oats, or a mixture of all three. Across all treatments, our analysis identified and quantified 1,766 different fungal proteins, including 161 putative biomass-degrading enzymes. We found significant differences in the protein profiles in the fungus gardens of sub-colonies fed different plant substrates. When provided with leaves or flowers, which contain the majority of their energy as recalcitrant plant polymers, the fungus gardens produced more proteins predicted to break down cellulose: endoglucanase, exoglucanase, and β-glucosidase. Further, the complete metaproteomes for the leaves and flowers treatments were very similar, while the mixed substrate treatment closely resembled the treatment with oats alone. This indicates that when provided a mixture of plant substrates, fungus gardens preferentially break down the simpler, more digestible substrates. This flexible, substrate-specific enzymatic response of the fungal cultivar allows leaf-cutter ants to derive energy from a wide range of substrates, which likely contributes to their ability to be dominant generalist herbivores. PMID:27696597

Functional associations between support use and forelimb shape in strepsirrhines and their relevance to inferring locomotor behavior in early primates.

PubMed

Fabre, Anne-Claire; Marigó, Judit; Granatosky, Michael C; Schmitt, Daniel

2017-07-01

The evolution of primates is intimately linked to their initial invasion of an arboreal environment. However, moving and foraging in this milieu creates significant mechanical challenges related to the presence of substrates differing in their size and orientation. It is widely assumed that primates are behaviorally and anatomically adapted to movement on specific substrates, but few explicit tests of this relationship in an evolutionary context have been conducted. Without direct tests of form-function relationships in living primates it is impossible to reliably infer behavior in fossil taxa. In this study, we test a hypothesis of co-variation between forelimb morphology and the type of substrates used by strepsirrhines. If associations between anatomy and substrate use exist, these can then be applied to better understand limb anatomy of extinct primates. The co-variation between each forelimb long bone and the type of substrate used was studied in a phylogenetic context. Our results show that despite the presence of significant phylogenetic signal for each long bone of the forelimb, clear support use associations are present. A strong co-variation was found between the type of substrate used and the shape of the radius, with and without taking phylogeny into account, whereas co-variation was significant for the ulna only when taking phylogeny into account. Species that use a thin branch milieu show radii that are gracile and straight and have a distal articular shape that allows for a wide range of movements. In contrast, extant species that commonly use large supports show a relatively robust and curved radius with an increased surface area available for forearm and hand muscles in pronated posture. These results, especially for the radius, support the idea that strepsirrhine primates exhibit specific skeletal adaptations associated with the supports that they habitually move on. With these robust associations in hand it will be possible to explore the same variables in extinct early primates and primate relatives and thus improve the reliability of inferences concerning substrate use in early primates. Copyright © 2017 Elsevier Ltd. All rights reserved.

Changes in the substrate of rivers in historic mining districts

USGS Publications Warehouse

Milhous, R.T.

2004-01-01

The restoration of rivers in watersheds with historic mining districts has become a topic of interest during the last decade. Rivers restoration in these areas is difficult because the mines and mills can be scattered over a wide area and often small. Many have also been abandoned. This paper presents two substrate related factors that are important in the evaluation of river restoration alternatives in watersheds with significance impacts from mines and mills most of which are old and abandoned. The two factors are 1) changes in the size distribution and specific weights of the substrate, and 2) the changes in quality of the interstecial waters caused by metals associated with the tailings in the substrate. The most important impacts of tailings from mills may be on the physical characteristics of the substrate (porosity) and on the quality of the pore waters. The measurements presented in this paper do show significant variation in the porosity in gravel bed rivers and in the quality of the pore waters. Copyright ASCE 2004.

High-efficiency robust perovskite solar cells on ultrathin flexible substrates

PubMed Central

Li, Yaowen; Meng, Lei; Yang, Yang (Michael); Xu, Guiying; Hong, Ziruo; Chen, Qi; You, Jingbi; Li, Gang; Yang, Yang; Li, Yongfang

2016-01-01

Wide applications of personal consumer electronics have triggered tremendous need for portable power sources featuring light-weight and mechanical flexibility. Perovskite solar cells offer a compelling combination of low-cost and high device performance. Here we demonstrate high-performance planar heterojunction perovskite solar cells constructed on highly flexible and ultrathin silver-mesh/conducting polymer substrates. The device performance is comparable to that of their counterparts on rigid glass/indium tin oxide substrates, reaching a power conversion efficiency of 14.0%, while the specific power (the ratio of power to device weight) reaches 1.96 kW kg−1, given the fact that the device is constructed on a 57-μm-thick polyethylene terephthalate based substrate. The flexible device also demonstrates excellent robustness against mechanical deformation, retaining >95% of its original efficiency after 5,000 times fully bending. Our results confirmed that perovskite thin films are fully compatible with our flexible substrates, and are thus promising for future applications in flexible and bendable solar cells. PMID:26750664

Conserved regions of ribonucleoprotein ribonuclease MRP are involved in interactions with its substrate.

PubMed

Esakova, Olga; Perederina, Anna; Berezin, Igor; Krasilnikov, Andrey S

2013-08-01

Ribonuclease (RNase) MRP is a ubiquitous and essential site-specific eukaryotic endoribonuclease involved in the metabolism of a wide range of RNA molecules. RNase MRP is a ribonucleoprotein with a large catalytic RNA moiety that is closely related to the RNA component of RNase P, and multiple proteins, most of which are shared with RNase P. Here, we report the results of an ultraviolet-cross-linking analysis of interactions between a photoreactive RNase MRP substrate and the Saccharomyces cerevisiae RNase MRP holoenzyme. The results show that the substrate interacts with phylogenetically conserved RNA elements universally found in all enzymes of the RNase P/MRP family, as well as with a phylogenetically conserved RNA region that is unique to RNase MRP, and demonstrate that four RNase MRP protein components, all shared with RNase P, interact with the substrate. Implications for the structural organization of RNase MRP and the roles of its components are discussed.

Glycosyltransferases in secondary plant metabolism: tranquilizers and stimulant controllers.

PubMed

Jones, P; Vogt, T

2001-06-01

Plants are exposed to a wide range of toxic and bioactive low-molecular-weight molecules from both exogenous and endogenous sources. Glycosylation is one of the primary sedative mechanisms that plants utilise in order to maintain metabolic homeostasis. Recently, a range of glycosyltransferases has been characterized in detail with regard to substrate specificity. The next step in increasing our understanding of the biology of glycosylation will require information regarding the exact role of individual glycosyltransferases in planta, as well as an insight into their potential involvement in metabolon-complexes. Hopefully, this will answer how a large number of glycosyltransferases with broad, rather than narrow, substrate specificity can be constrained in order to avoid interfering with other pathways of primary and secondary metabolism. These and other topics are discussed.

Real-time monitoring of hairpin ribozyme kinetics through base-specific quenching of fluorescein-labeled substrates.

PubMed Central

Walter, N G; Burke, J M

1997-01-01

Current methods for evaluating the kinetics of ribozyme-catalyzed reactions rely primarily on the use of radiolabeled RNA substrates, and so require tedious electrophoretic separation and quantitation of reaction products for each data point in any experiment. Here, we report the use of fluorescent substrates for real-time analysis of the time course of reactions of the hairpin ribozyme. Fluorescence of 3' fluorescein-labeled substrates was quenched upon binding to the hairpin ribozyme or its isolated substrate-binding strand (SBS), under conditions of ribozyme or SBS excess. This decrease was accompanied by an increase in anisotropy, and resulted from a base-specific quenching by a guanosine residue added to the 5' end of the SBS, close to fluorescein in the complex. Fluorescence quenching was used to determine rate constants for substrate binding (1.4 x 10(8) M(-1) min(-1)), cleavage (0.15 min(-1)), and substrate dissociation (0.010 min(-1)) by a structurally well-defined ribozyme at 25 degrees C in 50 mM Tris-HCI, pH 7.5, 12 mM MgCl2. These rates are in excellent agreement with those obtained using traditional radioisotopic methods. Measurements of dissociation rates provided physical support for interdomain interactions within the substrate-ribozyme complex. We estimate that 2.1 kcal/mol of additional substrate binding energy is provided by the B domain of the ribozyme. Part of this free energy apparently stems from coaxial stacking of helices in the hinge region between domains, and it is plausible that the remainder might be contributed by direct interactions with loop B. The fluorescence quenching and dequenching methods described here should be readily adaptable to studying a wide variety of RNA interactions and reactions using ribozymes and other model systems. PMID:9085846

A Novel Strategy to Isolate Ubiquitin Conjugates Reveals Wide Role for Ubiquitination during Neural Development*

PubMed Central

Franco, Maribel; Seyfried, Nicholas T.; Brand, Andrea H.; Peng, Junmin; Mayor, Ugo

2011-01-01

Ubiquitination has essential roles in neuronal development and function. Ubiquitin proteomics studies on yeast and HeLa cells have proven very informative, but there still is a gap regarding neuronal tissue-specific ubiquitination. In an organism context, direct evidence for the ubiquitination of neuronal proteins is even scarcer. Here, we report a novel proteomics strategy based on the in vivo biotinylation of ubiquitin to isolate ubiquitin conjugates from the neurons of Drosophila melanogaster embryos. We confidently identified 48 neuronal ubiquitin substrates, none of which was yet known to be ubiquitinated. Earlier proteomics and biochemical studies in non-neuronal cell types had identified orthologs to some of those but not to others. The identification here of novel ubiquitin substrates, those with no known ubiquitinated ortholog, suggests that proteomics studies must be performed on neuronal cells to identify ubiquitination pathways not shared by other cell types. Importantly, several of those newly found neuronal ubiquitin substrates are key players in synaptogenesis. Mass spectrometry results were validated by Western blotting to confirm that those proteins are indeed ubiquitinated in the Drosophila embryonic nervous system and to elucidate whether they are mono- or polyubiquitinated. In addition to the ubiquitin substrates, we also identified the ubiquitin carriers that are active during synaptogenesis. Identifying endogenously ubiquitinated proteins in specific cell types, at specific developmental stages, and within the context of a living organism will allow understanding how the tissue-specific function of those proteins is regulated by the ubiquitin system. PMID:20861518

Preparation of molecularly imprinted polymers specific to glycoproteins, glycans and monosaccharides via boronate affinity controllable-oriented surface imprinting.

PubMed

Xing, Rongrong; Wang, Shuangshou; Bie, Zijun; He, Hui; Liu, Zhen

2017-05-01

Molecularly imprinted polymers (MIPs) are materials that are designed to be receptors for a template molecule (e.g., a protein). They are made by polymerizing the polymerizable reagents in the presence of the template; when the template is removed, the material can be used for many applications that would traditionally use antibodies. Thus, MIPs are biomimetic of antibodies and in this capacity have found wide applications, such as sensing, separation and diagnosis. However, many imprinting approaches are uncontrollable, and facile imprinting approaches widely applicable to a large variety of templates remain limited. We developed an approach called boronate affinity controllable-oriented surface imprinting, which allows for easy and efficient preparation of MIPs specific to glycoproteins, glycans and monosaccharides. This approach relies on immobilization of a template (glycoprotein, glycan or monosaccharide) on a boronic-acid-functionalized substrate through boronate affinity interaction, followed by self-polymerization of biocompatible monomer(s) to form an imprinting layer on the substrate with appropriate thickness. Imprinting in this approach is performed in a controllable manner, permitting the thickness of the imprinting layer to be fine-tuned according to the molecular size of the template by adjusting the imprinting time. This not only simplifies the imprinting procedure but also makes the approach widely applicable to a large range of sugar-containing biomolecules. MIPs prepared by this approach exhibit excellent binding properties and can be applied to complex real samples. The MIPs prepared by this protocol have been used in affinity separation, disease diagnosis and bioimaging. The entire protocol, including preparation, property characterization and performance evaluation, takes ∼3-8 d, depending on the type of substrate and template used.

Impairments in Site-Specific AS160 Phosphorylation and Effects of Exercise Training

PubMed Central

Consitt, Leslie A.; Van Meter, Jessica; Newton, Christopher A.; Collier, David N.; Dar, Moahad S.; Wojtaszewski, Jørgen F.P.; Treebak, Jonas T.; Tanner, Charles J.; Houmard, Joseph A.

2013-01-01

The purpose of this study was to determine if site-specific phosphorylation at the level of Akt substrate of 160 kDa (AS160) is altered in skeletal muscle from sedentary humans across a wide range of the adult life span (18–84 years of age) and if endurance- and/or strength-oriented exercise training could rescue decrements in insulin action and skeletal muscle AS160 phosphorylation. A euglycemic-hyperinsulinemic clamp and skeletal muscle biopsies were performed in 73 individuals encompassing a wide age range (18–84 years of age), and insulin-stimulated AS160 phosphorylation was determined. Decrements in whole-body insulin action were associated with impairments in insulin-induced phosphorylation of skeletal muscle AS160 on sites Ser-588, Thr-642, Ser-666, and phospho-Akt substrate, but not Ser-318 or Ser-751. Twelve weeks of endurance- or strength-oriented exercise training increased whole-body insulin action and reversed impairments in AS160 phosphorylation evident in insulin-resistant aged individuals. These findings suggest that a dampening of insulin-induced phosphorylation of AS160 on specific sites in skeletal muscle contributes to the insulin resistance evident in a sedentary aging population and that exercise training is an effective intervention for treating these impairments. PMID:23801578

Crystal structure of a four-copper laccase complexed with an arylamine: insights into substrate recognition and correlation with kinetics.

PubMed

Bertrand, Thomas; Jolivalt, Claude; Briozzo, Pierre; Caminade, Eliane; Joly, Nathalie; Madzak, Catherine; Mougin, Christian

2002-06-11

Laccases are multicopper oxidases that catalyze the oxidation of a wide range of phenols or arylamines, and their use in industrial oxidative processes is increasing. We purified from the white rot fungus Trametes versicolor a laccase that exists as five different isozymes, depending on glycosylation. The 2.4 A resolution structure of the most abundant isozyme of the glycosylated enzyme was solved. The four copper atoms are present, and it is the first crystal structure of a laccase in its active form. The crystallized enzyme binds 2,5-xylidine, which was used as a laccase inducer in the fungus culture. This arylamine is a very weak reducing substrate of the enzyme. The cavity enclosing 2,5-xylidine is rather wide, allowing the accommodation of substrates of various sizes. Several amino acid residues make hydrophobic interactions with the aromatic ring of the ligand. In addition, two charged or polar residues interact with its amino group. The first one is an histidine that also coordinates the copper that functions as the primary electron acceptor. The second is an aspartate conserved among fungal laccases. The purified enzyme can oxidize various hydroxylated compounds of the phenylurea family of herbicides that we synthesized. These phenolic substrates have better affinities at pH 5 than at pH 3, which could be related to the 2,5-xylidine binding by the aspartate. This is the first high-resolution structure of a multicopper oxidase complexed to a reducing substrate. It provides a model for engineering laccases that are either more efficient or with a wider substrate specificity.

Functional and Structural Characterization of Purine Nucleoside Phosphorylase from Kluyveromyces lactis and Its Potential Applications in Reducing Purine Content in Food

PubMed Central

Mahor, Durga; Priyanka, Anu; Prasad, Gandham S; Thakur, Krishan Gopal

2016-01-01

Consumption of foods and beverages with high purine content increases the risk of hyperuricemia, which causes gout and can lead to cardiovascular, renal, and other metabolic disorders. As patients often find dietary restrictions challenging, enzymatically lowering purine content in popular foods and beverages offers a safe and attractive strategy to control hyperuricemia. Here, we report structurally and functionally characterized purine nucleoside phosphorylase (PNP) from Kluyveromyces lactis (KlacPNP), a key enzyme involved in the purine degradation pathway. We report a 1.97 Å resolution crystal structure of homotrimeric KlacPNP with an intrinsically bound hypoxanthine in the active site. KlacPNP belongs to the nucleoside phosphorylase-I (NP-I) family, and it specifically utilizes 6-oxopurine substrates in the following order: inosine > guanosine > xanthosine, but is inactive towards adenosine. To engineer enzymes with broad substrate specificity, we created two point variants, KlacPNPN256D and KlacPNPN256E, by replacing the catalytically active Asn256 with Asp and Glu, respectively, based on structural and comparative sequence analysis. KlacPNPN256D not only displayed broad substrate specificity by utilizing both 6-oxopurines and 6-aminopurines in the order adenosine > inosine > xanthosine > guanosine, but also displayed reversal of substrate specificity. In contrast, KlacPNPN256E was highly specific to inosine and could not utilize other tested substrates. Beer consumption is associated with increased risk of developing gout, owing to its high purine content. Here, we demonstrate that KlacPNP and KlacPNPN256D could be used to catalyze a key reaction involved in lowering beer purine content. Biochemical properties of these enzymes such as activity across a wide pH range, optimum activity at about 25°C, and stability for months at about 8°C, make them suitable candidates for food and beverage industries. Since KlacPNPN256D has broad substrate specificity, a combination of engineered KlacPNP and other enzymes involved in purine degradation could effectively lower the purine content in foods and beverages. PMID:27768715

A novel esterase gene cloned from a metagenomic library from neritic sediments of the South China Sea

PubMed Central

2011-01-01

Background Marine microbes are a large and diverse group, which are exposed to a wide variety of pressure, temperature, salinity, nutrient availability and other environmental conditions. They provide a huge potential source of novel enzymes with unique properties that may be useful in industry and biotechnology. To explore the lipolytic genetic resources in the South China Sea, 23 sediment samples were collected in the depth < 100 m marine areas. Results A metagenomic library of South China Sea sediments assemblage in plasmid vector containing about 194 Mb of community DNA was prepared. Screening of a part of the unamplified library resulted in isolation of 15 unique lipolytic clones with the ability to hydrolyze tributyrin. A positive recombinant clone (pNLE1), containing a novel esterase (Est_p1), was successfully expressed in E. coli and purified. In a series of assays, Est_p1 displayed maximal activity at pH 8.57, 40°C, with ρ-Nitrophenyl butyrate (C4) as substrate. Compared to other metagenomic esterases, Est_p1 played a notable role in specificity for substrate C4 (kcat/Km value 11,500 S-1m M-1) and showed no inhibited by phenylmethylsulfonyl fluoride, suggested that the substrate binding pocket was suitable for substrate C4 and the serine active-site residue was buried at the bottom of substrate binding pocket which sheltered by a lid structure. Conclusions Esterase, which specificity towards short chain fatty acids, especially butanoic acid, is commercially available as potent flavoring tools. According the outstanding activity and specificity for substrate C4, Est_p1 has potential application in flavor industries requiring hydrolysis of short chain esters. PMID:22067554

Controlling the preferential orientation in sol-gel prepared CaCu3Ti4O12 thin films by LaAlO3 and NdGaO3 substrates

NASA Astrophysics Data System (ADS)

Pongpaiboonkul, Suriyong; Kasa, Yumairah; Phokharatkul, Ditsayut; Putasaeng, Bundit; Hodak, Jose H.; Wisitsoraat, Anurat; Hodak, Satreerat K.

2016-11-01

Researchers have paid considerable attention to CaCu3Ti4O12 (CCTO) due to the colossal dielectric constant over a wide range of frequency and temperature. Despite of the growing number of works dealing with CCTO, there have been few studies of the role played by the substrate in inducing structural and dielectric effects of this material. In this work, highly-oriented CCTO thin films have been deposited on LaAlO3(100), NdGaO3(100) and NdGaO3(110) substrates using a sol-gel method. These single crystal substrates were chosen in terms of small lattice mismatch between CCTO and the substrate. The X-ray diffraction patterns showed that the CCTO film layers grow with different orientations depending upon the substrate used. We show that the preferred orientation of CCTO thin films can be manipulated to a high degree by growing it on specific crystal planes of the substrates without the use of buffer layers. Colossal dielectric constants are observed in our films which appear to correlate with the film crystallinity and preferred orientation.

Molecular Determinants for Substrate Interactions with the Glycine Transporter GlyT2.

PubMed

Carland, Jane E; Thomas, Michael; Mostyn, Shannon N; Subramanian, Nandhitha; O'Mara, Megan L; Ryan, Renae M; Vandenberg, Robert J

2018-03-21

Transporters in the SLC6 family play key roles in regulating neurotransmission and are the targets for a wide range of therapeutics. Important insights into the transport mechanisms and the specificity of drug interactions of SLC6 transporters have been obtained from the crystal structures of a bacterial homologue of the family, LeuT Aa , and more recently the Drosophila dopamine transporter and the human serotonin transporter. However, there is disputed evidence that the bacterial leucine transporter, LeuT Aa , contains two substrate binding sites that work cooperatively in the mechanism of transport, with the binding of a second substrate being required for the release of the substrate from the primary site. An alternate proposal is that there may be low affinity binding sites that serve to direct the flow of substrates to the primary site. We have used a combination of molecular dynamics simulations of substrate interactions with a homology model of GlyT2, together with radiolabeled amino acid uptake assays and electrophysiological analysis of wild-type and mutant transporters, to provide evidence that substrate selectivity of GlyT2 is determined entirely by the primary substrate binding site and, furthermore, if a secondary site exists then it is a low affinity nonselective amino acid binding site.

A pharmacophore model specific to active site of CYP1A2 with a novel molecular modeling explorer and CoMFA.

PubMed

Zhang, Tao; Wei, Dong-Qing; Chou, Kuo-Chen

2012-03-01

Comparative molecular field analysis (CoMFA) is a widely used 3D-QSAR method by which we can investigate the potential relation between biological activity of compounds and their structural features. In this study, a new application of this approach is presented by combining the molecular modeling with a new developed pharmacophore model specific to CYP1A2 active site. During constructing the model, we used the molecular dynamics simulation and molecular docking method to select the sensible binding conformations for 17 CYP1A2 substrates based on the experimental data. Subsequently, the results obtained via the alignment of binding conformations of substrates were projected onto the active- site residues, upon which a simple blueprint of active site was produced. It was validated by the experimental and computational results that the model did exhibit the high degree of rationality and provide useful insights into the substrate binding. It is anticipated that our approach can be extended to investigate the protein-ligand interactions for many other enzyme-catalyzed systems as well.

Dissecting the functional significance of non-catalytic carbohydrate binding modules in the deconstruction of plant cell walls

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hahn, Michael G.

The project seeks to investigate the mechanism by which CBMs potentiate the activity of glycoside hydrolases against complete plant cell walls. The project is based on the hypothesis that the wide range of CBMs present in bacterial enzymes maximize the potential target substrates by directing the cognate enzymes not only to different regions of a specific plant cell wall, but also increases the range of plant cell walls that can be degraded. In addition to maximizing substrate access, it was also proposed that CBMs can target specific subsets of hydrolases with complementary activities to the same region of the plantmore » cell wall, thereby maximizing the synergistic interactions between these enzymes. This synergy is based on the premise that the hydrolysis of a specific polysaccharide will increase the access of closely associated polymers to enzyme attack. In addition, it is unclear whether the catalytic module and appended CBM of modular enzymes have evolved unique complementary activities.« less

A Versatile Method of Patterning Proteins and Cells.

PubMed

Shrirao, Anil B; Kung, Frank H; Yip, Derek; Firestein, Bonnie L; Cho, Cheul H; Townes-Anderson, Ellen

2017-02-26

Substrate and cell patterning techniques are widely used in cell biology to study cell-to-cell and cell-to-substrate interactions. Conventional patterning techniques work well only with simple shapes, small areas and selected bio-materials. This article describes a method to distribute cell suspensions as well as substrate solutions into complex, long, closed (dead-end) polydimethylsiloxane (PDMS) microchannels using negative pressure. This method enables researchers to pattern multiple substrates including fibronectin, collagen, antibodies (Sal-1), poly-D-lysine (PDL), and laminin. Patterning of substrates allows one to indirectly pattern a variety of cells. We have tested C2C12 myoblasts, the PC12 neuronal cell line, embryonic rat cortical neurons, and amphibian retinal neurons. In addition, we demonstrate that this technique can directly pattern fibroblasts in microfluidic channels via brief application of a low vacuum on cell suspensions. The low vacuum does not significantly decrease cell viability as shown by cell viability assays. Modifications are discussed for application of the method to different cell and substrate types. This technique allows researchers to pattern cells and proteins in specific patterns without the need for exotic materials or equipment and can be done in any laboratory with a vacuum.

Patterns and Variation in Benthic Biodiversity in a Large Marine Ecosystem.

PubMed

Piacenza, Susan E; Barner, Allison K; Benkwitt, Cassandra E; Boersma, Kate S; Cerny-Chipman, Elizabeth B; Ingeman, Kurt E; Kindinger, Tye L; Lee, Jonathan D; Lindsley, Amy J; Reimer, Jessica N; Rowe, Jennifer C; Shen, Chenchen; Thompson, Kevin A; Thurman, Lindsey L; Heppell, Selina S

2015-01-01

While there is a persistent inverse relationship between latitude and species diversity across many taxa and ecosystems, deviations from this norm offer an opportunity to understand the conditions that contribute to large-scale diversity patterns. Marine systems, in particular, provide such an opportunity, as marine diversity does not always follow a strict latitudinal gradient, perhaps because several hypothesized drivers of the latitudinal diversity gradient are uncorrelated in marine systems. We used a large scale public monitoring dataset collected over an eight year period to examine benthic marine faunal biodiversity patterns for the continental shelf (55-183 m depth) and slope habitats (184-1280 m depth) off the US West Coast (47°20'N-32°40'N). We specifically asked whether marine biodiversity followed a strict latitudinal gradient, and if these latitudinal patterns varied across depth, in different benthic substrates, and over ecological time scales. Further, we subdivided our study area into three smaller regions to test whether coast-wide patterns of biodiversity held at regional scales, where local oceanographic processes tend to influence community structure and function. Overall, we found complex patterns of biodiversity on both the coast-wide and regional scales that differed by taxonomic group. Importantly, marine biodiversity was not always highest at low latitudes. We found that latitude, depth, substrate, and year were all important descriptors of fish and invertebrate diversity. Invertebrate richness and taxonomic diversity were highest at high latitudes and in deeper waters. Fish richness also increased with latitude, but exhibited a hump-shaped relationship with depth, increasing with depth up to the continental shelf break, ~200 m depth, and then decreasing in deeper waters. We found relationships between fish taxonomic and functional diversity and latitude, depth, substrate, and time at the regional scale, but not at the coast-wide scale, suggesting that coast-wide patterns can obscure important correlates at smaller scales. Our study provides insight into complex diversity patterns of the deep water soft substrate benthic ecosystems off the US West Coast.

Structure and Functional Diversity of GCN5-Related N-Acetyltransferases (GNAT)

PubMed Central

Salah Ud-Din, Abu Iftiaf Md; Tikhomirova, Alexandra; Roujeinikova, Anna

2016-01-01

General control non-repressible 5 (GCN5)-related N-acetyltransferases (GNAT) catalyze the transfer of an acyl moiety from acyl coenzyme A (acyl-CoA) to a diverse group of substrates and are widely distributed in all domains of life. This review of the currently available data acquired on GNAT enzymes by a combination of structural, mutagenesis and kinetic methods summarizes the key similarities and differences between several distinctly different families within the GNAT superfamily, with an emphasis on the mechanistic insights obtained from the analysis of the complexes with substrates or inhibitors. It discusses the structural basis for the common acetyltransferase mechanism, outlines the factors important for the substrate recognition, and describes the mechanism of action of inhibitors of these enzymes. It is anticipated that understanding of the structural basis behind the reaction and substrate specificity of the enzymes from this superfamily can be exploited in the development of novel therapeutics to treat human diseases and combat emerging multidrug-resistant microbial infections. PMID:27367672

Novel inexpensive fungi proteases: Production by solid state fermentation and characterization.

PubMed

Novelli, Paula Kern; Barros, Margarida Maria; Fleuri, Luciana Francisco

2016-05-01

A comparative study was carried out for proteases production using agroindustrial residues as substrate for solid state fermentation (SSF) of several fungal strains. High protease production was observed for most of the microorganisms studied, as well as very different biochemical characteristics, including activities at specific temperatures and a wide range of pH values. The enzymes produced were very different regarding optimum pH and they showed stability at 50 °C. Aspergillus oryzae showed stability at all pH values studied. Penicillium roquefortii and Aspergillus flavipes presented optimum activity at temperatures of 50 °C and 90 °C, respectively. Lyophilized protease from A. oryzae reached 1251.60 U/g and yield of 155010.66 U/kg of substrate. Therefore, the substrate as well as the microorganism strain can modify the biochemical character of the enzyme produced. The high protease activity and stability established plus the low cost of substrates, make these fungal proteases potential alternatives for the biotechnological industry. Copyright © 2015 Elsevier Ltd. All rights reserved.

Conserved regions of ribonucleoprotein ribonuclease MRP are involved in interactions with its substrate

PubMed Central

Esakova, Olga; Perederina, Anna; Berezin, Igor; Krasilnikov, Andrey S.

2013-01-01

Ribonuclease (RNase) MRP is a ubiquitous and essential site-specific eukaryotic endoribonuclease involved in the metabolism of a wide range of RNA molecules. RNase MRP is a ribonucleoprotein with a large catalytic RNA moiety that is closely related to the RNA component of RNase P, and multiple proteins, most of which are shared with RNase P. Here, we report the results of an ultraviolet-cross-linking analysis of interactions between a photoreactive RNase MRP substrate and the Saccharomyces cerevisiae RNase MRP holoenzyme. The results show that the substrate interacts with phylogenetically conserved RNA elements universally found in all enzymes of the RNase P/MRP family, as well as with a phylogenetically conserved RNA region that is unique to RNase MRP, and demonstrate that four RNase MRP protein components, all shared with RNase P, interact with the substrate. Implications for the structural organization of RNase MRP and the roles of its components are discussed. PMID:23700311

Design of a glutamine substrate tag enabling protein labelling mediated by Bacillus subtilis transglutaminase.

PubMed

Oteng-Pabi, Samuel K; Clouthier, Christopher M; Keillor, Jeffrey W

2018-01-01

Transglutaminases (TGases) are enzymes that catalyse protein cross-linking through a transamidation reaction between the side chain of a glutamine residue on one protein and the side chain of a lysine residue on another. Generally, TGases show low substrate specificity with respect to their amine substrate, such that a wide variety of primary amines can participate in the modification of specific glutamine residue. Although a number of different TGases have been used to mediate these bioconjugation reactions, the TGase from Bacillus subtilis (bTG) may be particularly suited to this application. It is smaller than most TGases, can be expressed in a soluble active form, and lacks the calcium dependence of its mammalian counterparts. However, little is known regarding this enzyme and its glutamine substrate specificity, limiting the scope of its application. In this work, we designed a FRET-based ligation assay to monitor the bTG-mediated conjugation of the fluorescent proteins Clover and mRuby2. This assay allowed us to screen a library of random heptapeptide glutamine sequences for their reactivity with recombinant bTG in bacterial cells, using fluorescence assisted cell sorting. From this library, several reactive sequences were identified and kinetically characterized, with the most reactive sequence (YAHQAHY) having a kcat/KM value of 19 ± 3 μM-1 min-1. This sequence was then genetically appended onto a test protein as a reactive 'Q-tag' and fluorescently labelled with dansyl-cadaverine, in the first demonstration of protein labelling mediated by bTG.

A new fusion protein platform for quantitatively measuring activity of multiple proteases

PubMed Central

2014-01-01

Background Recombinant proteins fused with specific cleavage sequences are widely used as substrate for quantitatively analyzing the activity of proteases. Here we propose a new fusion platform for multiple proteases, by using diaminopropionate ammonia-lyase (DAL) as the fusion protein. It was based on the finding that a fused His6-tag could significantly decreases the activities of DAL from E. coli (eDAL) and Salmonella typhimurium (sDAL). Previously, we have shown that His6GST-tagged eDAL could be used to determine the activity of tobacco etch virus protease (TEVp) under different temperatures or in the denaturant at different concentrations. In this report, we will assay different tags and cleavage sequences on DAL for expressing yield in E. coli, stability of the fused proteins and performance of substrate of other common proteases. Results We tested seven different protease cleavage sequences (rhinovirus 3C, TEV protease, factor Xa, Ssp DnaB intein, Sce VMA1 intein, thrombin and enterokinase), three different tags (His6, GST, CBD and MBP) and two different DALs (eDAL and sDAL), for their performance as substrate to the seven corresponding proteases. Among them, we found four active DAL-fusion substrates suitable for TEVp, factor Xa, thrombin and DnaB intein. Enterokinase cleaved eDAL at undesired positions and did not process sDAL. Substitution of GST with MBP increase the expression level of the fused eDAL and this fusion protein was suitable as a substrate for analyzing activity of rhinovirus 3C. We demonstrated that SUMO protease Ulp1 with a N-terminal His6-tag or MBP tag displayed different activity using the designed His6SUMO-eDAL as substrate. Finally, owing to the high level of the DAL-fusion protein in E. coli, these protein substrates can also be detected directly from the crude extract. Conclusion The results show that our designed DAL-fusion proteins can be used to quantify the activities of both sequence- and conformational-specific proteases, with sufficient substrate specificity. PMID:24649897

Structural determinants of tobacco vein mottling virus protease substrate specificity

PubMed Central

Sun, Ping; Austin, Brian P; Tözsér, József; Waugh, David S

2010-01-01

Tobacco vein mottling virus (TVMV) is a member of the Potyviridae, one of the largest families of plant viruses. The TVMV genome is translated into a single large polyprotein that is subsequently processed by three virally encoded proteases. Seven of the nine cleavage events are carried out by the NIa protease. Its homolog from the tobacco etch virus (TEV) is a widely used reagent for the removal of affinity tags from recombinant proteins. Although TVMV protease is a close relative of TEV protease, they exhibit distinct sequence specificities. We report here the crystal structure of a catalytically inactive mutant TVMV protease (K65A/K67A/C151A) in complex with a canonical peptide substrate (Ac-RETVRFQSD) at 1.7-Å resolution. As observed in several crystal structures of TEV protease, the C-terminus (∼20 residues) of TVMV protease is disordered. Unexpectedly, although deleting the disordered residues from TEV protease reduces its catalytic activity by ∼10-fold, an analogous truncation mutant of TVMV protease is significantly more active. Comparison of the structures of TEV and TVMV protease in complex with their respective canonical substrate peptides reveals that the S3 and S4 pockets are mainly responsible for the differing substrate specificities. The structure of TVMV protease suggests that it is less tolerant of variation at the P1′ position than TEV protease. This conjecture was confirmed experimentally by determining kinetic parameters kcat and Km for a series of oligopeptide substrates. Also, as predicted by the cocrystal structure, we confirm that substitutions in the P6 position are more readily tolerated by TVMV than TEV protease. PMID:20862670

Structural determinants of tobacco vein mottling virus protease substrate specificity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sun, Ping; Austin, Brian P.; Tozer, Jozsef

2010-10-28

Tobacco vein mottling virus (TVMV) is a member of the Potyviridae, one of the largest families of plant viruses. The TVMV genome is translated into a single large polyprotein that is subsequently processed by three virally encoded proteases. Seven of the nine cleavage events are carried out by the NIa protease. Its homolog from the tobacco etch virus (TEV) is a widely used reagent for the removal of affinity tags from recombinant proteins. Although TVMV protease is a close relative of TEV protease, they exhibit distinct sequence specificities. We report here the crystal structure of a catalytically inactive mutant TVMVmore » protease (K65A/K67A/C151A) in complex with a canonical peptide substrate (Ac-RETVRFQSD) at 1.7-{angstrom} resolution. As observed in several crystal structures of TEV protease, the C-terminus ({approx}20 residues) of TVMV protease is disordered. Unexpectedly, although deleting the disordered residues from TEV protease reduces its catalytic activity by {approx}10-fold, an analogous truncation mutant of TVMV protease is significantly more active. Comparison of the structures of TEV and TVMV protease in complex with their respective canonical substrate peptides reveals that the S3 and S4 pockets are mainly responsible for the differing substrate specificities. The structure of TVMV protease suggests that it is less tolerant of variation at the P1{prime} position than TEV protease. This conjecture was confirmed experimentally by determining kinetic parameters k{sub cat} and K{sub m} for a series of oligopeptide substrates. Also, as predicted by the cocrystal structure, we confirm that substitutions in the P6 position are more readily tolerated by TVMV than TEV protease.« less

New class of radioenzymatic assay for the quantification of p-tyramine and phenylethylamine

DOE Office of Scientific and Technical Information (OSTI.GOV)

Henry, D.P.; Van Huysse, J.W.; Bowsher, R.R.

Radioenzymatic assays are widely used for the quantification of a number of biogenic amines. All previous procedures have utilized methyltransferases derived from mammalian tissues. In this assay for the quantification of the trace aralkylamines, p-tyramine (p-tym) and phenylethylamine (PEA), an enzyme, tyramine N-methyltransferase isolated from sprouted barley roots was used. The enzyme was specific for phenylethylamines. Of 26 structurally-related compounds, only p-tym, PEA, m-tym and amphetamine were substrates in vitro. Theoretic maximal methylation of substrates occurred at 10-20/sup 0/C. When TLC was used to separate the radiolabeled reaction products, a specific method was developed for p-tym and PEA. The assaymore » had a sensitivity of 0.8 and 2.8 pg/tube with a C.V. < 5% and was applicable to human plasma and urine. Assay throughput is similar to that of other TLC based radioenzymatic assays.« less

Single-Molecule Probing the Energy Landscape of Enzymatic Reaction and Non-Covalent Interactions

NASA Astrophysics Data System (ADS)

Lu, H. Peter; Hu, Dehong; Chen, Yu; Vorpagel, Erich R.

2002-03-01

We have applied single-molecule spectroscopy under physiological conditions to study the mechanisms and dynamics of T4 lysozyme enzymatic reactions, characterizing mode-specific protein conformational dynamics. Enzymatic reaction turnovers and the associated structure changes of individual protein molecules were observed simultaneously in real-time. The overall reaction rates were found to vary widely from molecule-to-molecule, and the initial non-specific binding of the enzyme to the substrate was seen to dominate this inhomogeneity. The reaction steps subsequent to the initial binding were found to have homogeneous rates. Molecular dynamics simulation has been applied to elucidate the mechanism and intermediate states of the single-molecule enzymatic reaction. Combining the analysis of single-molecule experimental trajectories, MD simulation trajectories, and statistical modeling, we have revealed the nature of multiple intermediate states involved in the active enzyme-substrate complex formation and the associated conformational change mechanism and dynamics.

Prospecting Biotechnologically-Relevant Monooxygenases from Cold Sediment Metagenomes: An In Silico Approach

DOE PAGES

Musumeci, Matias A.; Lozada, Mariana; Rial, Daniela V.; ...

2017-04-09

The goal of this work was to identify sequences encoding monooxygenase biocatalysts with novel features by in silico mining an assembled metagenomic dataset of polar and subpolar marine sediments. The targeted enzyme sequences were Baeyer-Villiger and bacterial cytochrome P450 monooxygenases (CYP153). These enzymes have wide-ranging applications, from the synthesis of steroids, antibiotics, mycotoxins and pheromones to the synthesis of monomers for polymerization and anticancer precursors, due to their extraordinary enantio-, regio-, and chemo- selectivity that are valuable features for organic synthesis. Phylogenetic analyses were used to select the most divergent sequences affiliated to these enzyme families among the 264 putativemore » monooxygenases recovered from the ~14 million protein-coding sequences in the assembled metagenome dataset. Three-dimensional structure modeling and docking analysis suggested features useful in biotechnological applications in five metagenomic sequences, such as wide substrate range, novel substrate specificity or regioselectivity. Further analysis revealed structural features associated with psychrophilic enzymes, such as broader substrate accessibility, larger catalytic pockets or low domain interactions, suggesting that they could be applied in biooxidations at room or low temperatures, saving costs inherent to energy consumption. As a result, this work allowed the identification of putative enzyme candidates with promising features from metagenomes, providing a suitable starting point for further developments.« less

Prospecting Biotechnologically-Relevant Monooxygenases from Cold Sediment Metagenomes: An In Silico Approach

DOE Office of Scientific and Technical Information (OSTI.GOV)

Musumeci, Matias A.; Lozada, Mariana; Rial, Daniela V.

The goal of this work was to identify sequences encoding monooxygenase biocatalysts with novel features by in silico mining an assembled metagenomic dataset of polar and subpolar marine sediments. The targeted enzyme sequences were Baeyer-Villiger and bacterial cytochrome P450 monooxygenases (CYP153). These enzymes have wide-ranging applications, from the synthesis of steroids, antibiotics, mycotoxins and pheromones to the synthesis of monomers for polymerization and anticancer precursors, due to their extraordinary enantio-, regio-, and chemo- selectivity that are valuable features for organic synthesis. Phylogenetic analyses were used to select the most divergent sequences affiliated to these enzyme families among the 264 putativemore » monooxygenases recovered from the ~14 million protein-coding sequences in the assembled metagenome dataset. Three-dimensional structure modeling and docking analysis suggested features useful in biotechnological applications in five metagenomic sequences, such as wide substrate range, novel substrate specificity or regioselectivity. Further analysis revealed structural features associated with psychrophilic enzymes, such as broader substrate accessibility, larger catalytic pockets or low domain interactions, suggesting that they could be applied in biooxidations at room or low temperatures, saving costs inherent to energy consumption. As a result, this work allowed the identification of putative enzyme candidates with promising features from metagenomes, providing a suitable starting point for further developments.« less

Prospecting Biotechnologically-Relevant Monooxygenases from Cold Sediment Metagenomes: An In Silico Approach.

PubMed

Musumeci, Matías A; Lozada, Mariana; Rial, Daniela V; Mac Cormack, Walter P; Jansson, Janet K; Sjöling, Sara; Carroll, JoLynn; Dionisi, Hebe M

2017-04-09

The goal of this work was to identify sequences encoding monooxygenase biocatalysts with novel features by in silico mining an assembled metagenomic dataset of polar and subpolar marine sediments. The targeted enzyme sequences were Baeyer-Villiger and bacterial cytochrome P450 monooxygenases (CYP153). These enzymes have wide-ranging applications, from the synthesis of steroids, antibiotics, mycotoxins and pheromones to the synthesis of monomers for polymerization and anticancer precursors, due to their extraordinary enantio-, regio-, and chemo- selectivity that are valuable features for organic synthesis. Phylogenetic analyses were used to select the most divergent sequences affiliated to these enzyme families among the 264 putative monooxygenases recovered from the ~14 million protein-coding sequences in the assembled metagenome dataset. Three-dimensional structure modeling and docking analysis suggested features useful in biotechnological applications in five metagenomic sequences, such as wide substrate range, novel substrate specificity or regioselectivity. Further analysis revealed structural features associated with psychrophilic enzymes, such as broader substrate accessibility, larger catalytic pockets or low domain interactions, suggesting that they could be applied in biooxidations at room or low temperatures, saving costs inherent to energy consumption. This work allowed the identification of putative enzyme candidates with promising features from metagenomes, providing a suitable starting point for further developments.

Prospecting Biotechnologically-Relevant Monooxygenases from Cold Sediment Metagenomes: An In Silico Approach

PubMed Central

Musumeci, Matías A.; Lozada, Mariana; Rial, Daniela V.; Mac Cormack, Walter P.; Jansson, Janet K.; Sjöling, Sara; Carroll, JoLynn; Dionisi, Hebe M.

2017-01-01

The goal of this work was to identify sequences encoding monooxygenase biocatalysts with novel features by in silico mining an assembled metagenomic dataset of polar and subpolar marine sediments. The targeted enzyme sequences were Baeyer–Villiger and bacterial cytochrome P450 monooxygenases (CYP153). These enzymes have wide-ranging applications, from the synthesis of steroids, antibiotics, mycotoxins and pheromones to the synthesis of monomers for polymerization and anticancer precursors, due to their extraordinary enantio-, regio-, and chemo- selectivity that are valuable features for organic synthesis. Phylogenetic analyses were used to select the most divergent sequences affiliated to these enzyme families among the 264 putative monooxygenases recovered from the ~14 million protein-coding sequences in the assembled metagenome dataset. Three-dimensional structure modeling and docking analysis suggested features useful in biotechnological applications in five metagenomic sequences, such as wide substrate range, novel substrate specificity or regioselectivity. Further analysis revealed structural features associated with psychrophilic enzymes, such as broader substrate accessibility, larger catalytic pockets or low domain interactions, suggesting that they could be applied in biooxidations at room or low temperatures, saving costs inherent to energy consumption. This work allowed the identification of putative enzyme candidates with promising features from metagenomes, providing a suitable starting point for further developments. PMID:28397770

Regulator and enzyme specificities of the TOL plasmid-encoded upper pathway for degradation of aromatic hydrocarbons and expansion of the substrate range of the pathway.

PubMed Central

Abril, M A; Michan, C; Timmis, K N; Ramos, J L

1989-01-01

The TOL plasmid upper pathway operon encodes enzymes involved in the catabolism of aromatic hydrocarbons such as toluene and xylenes. The regulator of the gene pathway, the XylR protein, exhibits a very broad effector specificity, being able to recognize as effectors not only pathway substrates but also a wide variety of mono- and disubstituted methyl-, ethyl-, and chlorotoluenes, benzyl alcohols, and p-chlorobenzaldehyde. Benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase, two upper pathway enzymes, exhibit very broad substrate specificities and transform unsubstituted substrates and m- and p-methyl-, m- and p-ethyl-, and m- and p-chloro-substituted benzyl alcohols and benzaldehydes, respectively, at a high rate. In contrast, toluene oxidase only oxidizes toluene, m- and p-xylene, m-ethyltoluene, and 1,2,4-trimethylbenzene [corrected], also at a high rate. A biological test showed that toluene oxidase attacks m- and p-chlorotoluene, albeit at a low rate. No evidence for the transformation of p-ethyltoluene by toluene oxidase has been found. Hence, toluene oxidase acts as the bottleneck step for the catabolism of p-ethyl- and m- and p-chlorotoluene through the TOL upper pathway. A mutant toluene oxidase able to transform p-ethyltoluene was isolated, and a mutant strain capable of fully degrading p-ethyltoluene was constructed with a modified TOL plasmid meta-cleavage pathway able to mineralize p-ethylbenzoate. By transfer of a TOL plasmid into Pseudomonas sp. strain B13, a clone able to slowly degrade m-chlorotoluene was also obtained. PMID:2687253

Broadening the functionality of a J-protein/Hsp70 molecular chaperone system.

PubMed

Schilke, Brenda A; Ciesielski, Szymon J; Ziegelhoffer, Thomas; Kamiya, Erina; Tonelli, Marco; Lee, Woonghee; Cornilescu, Gabriel; Hines, Justin K; Markley, John L; Craig, Elizabeth A

2017-10-01

By binding to a multitude of polypeptide substrates, Hsp70-based molecular chaperone systems perform a range of cellular functions. All J-protein co-chaperones play the essential role, via action of their J-domains, of stimulating the ATPase activity of Hsp70, thereby stabilizing its interaction with substrate. In addition, J-proteins drive the functional diversity of Hsp70 chaperone systems through action of regions outside their J-domains. Targeting to specific locations within a cellular compartment and binding of specific substrates for delivery to Hsp70 have been identified as modes of J-protein specialization. To better understand J-protein specialization, we concentrated on Saccharomyces cerevisiae SIS1, which encodes an essential J-protein of the cytosol/nucleus. We selected suppressors that allowed cells lacking SIS1 to form colonies. Substitutions changing single residues in Ydj1, a J-protein, which, like Sis1, partners with Hsp70 Ssa1, were isolated. These gain-of-function substitutions were located at the end of the J-domain, suggesting that suppression was connected to interaction with its partner Hsp70, rather than substrate binding or subcellular localization. Reasoning that, if YDJ1 suppressors affect Ssa1 function, substitutions in Hsp70 itself might also be able to overcome the cellular requirement for Sis1, we carried out a selection for SSA1 suppressor mutations. Suppressing substitutions were isolated that altered sites in Ssa1 affecting the cycle of substrate interaction. Together, our results point to a third, additional means by which J-proteins can drive Hsp70's ability to function in a wide range of cellular processes-modulating the Hsp70-substrate interaction cycle.

A novel serine protease from strawberry (Fragaria ananassa): Purification and biochemical characterization.

PubMed

Alici, Esma Hande; Arabaci, Gulnur

2018-03-27

In this study, a protease enzyme was purified from strawberry by using Sepharose-4B-l-tyrosine-p-amino benzoic acid affinity chromatography. The molecular weight of pure protease was determined 65.8 kDa by SDS-PAGE. The single band observed on the gel showed that the enzyme had a single polypeptide chain and was successfully purified. Purification of the protease by the chromatographic method resulted in a 395.6-fold increase in specific activity (3600 U/mg). Optimum pH and temperature for the enzyme were 6 and 40 °C, respectively. The protease was stable at a wide temperature range of 40 to 70 °C and a pH range of 3.0 to 9.0. Co 2+ ions stimulated protease activity very strongly. Cu 2+ , Hg 2+ , Cd 2+ and Mn 2+ ions significantly inhibited protease activity. While 2-propanol completely inhibited the enzyme, the enzyme maintained its activity better in the presence of ethanol and methanol. The strawberry protease showed the highest specificity towards hemoglobin among all the natural substrates tested. The specificity of the enzyme towards synthetic substrates was also investigated and it was concluded that it has broad substrate specificity. The obtained results indicated that this purified protease was highly-likely a serine protease and its activity was significantly affected by the presence of metal ions. Copyright © 2018. Published by Elsevier B.V.

Mouse d-Amino-Acid Oxidase: Distribution and Physiological Substrates

PubMed Central

Koga, Reiko; Miyoshi, Yurika; Sakaue, Hiroaki; Hamase, Kenji; Konno, Ryuichi

2017-01-01

d-Amino-acid oxidase (DAO) catalyzes the oxidative deamination of d-amino acids. DAO is present in a wide variety of organisms and has important roles. Here, we review the distribution and physiological substrates of mouse DAO. Mouse DAO is present in the kidney, brain, and spinal cord, like DAOs in other mammals. However, in contrast to other animals, it is not present in the mouse liver. Recently, DAO has been detected in the neutrophils, retina, and small intestine in mice. To determine the physiological substrates of mouse DAO, mutant mice lacking DAO activity are helpful. As DAO has wide substrate specificity and degrades various d-amino acids, many d-amino acids accumulate in the tissues and body fluids of the mutant mice. These amino acids are d-methionine, d-alanine, d-serine, d-leucine, d-proline, d-phenylalanine, d-tyrosine, and d-citrulline. Even in wild-type mice, administration of DAO inhibitors elevates D-serine levels in the plasma and brain. Among the above d-amino acids, the main physiological substrates of mouse DAO are d-alanine and d-serine. These two d-amino acids are most abundant in the tissues and body fluids of mice. d-Alanine derives from bacteria and produces bactericidal reactive oxygen species by the action of DAO. d-Serine is synthesized by serine racemase and is present especially in the central nervous system, where it serves as a neuromodulator. DAO is responsible for the metabolism of d-serine. Since DAO has been implicated in the etiology of neuropsychiatric diseases, mouse DAO has been used as a representative model. Recent reports, however, suggest that mouse DAO is different from human DAO with respect to important properties. PMID:29255714

Substrate inhibition kinetics of phenol biodegradation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Goudar, C.T.; Ganji, S.H.; Pujar, B.G.

Phenol biodegradation was studied in batch experiments using an acclimated inoculum and initial phenol concentrations ranging from 0.1 to 1.3 g/L. Phenol depletion an associated microbial growth were monitored over time to provide information that was used to estimate the kinetics of phenol biodegradation. Phenol inhibited biodegradation at high concentrations, and a generalized substrate inhibition model based on statistical thermodynamics was used to describe the dynamics of microbial growth in phenol. For experimental data obtained in this study, the generalized substrate inhibition model reduced to a form that is analogous to the Andrews equation, and the biokinetic parameters {micro}{sub max},more » maximum specific growth; K{sub s}, saturation constant; and K{sub i}, inhibition constant were estimated as 0.251 h{sup {minus}1}, 0.011 g/L, and 0.348 g/L, respectively, using a nonlinear least squares technique. Given the wide variability in substrate inhibition models used to describe phenol biodegradation, an attempt was made to justify selection of particular model based on theoretical considerations. Phenol biodegradation data from nine previously published studies were used in the generalized substrate inhibition model to determine the appropriate form of the substrate inhibition model. In all nine cases, the generalized substrate inhibition model reduced to a form analogous to the Andrews equation suggesting the suitability of the Andrews equation to describe phenol biodegradation data.« less

Identification of putative substrates for cynomolgus monkey cytochrome P450 2C8 by substrate depletion assays with 22 human P450 substrates and inhibitors.

PubMed

Hosaka, Shinya; Murayama, Norie; Satsukawa, Masahiro; Uehara, Shotaro; Shimizu, Makiko; Iwasaki, Kazuhide; Iwano, Shunsuke; Uno, Yasuhiro; Yamazaki, Hiroshi

2016-07-01

Cynomolgus monkeys are widely used in drug developmental stages as non-human primate models. Previous studies used 89 compounds to investigate species differences associated with cytochrome P450 (P450 or CYP) function that reported monkey specific CYP2C76 cleared 19 chemicals, and homologous CYP2C9 and CYP2C19 metabolized 17 and 30 human CYP2C9 and/or CYP2C19 substrates/inhibitors, respectively. In the present study, 22 compounds selected from viewpoints of global drug interaction guidances and guidelines were further evaluated to seek potential substrates for monkey CYP2C8, which is highly homologous to human CYP2C8 (92%). Amodiaquine, montelukast, quercetin and rosiglitazone, known as substrates or competitive inhibitors of human CYP2C8, were metabolically depleted by recombinant monkey CYP2C8 at relatively high rates. Taken together with our reported findings of the slow eliminations of amodiaquine and montelukast by monkey CYP2C9, CYP2C19 and CYP2C76, the present results suggest that these at least four chemicals may be good marker substrates for monkey CYP2C8. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

Carbon utilization profile of the filamentous fungal species Fusarium fujikuroi, Penicillium decumbens, and Sarocladium strictum isolated from marine coastal environments.

PubMed

Fuentes, Marcelo E; Quiñones, Renato A

Facultative marine filamentous fungi have recently emerged as a functional component in coastal marine systems. However, little is known about their ecological role and functions in biogeochemical cycles. Penicillium decumbens, S. strictum, and F. fujikuroi were isolated from the coastal upwelling zone off south-central Chile. Their carbon profiles were characterized using Biolog FF MicroPlates. These species used a wide range of carbon sources, mainly carbohydrates, but also amino acids, suggesting the use of metabolic routes that include glycolysis/gluconeogenesis. Substrate richness revealed a great capacity for the utilization of nutritional sources, reflected by the following Shannon Indices of utilization of specific substrates: 4.02 for S. strictum, 4.01 for P. decumbes, and 3.91 for F. fujikuroi, which reveals a high physiological capacity for oxidizing different substrates. Significant differences were found between 18 substrates utilized by all three species. Results suggest that filamentous fungi should be considered an integral part of the marine microbial community and included in biogeochemical cycling models of upwelling ecosystems.

Introducing N-glycans into natural products through a chemoenzymatic approach.

PubMed

Huang, Wei; Ochiai, Hirofumi; Zhang, Xinyu; Wang, Lai-Xi

2008-11-24

The present study describes an efficient chemoenzymatic method for introducing a core N-glycan of glycoprotein origin into various lipophilic natural products. It was found that the endo-beta-N-acetylglucosaminidase from Arthrobactor protophormiae (Endo-A) had broad substrate specificity and can accommodate a wide range of glucose (Glc)- or N-acetylglucosamine (GlcNAc)-containing natural products as acceptors for transglycosylation, when an N-glycan oxazoline was used as a donor substrate. Using lithocholic acid as a model compound, we have shown that introduction of an N-glycan could be achieved by a two-step approach: chemical glycosylation to introduce a monosaccharide (Glc or GlcNAc) as a handle, and then Endo-A catalyzed transglycosylation to accomplish the site-specific N-glycan attachment. For those natural products that already carry terminal Glc or GlcNAc residues, direct enzymatic transglycosylation using sugar oxazoline as the donor substrate was achievable to introduce an N-glycan. It was also demonstrated that simultaneous double glycosylation could be fulfilled when the natural product contains two Glc residues. This chemoenzymatic method is concise, site-specific, and highly convergent. Because N-glycans of glycoprotein origin can serve as ligands for diverse lectins and cell-surface receptors, introduction of a defined N-glycan into biologically significant natural products may bestow novel properties onto these natural products for drug discovery and development.

Prolonged Maltose-Limited Cultivation of Saccharomyces cerevisiae Selects for Cells with Improved Maltose Affinity and Hypersensitivity

PubMed Central

Jansen, Mickel L. A.; Daran-Lapujade, Pascale; de Winde, Johannes H.; Piper, Matthew D. W.; Pronk, Jack T.

2004-01-01

Prolonged cultivation (>25 generations) of Saccharomyces cerevisiae in aerobic, maltose-limited chemostat cultures led to profound physiological changes. Maltose hypersensitivity was observed when cells from prolonged cultivations were suddenly exposed to excess maltose. This substrate hypersensitivity was evident from massive cell lysis and loss of viability. During prolonged cultivation at a fixed specific growth rate, the affinity for the growth-limiting nutrient (i.e., maltose) increased, as evident from a decreasing residual maltose concentration. Furthermore, the capacity of maltose-dependent proton uptake increased up to 2.5-fold during prolonged cultivation. Genome-wide transcriptome analysis showed that the increased maltose transport capacity was not primarily due to increased transcript levels of maltose-permease genes upon prolonged cultivation. We propose that selection for improved substrate affinity (ratio of maximum substrate consumption rate and substrate saturation constant) in maltose-limited cultures leads to selection for cells with an increased capacity for maltose uptake. At the same time, the accumulative nature of maltose-proton symport in S. cerevisiae leads to unrestricted uptake when maltose-adapted cells are exposed to a substrate excess. These changes were retained after isolation of individual cell lines from the chemostat cultures and nonselective cultivation, indicating that mutations were involved. The observed trade-off between substrate affinity and substrate tolerance may be relevant for metabolic engineering and strain selection for utilization of substrates that are taken up by proton symport. PMID:15066785

Crystal Structure and Substrate Specificity Modification of Acetyl Xylan Esterase from Aspergillus luchuensis.

PubMed

Komiya, Dai; Hori, Akane; Ishida, Takuya; Igarashi, Kiyohiko; Samejima, Masahiro; Koseki, Takuya; Fushinobu, Shinya

2017-10-15

Acetyl xylan esterase (AXE) catalyzes the hydrolysis of the acetyl bonds present in plant cell wall polysaccharides. Here, we determined the crystal structure of AXE from Aspergillus luchuensis ( Al AXEA), providing the three-dimensional structure of an enzyme in the Esterase_phb family. Al AXEA shares its core α/β-hydrolase fold structure with esterases in other families, but it has an extended central β-sheet at both its ends and an extra loop. Structural comparison with a ferulic acid esterase (FAE) from Aspergillus niger indicated that Al AXEA has a conserved catalytic machinery: a catalytic triad (Ser119, His259, and Asp202) and an oxyanion hole (Cys40 and Ser120). Near the catalytic triad of A lAXEA, two aromatic residues (Tyr39 and Trp160) form small pockets at both sides. Homology models of fungal FAEs in the same Esterase_phb family have wide pockets at the corresponding sites because they have residues with smaller side chains (Pro, Ser, and Gly). Mutants with site-directed mutations at Tyr39 showed a substrate specificity similar to that of the wild-type enzyme, whereas those with mutations at Trp160 acquired an expanded substrate specificity. Interestingly, the Trp160 mutants acquired weak but significant type B-like FAE activity. Moreover, the engineered enzymes exhibited ferulic acid-releasing activity from wheat arabinoxylan. IMPORTANCE Hemicelluloses in the plant cell wall are often decorated by acetyl and ferulic acid groups. Therefore, complete and efficient degradation of plant polysaccharides requires the enzymes for cleaving the side chains of the polymer. Since the Esterase_phb family contains a wide array of fungal FAEs and AXEs from fungi and bacteria, our study will provide a structural basis for the molecular mechanism of these industrially relevant enzymes in biopolymer degradation. The structure of the Esterase_phb family also provides information for bacterial polyhydroxyalkanoate depolymerases that are involved in biodegradation of thermoplastic polymers. Copyright © 2017 American Society for Microbiology.

Characterization of a digestive carboxypeptidase from the insect pest corn earworm (Helicoverpa armigera) with novel specificity towards C-terminal glutamate residues.

PubMed

Bown, David P; Gatehouse, John A

2004-05-01

Carboxypeptidases were purified from guts of larvae of corn earworm (Helicoverpa armigera), a lepidopteran crop pest, by affinity chromatography on immobilized potato carboxypeptidase inhibitor, and characterized by N-terminal sequencing. A larval gut cDNA library was screened using probes based on these protein sequences. cDNA HaCA42 encoded a carboxypeptidase with sequence similarity to enzymes of clan MC [Barrett, A. J., Rawlings, N. D. & Woessner, J. F. (1998) Handbook of Proteolytic Enzymes. Academic Press, London.], but with a novel predicted specificity towards C-terminal acidic residues. This carboxypeptidase was expressed as a recombinant proprotein in the yeast Pichia pastoris. The expressed protein could be activated by treatment with bovine trypsin; degradation of bound pro-region, rather than cleavage of pro-region from mature protein, was the rate-limiting step in activation. Activated HaCA42 carboxypeptidase hydrolysed a synthetic substrate for glutamate carboxypeptidases (FAEE, C-terminal Glu), but did not hydrolyse substrates for carboxypeptidase A or B (FAPP or FAAK, C-terminal Phe or Lys) or methotrexate, cleaved by clan MH glutamate carboxypeptidases. The enzyme was highly specific for C-terminal glutamate in peptide substrates, with slow hydrolysis of C-terminal aspartate also observed. Glutamate carboxypeptidase activity was present in larval gut extract from H. armigera. The HaCA42 protein is the first glutamate-specific metallocarboxypeptidase from clan MC to be identified and characterized. The genome of Drosophila melanogaster contains genes encoding enzymes with similar sequences and predicted specificity, and a cDNA encoding a similar enzyme has been isolated from gut tissue in tsetse fly. We suggest that digestive carboxypeptidases with sequence similarity to the classical mammalian enzymes, but with specificity towards C-terminal glutamate, are widely distributed in insects.

Patterns and Variation in Benthic Biodiversity in a Large Marine Ecosystem

PubMed Central

Lee, Jonathan D.

2015-01-01

While there is a persistent inverse relationship between latitude and species diversity across many taxa and ecosystems, deviations from this norm offer an opportunity to understand the conditions that contribute to large-scale diversity patterns. Marine systems, in particular, provide such an opportunity, as marine diversity does not always follow a strict latitudinal gradient, perhaps because several hypothesized drivers of the latitudinal diversity gradient are uncorrelated in marine systems. We used a large scale public monitoring dataset collected over an eight year period to examine benthic marine faunal biodiversity patterns for the continental shelf (55–183 m depth) and slope habitats (184–1280 m depth) off the US West Coast (47°20′N—32°40′N). We specifically asked whether marine biodiversity followed a strict latitudinal gradient, and if these latitudinal patterns varied across depth, in different benthic substrates, and over ecological time scales. Further, we subdivided our study area into three smaller regions to test whether coast-wide patterns of biodiversity held at regional scales, where local oceanographic processes tend to influence community structure and function. Overall, we found complex patterns of biodiversity on both the coast-wide and regional scales that differed by taxonomic group. Importantly, marine biodiversity was not always highest at low latitudes. We found that latitude, depth, substrate, and year were all important descriptors of fish and invertebrate diversity. Invertebrate richness and taxonomic diversity were highest at high latitudes and in deeper waters. Fish richness also increased with latitude, but exhibited a hump-shaped relationship with depth, increasing with depth up to the continental shelf break, ~200 m depth, and then decreasing in deeper waters. We found relationships between fish taxonomic and functional diversity and latitude, depth, substrate, and time at the regional scale, but not at the coast-wide scale, suggesting that coast-wide patterns can obscure important correlates at smaller scales. Our study provides insight into complex diversity patterns of the deep water soft substrate benthic ecosystems off the US West Coast. PMID:26308521

Molecular Basis of Signaling Specificity of Insulin and IGF Receptors: Neglected Corners and Recent Advances

PubMed Central

Siddle, Kenneth

2011-01-01

Insulin and insulin-like growth factor (IGF) receptors utilize common phosphoinositide 3-kinase/Akt and Ras/extracellular signal-regulated kinase signaling pathways to mediate a broad spectrum of “metabolic” and “mitogenic” responses. Specificity of insulin and IGF action in vivo must in part reflect expression of receptors and responsive pathways in different tissues but it is widely assumed that it is also determined by the ligand binding and signaling mechanisms of the receptors. This review focuses on receptor-proximal events in insulin/IGF signaling and examines their contribution to specificity of downstream responses. Insulin and IGF receptors may differ subtly in the efficiency with which they recruit their major substrates (IRS-1 and IRS-2 and Shc) and this could influence effectiveness of signaling to “metabolic” and “mitogenic” responses. Other substrates (Grb2-associated binder, downstream of kinases, SH2Bs, Crk), scaffolds (RACK1, β-arrestins, cytohesins), and pathways (non-receptor tyrosine kinases, phosphoinositide kinases, reactive oxygen species) have been less widely studied. Some of these components appear to be specifically involved in “metabolic” or “mitogenic” signaling but it has not been shown that this reflects receptor-preferential interaction. Very few receptor-specific interactions have been characterized, and their roles in signaling are unclear. Signaling specificity might also be imparted by differences in intracellular trafficking or feedback regulation of receptors, but few studies have directly addressed this possibility. Although published data are not wholly conclusive, no evidence has yet emerged for signaling mechanisms that are specifically engaged by insulin receptors but not IGF receptors or vice versa, and there is only limited evidence for differential activation of signaling mechanisms that are common to both receptors. Cellular context, rather than intrinsic receptor activity, therefore appears to be the major determinant of whether responses to insulin and IGFs are perceived as “metabolic” or “mitogenic.” PMID:22649417

Bulk growth and surface characterization of epitaxy ready cadmium zinc telluride substrates for use in IR imaging applications

NASA Astrophysics Data System (ADS)

Flint, J. P.; Martinez, B.; Betz, T. E. M.; Mackenzie, J.; Kumar, F. J.; Burgess, L.

2017-02-01

Cadmium Zinc Telluride (Cd1-xZnxTe or CZT) is a compound semiconductor substrate material that has been used for infrared detector (IR) applications for many years. CZT is a perfect substrate for the epitaxial growth of Mercury Cadmium Telluride (Hg1-xCdxTe or MCT) epitaxial layers and remains the material of choice for many high performance IR detectors and focal plane arrays that are used to detect across wide IR spectral bands. Critical to the fabrication of high performance MCT IR detectors is a high quality starting CZT substrate, this being a key determinant of epitaxial layer crystallinity, defectivity and ultimately device electro-optical performance. In this work we report on a new source of substrates suitable for IR detector applications, grown using the Travelling Heater Method (THM). This proven method of crystal growth has been used to manufacture high quality IR specification CZT substrates where industry requirements for IR transmission, dislocations, tellurium precipitates and copper impurity levels have been met. Results will be presented for the chemo-mechanical (CMP) polishing of CZT substrates using production tool sets that are identical to those that are used to produce epitaxy-ready surface finishes on related IR compound semiconductor materials such as GaSb and InSb. We will also discuss the requirements to scale CZT substrate manufacture and how with a new III-V like approach to both CZT crystal growth and substrate polishing, we can move towards a more standardized product and one that can ultimately deliver a standard round CZT substrate, as is the case for competing IR materials such as GaSb, InSb and InP.

Engineering Neprilysin Activity and Specificity to Create a Novel Therapeutic for Alzheimer’s Disease

PubMed Central

Webster, Carl I.; Burrell, Matthew; Olsson, Lise-Lotte; Fowler, Susan B.; Digby, Sarah; Sandercock, Alan; Snijder, Arjan; Tebbe, Jan; Haupts, Ulrich; Grudzinska, Joanna; Jermutus, Lutz; Andersson, Christin

2014-01-01

Neprilysin is a transmembrane zinc metallopeptidase that degrades a wide range of peptide substrates. It has received attention as a potential therapy for Alzheimer’s disease due to its ability to degrade the peptide amyloid beta. However, its broad range of peptide substrates has the potential to limit its therapeutic use due to degradation of additional peptides substrates that tightly regulate many physiological processes. We sought to generate a soluble version of the ectodomain of neprilysin with improved activity and specificity towards amyloid beta as a potential therapeutic for Alzheimer’s disease. Extensive amino acid substitutions were performed at positions surrounding the active site and inner surface of the enzyme and variants screened for activity on amyloid beta 1–40, 1–42 and a variety of other physiologically relevant peptides. We identified several mutations that modulated and improved both enzyme selectivity and intrinsic activity. Neprilysin variant G399V/G714K displayed an approximately 20-fold improved activity on amyloid beta 1–40 and up to a 3,200-fold reduction in activity on other peptides. Along with the altered peptide substrate specificity, the mutant enzyme produced a markedly altered series of amyloid beta cleavage products compared to the wild-type enzyme. Crystallisation of the mutant enzyme revealed that the amino acid substitutions result in alteration of the shape and size of the pocket containing the active site compared to the wild-type enzyme. The mutant enzyme offers the potential for the more efficient degradation of amyloid beta in vivo as a therapeutic for the treatment of Alzheimer’s disease. PMID:25089527

Development of 66 kV class REBCO superconducting cable

NASA Astrophysics Data System (ADS)

Ohya, M.; Masuda, T.; Amemiya, N.; Ishiyama, A.; Ohkuma, T.

Sumitomo Electric Industries (SEI) has been involved in the development of 66 kV/5 kA-class HTS cables using REBCO wires. One of the technical targets was to reduce the AC loss to less than 2 W/m/phase at 5 kA. SEI developed a clad-type textured metal substrate with lower magnetization loss than NiW substrates. REBCO wires of 30 mm wide were slit into 4 mm-wide strips, and these strips were wound spirally on a former with small gaps. The measured AC loss of the manufactured cable was 1.8 W/m/phase at 5 kA, achieving the AC loss goal. Another important target was to manage fault current. The copper protection layers were designed based on simulation findings. Fault current tests (max. 31.5 kA, 2 sec) showed that the designed HTS cable has the required withstanding performance. The development of the elemental technologies was finished on schedule, and a 15 m-long HTS cable system will be constructed to demonstrate that it meets all the required specifications.

Genome-wide identification of ATP-binding cassette (ABC) transporters and their roles in response to polycyclic aromatic hydrocarbons (PAHs) in the copepod Paracyclopina nana.

PubMed

Jeong, Chang-Bum; Kim, Duck-Hyun; Kang, Hye-Min; Lee, Young Hwan; Kim, Hui-Su; Kim, Il-Chan; Lee, Jae-Seong

2017-02-01

The ATP-binding cassette (ABC) protein superfamily is one of the largest gene families and is highly conserved in all domains. The ABC proteins play roles in several biological processes, including multi-xenobiotic resistance (MXR), by functioning as transporters in the cellular membrane. They also mediate the cellular efflux of a wide range of substrates against concentration gradients. In this study, 37 ABC genes belonging to eight distinct subfamilies were identified in the marine copepod Paracyclopina nana and annotated based on a phylogenetic analysis. Also, the functions of P-glycoproteins (P-gp) and multidrug resistance-associated proteins (MRPs), conferring MXR, were verified using fluorescent substrates and specific inhibitors. The activities of MXR-mediated ABC proteins and their transcriptional level were examined in response to polyaromatic hydrocarbons (PAHs), main components of the water-accommodated fraction. This study increases the understanding of the protective role of MXR in response to PAHs over the comparative evolution of ABC gene families. Copyright © 2016 Elsevier B.V. All rights reserved.

Membrane-bound dd-carboxypeptidases from Bacillus megaterium KM. General properties, substrate specificity and sensitivity to penicillins, cephalosporins and peptide inhibitors of the activity at pH5

PubMed Central

Diaz-Mauriño, Teresa; Nieto, Manuel; Perkins, Harold R.

1974-01-01

1. The membrane from Bacillus megaterium KM contained a dd-carboxypeptidase with optimum activity under the following conditions: pH5.2, bivalent cation, 3mm; ionic strength, 40mm; temperature, 35°C. It was inactivated by treatment with p-chloromercuribenzoate but was fairly insensitive to 2-mercaptoethanol. 2. The enzyme was inhibited by penicillins and cephalosporins. The inhibition of this enzyme was partially reversed on dialysis but 0.2m-2-mercaptoethanol could neither prevent nor reverse the inhibition. 3. The enzyme was extremely sensitive to changes in the configuration and size of the side chain of the C-terminal dipeptide of the substrate. An aliphatic side chain of a well-defined length and polarity was required in the residue that precedes the C-terminal dipeptide. 4. The enzyme was inhibited by a wide range of analogues of the peptidic portion of the natural substrate. PMID:4218954

Variable substrate preference among phospholipase D toxins from sicariid spiders

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lajoie, Daniel M.; Roberts, Sue A.; Zobel-Thropp, Pamela A.

Venoms of the sicariid spiders contain phospholipase D enzyme toxins that can cause severe dermonecrosis and even death in humans. These enzymes convert sphingolipid and lysolipid substrates to cyclic phosphates by activating a hydroxyl nucleophile present in both classes of lipid. The most medically relevant substrates are thought to be sphingomyelin and/or lysophosphatidylcholine. To better understand the substrate preference of these toxins, we used 31P NMR to compare the activity of three related but phylogenetically diverse sicariid toxins against a diverse panel of sphingolipid and lysolipid substrates. Two of the three showed significantly faster turnover of sphingolipids over lysolipids, andmore » all three showed a strong preference for positively charged (choline and/or ethanolamine) over neutral (glycerol and serine) headgroups. Strikingly, however, the enzymes vary widely in their preference for choline, the headgroup of both sphingomyelin and lysophosphatidylcholine, versus ethanolamine. An enzyme from Sicarius terrosus showed a strong preference for ethanolamine over choline, whereas two paralogous enzymes from Loxosceles arizonica either preferred choline or showed no significant preference. Intrigued by the novel substrate preference of the Sicarius enzyme, we solved its crystal structure at 2.1 Å resolution. Lastly, the evolution of variable substrate specificity may help explain the reduced dermonecrotic potential of some natural toxin variants, because mammalian sphingolipids use primarily choline as a positively charged headgroup; it may also be relevant for sicariid predatory behavior, because ethanolamine-containing sphingolipids are common in insect prey.« less

Variable substrate preference among phospholipase D toxins from sicariid spiders

DOE PAGES

Lajoie, Daniel M.; Roberts, Sue A.; Zobel-Thropp, Pamela A.; ...

2015-03-09

Venoms of the sicariid spiders contain phospholipase D enzyme toxins that can cause severe dermonecrosis and even death in humans. These enzymes convert sphingolipid and lysolipid substrates to cyclic phosphates by activating a hydroxyl nucleophile present in both classes of lipid. The most medically relevant substrates are thought to be sphingomyelin and/or lysophosphatidylcholine. To better understand the substrate preference of these toxins, we used 31P NMR to compare the activity of three related but phylogenetically diverse sicariid toxins against a diverse panel of sphingolipid and lysolipid substrates. Two of the three showed significantly faster turnover of sphingolipids over lysolipids, andmore » all three showed a strong preference for positively charged (choline and/or ethanolamine) over neutral (glycerol and serine) headgroups. Strikingly, however, the enzymes vary widely in their preference for choline, the headgroup of both sphingomyelin and lysophosphatidylcholine, versus ethanolamine. An enzyme from Sicarius terrosus showed a strong preference for ethanolamine over choline, whereas two paralogous enzymes from Loxosceles arizonica either preferred choline or showed no significant preference. Intrigued by the novel substrate preference of the Sicarius enzyme, we solved its crystal structure at 2.1 Å resolution. Lastly, the evolution of variable substrate specificity may help explain the reduced dermonecrotic potential of some natural toxin variants, because mammalian sphingolipids use primarily choline as a positively charged headgroup; it may also be relevant for sicariid predatory behavior, because ethanolamine-containing sphingolipids are common in insect prey.« less

Variable Substrate Preference among Phospholipase D Toxins from Sicariid Spiders*

PubMed Central

Lajoie, Daniel M.; Roberts, Sue A.; Zobel-Thropp, Pamela A.; Delahaye, Jared L.; Bandarian, Vahe; Binford, Greta J.; Cordes, Matthew H. J.

2015-01-01

Venoms of the sicariid spiders contain phospholipase D enzyme toxins that can cause severe dermonecrosis and even death in humans. These enzymes convert sphingolipid and lysolipid substrates to cyclic phosphates by activating a hydroxyl nucleophile present in both classes of lipid. The most medically relevant substrates are thought to be sphingomyelin and/or lysophosphatidylcholine. To better understand the substrate preference of these toxins, we used 31P NMR to compare the activity of three related but phylogenetically diverse sicariid toxins against a diverse panel of sphingolipid and lysolipid substrates. Two of the three showed significantly faster turnover of sphingolipids over lysolipids, and all three showed a strong preference for positively charged (choline and/or ethanolamine) over neutral (glycerol and serine) headgroups. Strikingly, however, the enzymes vary widely in their preference for choline, the headgroup of both sphingomyelin and lysophosphatidylcholine, versus ethanolamine. An enzyme from Sicarius terrosus showed a strong preference for ethanolamine over choline, whereas two paralogous enzymes from Loxosceles arizonica either preferred choline or showed no significant preference. Intrigued by the novel substrate preference of the Sicarius enzyme, we solved its crystal structure at 2.1 Å resolution. The evolution of variable substrate specificity may help explain the reduced dermonecrotic potential of some natural toxin variants, because mammalian sphingolipids use primarily choline as a positively charged headgroup; it may also be relevant for sicariid predatory behavior, because ethanolamine-containing sphingolipids are common in insect prey. PMID:25752604

Measurements of weak interactions between truncated substrates and a hammerhead ribozyme by competitive kinetic analyses: implications for the design of new and efficient ribozymes with high sequence specificity

PubMed Central

Kasai, Yasuhiro; Shizuku, Hideki; Takagi, Yasuomi; Warashina, Masaki; Taira, Kazunari

2002-01-01

Exploitation of ribozymes in a practical setting requires high catalytic activity and strong specificity. The hammerhead ribozyme R32 has considerable potential in this regard since it has very high catalytic activity. In this study, we have examined how R32 recognizes and cleaves a specific substrate, focusing on the mechanism behind the specificity. Comparing rates of cleavage of a substrate in a mixture that included the correct substrate and various substrates with point mutations, we found that R32 cleaved the correct substrate specifically and at a high rate. To clarify the source of this strong specificity, we quantified the weak interactions between R32 and various truncated substrates, using truncated substrates as competitive inhibitors since they were not readily cleaved during kinetic measurements of cleavage of the correct substrate, S11. We found that the strong specificity of the cleavage reaction was due to a closed form of R32 with a hairpin structure. The self-complementary structure within R32 enabled the ribozyme to discriminate between the correct substrate and a mismatched substrate. Since this hairpin motif did not increase the Km (it did not inhibit the binding interaction) or decrease the kcat (it did not decrease the cleavage rate), this kind of hairpin structure might be useful for the design of new ribozymes with strong specificity and high activity. PMID:12034825

15N and 31P solid-state NMR study of transmembrane domain alignment of M2 protein of influenza A virus in hydrated cylindrical lipid bilayers confined to anodic aluminum oxide nanopores.

PubMed

Chekmenev, Eduard Y; Hu, Jun; Gor'kov, Peter L; Brey, William W; Cross, Timothy A; Ruuge, Andres; Smirnov, Alex I

2005-04-01

This communication reports the first example of a high resolution solid-state 15N 2D PISEMA NMR spectrum of a transmembrane peptide aligned using hydrated cylindrical lipid bilayers formed inside nanoporous anodic aluminum oxide (AAO) substrates. The transmembrane domain SSDPLVVA(A-15N)SIIGILHLILWILDRL of M2 protein from influenza A virus was reconstituted in hydrated 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine bilayers that were macroscopically aligned by a conventional micro slide glass support or by the AAO nanoporous substrate. 15N and 31P NMR spectra demonstrate that both the phospholipids and the protein transmembrane domain are uniformly aligned in the nanopores. Importantly, nanoporous AAO substrates may offer several advantages for membrane protein alignment in solid-state NMR studies compared to conventional methods. Specifically, higher thermal conductivity of aluminum oxide is expected to suppress thermal gradients associated with inhomogeneous radio frequency heating. Another important advantage of the nanoporous AAO substrate is its excellent accessibility to the bilayer surface for exposure to solute molecules. Such high accessibility achieved through the substrate nanochannel network could facilitate a wide range of structure-function studies of membrane proteins by solid-state NMR.

Advanced Materials From Fungal Mycelium: Fabrication and Tuning of Physical Properties

NASA Astrophysics Data System (ADS)

Haneef, Muhammad; Ceseracciu, Luca; Canale, Claudio; Bayer, Ilker S.; Heredia-Guerrero, José A.; Athanassiou, Athanassia

2017-01-01

In this work is presented a new category of self-growing, fibrous, natural composite materials with controlled physical properties that can be produced in large quantities and over wide areas, based on mycelium, the main body of fungi. Mycelia from two types of edible, medicinal fungi, Ganoderma lucidum and Pleurotus ostreatus, have been carefully cultivated, being fed by two bio-substrates: cellulose and cellulose/potato-dextrose, the second being easier to digest by mycelium due to presence of simple sugars in its composition. After specific growing times the mycelia have been processed in order to cease their growth. Depending on their feeding substrate, the final fibrous structures showed different relative concentrations in polysaccharides, lipids, proteins and chitin. Such differences are reflected as alterations in morphology and mechanical properties. The materials grown on cellulose contained more chitin and showed higher Young’s modulus and lower elongation than those grown on dextrose-containing substrates, indicating that the mycelium materials get stiffer when their feeding substrate is harder to digest. All the developed fibrous materials were hydrophobic with water contact angles higher than 120°. The possibility of tailoring mycelium materials’ properties by properly choosing their nutrient substrates paves the way for their use in various scale applications.

Assessing Carbon-Based Anodes for Lithium-Ion Batteries: A Universal Description of Charge-Transfer Binding

DOE PAGES

Liu, Yuanyue; Wang, Y. Morris; Yakobson, Boris I.; ...

2014-07-11

Many key performance characteristics of carbon-based lithium-ion battery anodes are largely determined by the strength of binding between lithium (Li) and sp 2 carbon (C), which can vary significantly with subtle changes in substrate structure, chemistry, and morphology. We use density functional theory calculations to investigate the interactions of Li with a wide variety of sp 2 C substrates, including pristine, defective, and strained graphene, planar C clusters, nanotubes, C edges, and multilayer stacks. In almost all cases, we find a universal linear relation between the Li-C binding energy and the work required to fill previously unoccupied electronic states withinmore » the substrate. This suggests that Li capacity is predominantly determined by two key factors—namely, intrinsic quantum capacitance limitations and the absolute placement of the Fermi level. This simple descriptor allows for straightforward prediction of the Li-C binding energy and related battery characteristics in candidate C materials based solely on the substrate electronic structure. It further suggests specific guidelines for designing more effective C-based anodes. Furthermore, this method should be broadly applicable to charge-transfer adsorption on planar substrates, and provides a phenomenological connection to established principles in supercapacitor and catalyst design.« less

Similar substrate specificity of cynomolgus monkey cytochrome P450 2C19 to reported human P450 2C counterpart enzymes by evaluation of 89 drug clearances.

PubMed

Hosaka, Shinya; Murayama, Norie; Satsukawa, Masahiro; Uehara, Shotaro; Shimizu, Makiko; Iwasaki, Kazuhide; Iwano, Shunsuke; Uno, Yasuhiro; Yamazaki, Hiroshi

2015-12-01

Cynomolgus monkeys are used widely in preclinical studies as non-human primate species. The amino acid sequence of cynomolgus monkey cytochrome P450 (P450 or CYP) 2C19 is reportedly highly correlated to that of human CYP2C19 (92%) and CYP2C9 (93%). In the present study, 89 commercially available compounds were screened to find potential substrates for cynomolgus monkey CYP2C19. Of 89 drugs, 34 were metabolically depleted by cynomolgus monkey CYP2C19 with relatively high rates. Among them, 30 compounds have been reported as substrates or inhibitors of, either or both, human CYP2C19 and CYP2C9. Several compounds, including loratadine, showed high selectivity to cynomolgus monkey CYP2C19, and all of these have been reported as human CYP2C19 and/or CYP2C9 substrates. In addition, cynomolgus monkey CYP2C19 formed the same loratadine metabolite as human CYP2C19, descarboethoxyloratadine. These results suggest that cynomolgus monkey CYP2C19 is generally similar to human CYP2C19 and CYP2C9 in its substrate recognition functionality. Copyright © 2015 John Wiley & Sons, Ltd.

Simple method of DNA stretching on glass substrate for fluorescence image and spectroscopy

NASA Astrophysics Data System (ADS)

Neupane, Guru P.; Dhakal, Krishna P.; Lee, Hyunsoo; Guthold, Martin; Joseph, Vincent S.; Hong, Jong-Dal; Kim, Jeongyong

2013-05-01

Study of biological molecule DNA has contributed to developing many breaking thoughts and wide applications in multidisciplinary fields, such as genomic, medical, sensing and forensic fields. Stretching of DNA molecules is an important supportive tool for AFM or spectroscopic studies of DNA in a single molecular level. In this article, we established a simple method of DNA stretching (to its full length) that occurred on a rotating negatively-charged surface of glass substrate. The isolation of a single DNA molecule was attained by the two competitive forces on DNA molecules, that is, the electrostatic attraction developed between the positively charged YOYO-1 stained DNA and the negatively charged substrate, and the centrifugal force of the rotating substrate, which separates the DNA aggregates into the single molecule. Density of stretched DNA molecules was controlled by selecting the specific parameters such as spinning time and rates, loading volume of DNA-dye complex solution etc. The atomic force microscopy image exhibited a single DNA molecule on the negatively-charged substrate in an isolated state. Further, the photoluminescence spectra of a single DNA molecule stained with YOYO-1 were achieved using the method developed in the present study, which is strongly believed to effectively support the spectroscopic analysis of DNA in a single molecular level.

Structures of enzyme-intermediate complexes of yeast Nit2: insights into its catalytic mechanism and different substrate specificity compared with mammalian Nit2.

PubMed

Liu, Hejun; Gao, Yongxiang; Zhang, Mengying; Qiu, Xiaoting; Cooper, Arthur J L; Niu, Liwen; Teng, Maikun

2013-08-01

The Nit (nitrilase-like) protein subfamily constitutes branch 10 of the nitrilase superfamily. Nit proteins are widely distributed in nature. Mammals possess two members of the Nit subfamily, namely Nit1 and Nit2. Based on sequence similarity, yeast Nit2 (yNit2) is a homologue of mouse Nit1, a tumour-suppressor protein whose substrate specificity is not yet known. Previous studies have shown that mammalian Nit2 (also a putative tumour suppressor) is identical to ω-amidase, an enzyme that catalyzes the hydrolysis of α-ketoglutaramate (α-KGM) and α-ketosuccinamate (α-KSM) to α-ketoglutarate (α-KG) and oxaloacetate (OA), respectively. In the present study, crystal structures of wild-type (WT) yNit2 and of WT yNit2 in complex with α-KG and with OA were determined. In addition, the crystal structure of the C169S mutant of yNit2 (yNit2-C169S) in complex with an endogenous molecule of unknown structure was also solved. Analysis of the structures revealed that α-KG and OA are covalently bound to Cys169 by the formation of a thioester bond between the sulfhydryl group of the cysteine residue and the γ-carboxyl group of α-KG or the β-carboxyl group of OA, reflecting the presumed reaction intermediates. However, an enzymatic assay suggests that α-KGM is a relatively poor substrate of yNit2. Finally, a ligand was found in the active site of yNit2-C169S that may be a natural substrate of yNit2 or an endogenous regulator of enzyme activity. These crystallographic analyses provide information on the mode of substrate/ligand binding at the active site of yNit2 and insights into the catalytic mechanism. These findings suggest that yNit2 may have broad biological roles in yeast, especially in regard to nitrogen homeostasis, and provide a framework for the elucidation of the substrate specificity and biological role of mammalian Nit1.

Flow-through SIP - A novel stable isotope probing approach limiting cross-feeding

NASA Astrophysics Data System (ADS)

Mooshammer, Maria; Kitzinger, Katharina; Schintlmeister, Arno; Kjedal, Henrik; Nielsen, Jeppe Lund; Nielsen, Per; Wagner, Michael

2017-04-01

Stable isotope probing (SIP) is a widely applied tool to link specific microbial populations to metabolic processes in the environment without the prerequisite of cultivation, which has greatly advanced our understanding of the role of microorganisms in biogeochemical cycling. SIP relies on tracing specific isotopically labeled substrates (e.g., 13C, 15N, 18O) into cellular biomarkers, such as DNA, RNA or phospholipid fatty acids, and is considered to be a robust technique to identify microbial populations that assimilate the labeled substrate. However, cross-feeding can occur when labeled metabolites are released from a primary consumer and then used by other microorganisms. This leads to erroneous identification of organisms that are not directly responsible for the process of interest, but are rather connected to primary consumers via a microbial food web. Here, we introduce a new approach that has the potential to eliminate the effect of cross-feeding in SIP studies and can thus also be used to distinguish primary consumers from other members of microbial food webs. In this approach, a monolayer of microbial cells are placed on a filter membrane, and labeled substrates are supplied by a continuous flow. By means of flow-through, labeled metabolites and degradation products are constantly removed, preventing secondary consumption of the substrate. We present results from a proof-of-concept experiment using nitrifiers from activated sludge as model system, in which we used fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes for identification of nitrifiers in combination with nanoscale secondary ion mass spectrometry (NanoSIMS) for visualization of isotope incorporation at the single-cell level. Our results show that flow-through SIP is a promising approach to significantly reduce cross-feeding and secondary substrate consumption in SIP experiments.

Antibody conjugated glycine doped polyaniline nanofilms as efficient biosensor for atrazine

NASA Astrophysics Data System (ADS)

Bhardwaj, Sanjeev K.; Sharma, Amit L.; Kim, Ki-Hyun; Deep, Akash

2017-12-01

Atrazine is an important member of triazine family of pesticides. The development of its detection methods gained great attention due to the potential health risks associated with its contamination in various media including water, soil, and food. The contamination of atrazine in drinking water beyond the legal permissible limit of EPA (e.g. 3 ng ml-1) may cause various damages to living organisms (e.g. heart, urinary, and limb defects). In this research, we discuss the potential significance of a highly sensitive conductometric immunosensor for sensing the atrazine pesticide. To this end, electrochemical assembly of glycine doped polyaniline (PAni) nanofilms on silicon (Si) substrate was built and modified further with anti-atrazine antibodies. The herein developed immunosensor offered highly sensitive detection of atrazine with a low detection limit of 0.07 ng ml-1. The proposed biosensor was simple in design with excellent performance in terms of its sensitivity, stability and specificity. Highlights •Glycine doped PAni nanofilms have been electropolymerized on Silicon substrates. •Functionality of the above thin films provides opportunity to develop an immunosensing platform. •Highly sensitive and specific detection of atrazine has been realized over a wide concentration range with a LOD of 0.07 ng ml-1. Novelty statement Atrazine is a widely used pesticide in the agriculture sector. It is highly recommended to develop simple biosensing systems for enabling the prospect of routine monitoring. The present research for the first time proposes the design of a glycine doped PAni based simple and highly effective biosensor for the atrazine pesticide. The doping of glycine has easily generated functional groups on the nano-PAni material for further convenient immobilization of anti-atrazine antibodies. The proposed sensor can be highlighted with advantages like ease of fabrication, use of environment friendly functionalization agent, specificity, wide linearity, and good sensitivity with enhanced viability.

Detection of Fusarium oxysporum f.sp. basilici in substrates and roots by PCR.

PubMed

Pugliese, M; Ferrocino, I; Gullino, M L; Garibaldi, A

2013-01-01

Fusarium oxysporum is a soil-borne fungus that causes vascular wilts in a wide variety of plant species. Basil is recognized as an ecological niche for Fusarium oxysporum f.sp. basilici (FOB) and this fungus is now present in most countries where basil is cultivated. The rapid identification of the species affecting basil plants is necessary to define a successful method for crop protection. The aim of this study was to develop a PCR method for the rapid detection of Fusarium oxysporum f. sp. basilici in substrates. The specificity of the primers used was tested using the DNA extracted directly from substrate samples. Fusarium oxysporum f.sp. basilici was artificially inoculated with decreasing amounts in a commercial substrate (sphagnum peat moss) and in a mixture with 40% of municipal compost, after steam disinfestation. Basil seeds (cv. Fine verde) were sown in pots that were laid on a bench in the greenhouse. At time 0 and after 7, 14 and 21 days from the inoculation, substrate and root samples were collected and prepared for microbial analysis and for the DNA extraction. DNA extraction was carried out using NucleoSpin Soil Kit (Macherey-Nagel, Germany). PCR amplification for the specific detection was carried out using primer sets Bik 1 (5'-ATT CAA GAG CTA AAG GTC C-3') and Bik 4 (5'-TTT GAC CAA GAT AGA TGC C-3') for the first PCR, while primers Bik 1 + Bik 2 (5'-AAA GGT AGT ATA TCG GAG G-3') for the nested PCR to increase detection sensitivity. Disease incidence was also assessed 21 days after seeding. The results showed the presence of amplified fragments of the expected size when the concentration of F. oxysporum f.sp. basilici was at least 3.5 Log CFU g(-1) by using DNA extract directly from substrate, before roots were infected by the pathogen. The detection of Fusarium oxysporum f. sp. basilici by PCR method developed in this study is certainly simple and fast and can be useful for its reliable detection in substrate samples, but not to guarantee that the substrate is totally free of pathogens.

Steel slag affects pH and Si content of container substrates

USDA-ARS?s Scientific Manuscript database

A substrate representing a typical greenhouse potting mix was prepared using 85% sphagnum peat and 15% perlite. The substrate was filled into 10 cm wide containers. A pulverized steel slag (SS) from a basic oxygen furnace, and dolomitic limestone (DL) were amended to the base substrate at a rate o...

Final acceptance testing of the LSST monolithic primary/tertiary mirror

NASA Astrophysics Data System (ADS)

Tuell, Michael T.; Burge, James H.; Cuerden, Brian; Gressler, William; Martin, Hubert M.; West, Steven C.; Zhao, Chunyu

2014-07-01

The Large Synoptic Survey Telescope (LSST) is a three-mirror wide-field survey telescope with the primary and tertiary mirrors on one monolithic substrate1. This substrate is made of Ohara E6 borosilicate glass in a honeycomb sandwich, spin cast at the Steward Observatory Mirror Lab at The University of Arizona2. Each surface is aspheric, with the specification in terms of conic constant error, maximum active bending forces and finally a structure function specification on the residual errors3. There are high-order deformation terms, but with no tolerance, any error is considered as a surface error and is included in the structure function. The radii of curvature are very different, requiring two independent test stations, each with instantaneous phase-shifting interferometers with null correctors. The primary null corrector is a standard two-element Offner null lens. The tertiary null corrector is a phase-etched computer-generated hologram (CGH). This paper details the two optical systems and their tolerances, showing that the uncertainty in measuring the figure is a small fraction of the structure function specification. Additional metrology includes the radii of curvature, optical axis locations, and relative surface tilts. The methods for measuring these will also be described along with their tolerances.

Citrobacter diversus ULA-27 beta-lactamases. Improved purification and general properties.

PubMed Central

Amicosante, G; Oratore, A; Franceschini, N; Maccarrone, M; Strom, R; Galleni, M; Frère, J M

1988-01-01

Two chromosome-encoded beta-lactamases have been purified from Citrobacter diversus ULA-27. They exhibited slightly different isoelectric points (6.8 and 6.2) and very similar Mr values (congruent to 29,000). Their specificity spectrum was rather wide, since they hydrolysed some cephalosporins with kcat: values similar to those observed with the best penicillin substrates. Cloxacillin, methicillin and imipenem were hydrolysed very slowly. Hydrolysis of azthreonam could not be detected. Images Fig. 3. Fig. 4. PMID:3264152

Performance and Reliability of Bonded Interfaces for High-Temperature Packaging. Annual Report

DOE Office of Scientific and Technical Information (OSTI.GOV)

DeVoto, Douglas

2016-04-01

Current generation automotive power electronics packages utilize silicon devices and lead-free solder alloys. To meet stringent technical targets for 2020 and beyond (for cost, power density, specific power, efficiency and reliability), wide-bandgap devices are being considered since they offer advantages such as operation at higher frequencies, voltages, and temperatures. Traditional power electronics packages must be redesigned to utilize the full potential of wide-bandgap devices, and the die- and substrate-attach layers are key areas where new material development and validation is required. Present solder alloys do not meet the performance requirements for these new package designs while also meeting cost andmore » hazardous substance restrictions. Sintered silver (Ag) promises to meet the needs for die- and substrate-attach interfaces but synthesis optimization and reliability evaluation must be completed. Sintered Ag material was proposed as an alternative solution in power electronics packages almost 20 years back. However, synthesis pressure requirements up 40 MPa caused a higher complexity in the production process and more stringent flatness specifications for the substrates. Recently, several manufacturers have developed sintered Ag materials that require lower (3-5 MPa) or even no bonding pressures. Degradation mechanisms for these sintered Ag materials are not well known and need to be addressed. We are addressing these aspects to some extent in this project. We are developing generalized (i.e., independent of geometry) stress intensity factor versus cycles-to-failure relations for sintered Ag. Because sintered Ag is a relatively new material for automotive power electronics, the industry currently does not have a good understanding of recommended synthesis parameters or expected reliability under prescribed conditions. It is an important deliverable of this project to transfer findings to industry to eliminate barriers to using sintered Ag as a viable and commercialized die- and substrate-attach material. Only a few manufacturers produce sintered Ag pastes and may consider some processing conditions as proprietary. It is the goal of this project to openly explore and define best practices in order to impact the maximum number of power electronics module manufacturers and suppliers.« less

Both Intrinsic Substrate Preference and Network Context Contribute to Substrate Selection of Classical Tyrosine Phosphatases*

PubMed Central

Tinti, Michele; Paoluzi, Serena; Santonico, Elena; Masch, Antonia; Schutkowski, Mike

2017-01-01

Reversible tyrosine phosphorylation is a widespread post-translational modification mechanism underlying cell physiology. Thus, understanding the mechanisms responsible for substrate selection by kinases and phosphatases is central to our ability to model signal transduction at a system level. Classical protein-tyrosine phosphatases can exhibit substrate specificity in vivo by combining intrinsic enzymatic specificity with the network of protein-protein interactions, which positions the enzymes in close proximity to their substrates. Here we use a high throughput approach, based on high density phosphopeptide chips, to determine the in vitro substrate preference of 16 members of the protein-tyrosine phosphatase family. This approach helped identify one residue in the substrate binding pocket of the phosphatase domain that confers specificity for phosphopeptides in a specific sequence context. We also present a Bayesian model that combines intrinsic enzymatic specificity and interaction information in the context of the human protein interaction network to infer new phosphatase substrates at the proteome level. PMID:28159843

Phylogenetic and Functional Substrate Specificity for Endolithic Microbial Communities in Hyper-Arid Environments

PubMed Central

Crits-Christoph, Alexander; Robinson, Courtney K.; Ma, Bing; Ravel, Jacques; Wierzchos, Jacek; Ascaso, Carmen; Artieda, Octavio; Souza-Egipsy, Virginia; Casero, M. Cristina; DiRuggiero, Jocelyne

2016-01-01

Under extreme water deficit, endolithic (inside rock) microbial ecosystems are considered environmental refuges for life in cold and hot deserts, yet their diversity and functional adaptations remain vastly unexplored. The metagenomic analyses of the communities from two rock substrates, calcite and ignimbrite, revealed that they were dominated by Cyanobacteria, Actinobacteria, and Chloroflexi. The relative distribution of major phyla was significantly different between the two substrates and biodiversity estimates, from 16S rRNA gene sequences and from the metagenomic data, all pointed to a higher taxonomic diversity in the calcite community. While both endolithic communities showed adaptations to extreme aridity and to the rock habitat, their functional capabilities revealed significant differences. ABC transporters and pathways for osmoregulation were more diverse in the calcite chasmoendolithic community. In contrast, the ignimbrite cryptoendolithic community was enriched in pathways for secondary metabolites, such as non-ribosomal peptides (NRP) and polyketides (PK). Assemblies of the metagenome data produced population genomes for the major phyla found in both communities and revealed a greater diversity of Cyanobacteria population genomes for the calcite substrate. Draft genomes of the dominant Cyanobacteria in each community were constructed with more than 93% estimated completeness. The two annotated proteomes shared 64% amino acid identity and a significantly higher number of genes involved in iron update, and NRPS gene clusters, were found in the draft genomes from the ignimbrite. Both the community-wide and genome-specific differences may be related to higher water availability and the colonization of large fissures and cracks in the calcite in contrast to a harsh competition for colonization space and nutrient resources in the narrow pores of the ignimbrite. Together, these results indicated that the habitable architecture of both lithic substrates- chasmoendolithic versus cryptoendolithic – might be an essential element in determining the colonization and the diversity of the microbial communities in endolithic substrates at the dry limit for life. PMID:27014224

Substrate specificity of human metallocarboxypeptidase D: Comparison of the two active carboxypeptidase domains

PubMed Central

Tanco, Sebastian; Díaz, Lucía; Dasgupta, Sayani; Fernandez-Recio, Juan; Lorenzo, Julia; Aviles, Francesc X.; Fricker, Lloyd D.

2017-01-01

Metallocarboxypeptidase D (CPD) is a membrane-bound component of the trans-Golgi network that cycles to the cell surface through exocytic and endocytic pathways. Unlike other members of the metallocarboxypeptidase family, CPD is a multicatalytic enzyme with three carboxypeptidase-like domains, although only the first two domains are predicted to be enzymatically active. To investigate the enzymatic properties of each domain in human CPD, a critical active site Glu in domain I and/or II was mutated to Gln and the protein expressed, purified, and assayed with a wide variety of peptide substrates. CPD with all three domains intact displays >50% activity from pH 5.0 to 7.5 with a maximum at pH 6.5, as does CPD with mutation of domain I. In contrast, the domain II mutant displayed >50% activity from pH 6.5–7.5. CPD with mutations in both domains I and II was completely inactive towards all substrates and at all pH values. A quantitative peptidomics approach was used to compare the activities of CPD domains I and II towards a large number of peptides. CPD cleaved C-terminal Lys or Arg from a subset of the peptides. Most of the identified substrates of domain I contained C-terminal Arg, whereas comparable numbers of Lys- and Arg-containing peptides were substrates of domain II. We also report that some peptides with C-terminal basic residues were not cleaved by either domain I or II, showing the importance of the P1 position for CPD activity. Finally, the preference of domain I for C-terminal Arg was validated through molecular docking experiments. Together with the differences in pH optima, the different substrate specificities of CPD domains I and II allow the enzyme to perform distinct functions in the various locations within the cell. PMID:29131831

Oviposition substrate in Asian tiger mosquito surveillance: Do the sizes matter?

PubMed

Falsone, Luigi; Brianti, Emanuele; Severini, Francesco; Giannetto, Salvatore; Romi, Roberto

2015-12-01

Ovitraps are regarded as a reliable system to monitor Aedes albopictus dynamics. However, the dimensions of the oviposition substrate are not standardized, and no studies have investigated which should be the most effective sizes. In this study, the effect of paddle sizes in tiger mosquito egg collection was evaluated. Egg count and density on the wide surfaces and margins of different-sized oviposition substrates have been evaluated in two studies (A and B). In study A, a total of 29,995 Ae. albopictus eggs was counted in 250 classic oviposition substrates. Eggs were found on both wide surfaces (53.1%) and margins (46.9%). Egg density was significantly larger in margins compared to wide surfaces. Overall in study B, 983 Ae. albopictus eggs were collected. According to paddle sizes, 51.8% of eggs were on large and 48.2% on small paddles. Mean egg density of wide surfaces was significantly larger in small paddles (0.25 eggs/cm(2) ) compared to large paddles (0.06 eggs/cm(2) ). Results indicate that wider oviposition substrates do not mean larger number of Ae. albopictus eggs. Indeed, on paddles four times thinner than others, the number of eggs counted was not statistically different. These findings suggest that small paddles may be routinely employed in ovitraps, thus allowing savings of materials and money. © 2015 The Society for Vector Ecology.

Modelling anaerobic co-digestion in Benchmark Simulation Model No. 2: Parameter estimation, substrate characterisation and plant-wide integration.

PubMed

Arnell, Magnus; Astals, Sergi; Åmand, Linda; Batstone, Damien J; Jensen, Paul D; Jeppsson, Ulf

2016-07-01

Anaerobic co-digestion is an emerging practice at wastewater treatment plants (WWTPs) to improve the energy balance and integrate waste management. Modelling of co-digestion in a plant-wide WWTP model is a powerful tool to assess the impact of co-substrate selection and dose strategy on digester performance and plant-wide effects. A feasible procedure to characterise and fractionate co-substrates COD for the Benchmark Simulation Model No. 2 (BSM2) was developed. This procedure is also applicable for the Anaerobic Digestion Model No. 1 (ADM1). Long chain fatty acid inhibition was included in the ADM1 model to allow for realistic modelling of lipid rich co-substrates. Sensitivity analysis revealed that, apart from the biodegradable fraction of COD, protein and lipid fractions are the most important fractions for methane production and digester stability, with at least two major failure modes identified through principal component analysis (PCA). The model and procedure were tested on bio-methane potential (BMP) tests on three substrates, each rich on carbohydrates, proteins or lipids with good predictive capability in all three cases. This model was then applied to a plant-wide simulation study which confirmed the positive effects of co-digestion on methane production and total operational cost. Simulations also revealed the importance of limiting the protein load to the anaerobic digester to avoid ammonia inhibition in the digester and overloading of the nitrogen removal processes in the water train. In contrast, the digester can treat relatively high loads of lipid rich substrates without prolonged disturbances. Copyright © 2016 Elsevier Ltd. All rights reserved.

Kinetic characterization of factor Xa binding using a quenched fluorescent substrate based on the reactive site of factor Xa inhibitor from Bauhinia ungulata seeds.

PubMed

Oliva, M L V; Andrade, S A; Juliano, M A; Sallai, R C; Torquato, R J; Sampaio, M U; Pott, V J; Sampaio, C A M

2003-07-01

The specific Kunitz Bauhinia ungulata factor Xa inhibitor (BuXI) and the Bauhinia variegata trypsin inhibitor (BvTI) blocked the activity of trypsin, chymotrypsin, plasmin, plasma kallikrein and factor XIIa, and factor Xa inhibition was achieved only by BuXI (K(i) 14 nM). BuXI and BvTI are highly homologous (70%). The major differences are the methionine residues at BuXI reactive site, which are involved in the inhibition, since the oxidized protein no longer inhibits factor Xa but maintains the trypsin inhibition. Quenched fluorescent substrates based on the reactive site sequence of the inhibitors were synthesized and the kinetic parameters of the hydrolysis were determined using factor Xa and trypsin. The catalytic efficiency k(cat)/K(m) 4.3 x 10(7) M(-1)sec(>-1) for Abz-VMIAALPRTMFIQ-EDDnp (lead peptide) hydrolysis by factor Xa was 10(4)-fold higher than that of Boc-Ile-Glu-Gly-Arg-AMC, widely used as factor Xa substrate. Lengthening of the substrate changed its susceptibility to factor Xa hydrolysis. Both methionine residues in the substrate influence the binding to factor Xa. Serine replacement of threonine (P(1)') decreases the catalytic efficiency by four orders of magnitude. Factor Xa did not hydrolyze the substrate containing the reactive site sequence of BvTI, that inhibits trypsin inhibitor but not factor Xa. Abz-VMIAALPRTMFIQ-EDDnp prolonged both the prothrombin time and the activated partial thromboplastin time, and the other modified substrates used in this experiment altered blood-clotting assays.

An Alternative Wearable Tracking System Based on a Low-Power Wide-Area Network.

PubMed

Fernández-Garcia, Raul; Gil, Ignacio

2017-03-14

This work presents an alternative wearable tracking system based on a low-power wide area network. A complete GPS receiver was integrated with a textile substrate, and the latitude and longitude coordinates were sent to the cloud by means of the SIM-less SIGFOX network. To send the coordinates over SIGFOX protocol, a specific codification algorithm was used and a customized UHF antenna on jeans fabric was designed, simulated and tested. Moreover, to guarantee the compliance to international regulations for human body exposure to electromagnetic radiation, the electromagnetic specific absorption rate of this antenna was analyzed. A specific remote server was developed to decode the latitude and longitude coordinates. Once the coordinates have been decoded, the remote server sends this information to the open source data viewer SENTILO to show the location of the sensor node in a map. The functionality of this system has been demonstrated experimentally. The results guarantee the utility and wearability of the proposed tracking system for the development of sensor nodes and point out that it can be a low cost alternative to other commercial products based on GSM networks.

Fine-mapping inflammatory bowel disease loci to single-variant resolution.

PubMed

Huang, Hailiang; Fang, Ming; Jostins, Luke; Umićević Mirkov, Maša; Boucher, Gabrielle; Anderson, Carl A; Andersen, Vibeke; Cleynen, Isabelle; Cortes, Adrian; Crins, François; D'Amato, Mauro; Deffontaine, Valérie; Dmitrieva, Julia; Docampo, Elisa; Elansary, Mahmoud; Farh, Kyle Kai-How; Franke, Andre; Gori, Ann-Stephan; Goyette, Philippe; Halfvarson, Jonas; Haritunians, Talin; Knight, Jo; Lawrance, Ian C; Lees, Charlie W; Louis, Edouard; Mariman, Rob; Meuwissen, Theo; Mni, Myriam; Momozawa, Yukihide; Parkes, Miles; Spain, Sarah L; Théâtre, Emilie; Trynka, Gosia; Satsangi, Jack; van Sommeren, Suzanne; Vermeire, Severine; Xavier, Ramnik J; Weersma, Rinse K; Duerr, Richard H; Mathew, Christopher G; Rioux, John D; McGovern, Dermot P B; Cho, Judy H; Georges, Michel; Daly, Mark J; Barrett, Jeffrey C

2017-07-13

Inflammatory bowel diseases are chronic gastrointestinal inflammatory disorders that affect millions of people worldwide. Genome-wide association studies have identified 200 inflammatory bowel disease-associated loci, but few have been conclusively resolved to specific functional variants. Here we report fine-mapping of 94 inflammatory bowel disease loci using high-density genotyping in 67,852 individuals. We pinpoint 18 associations to a single causal variant with greater than 95% certainty, and an additional 27 associations to a single variant with greater than 50% certainty. These 45 variants are significantly enriched for protein-coding changes (n = 13), direct disruption of transcription-factor binding sites (n = 3), and tissue-specific epigenetic marks (n = 10), with the last category showing enrichment in specific immune cells among associations stronger in Crohn's disease and in gut mucosa among associations stronger in ulcerative colitis. The results of this study suggest that high-resolution fine-mapping in large samples can convert many discoveries from genome-wide association studies into statistically convincing causal variants, providing a powerful substrate for experimental elucidation of disease mechanisms.

Enzyme specificity under dynamic control

NASA Astrophysics Data System (ADS)

Ota, Nobuyuki; Agard, David A.

2002-03-01

The contributions of conformational dynamics to substrate specificity have been examined by the application of principal component analysis to molecular dynamics trajectories of alpha-lytic protease. The wild-type alpha-lytic protease is highly specific for substrates with small hydrophobic side chains at the specificity pocket, while the Met190Ala binding pocket mutant has a much broader specificity, actively hydrolyzing substrates ranging from Ala to Phe. We performed a principal component analysis using 1-nanosecond molecular dynamics simulations using solvent boundary condition. We found that the walls of the wild-type substrate binding pocket move in tandem with one another, causing the pocket size to remain fixed so that only small substrates are recognized. In contrast, the M190A mutant shows uncoupled movement of the binding pocket walls, allowing the pocket to sample both smaller and larger sizes, which appears to be the cause of the observed broad specificity. The results suggest that the protein dynamics of alpha-lytic protease may play a significant role in defining the patterns of substrate specificity.

Process for applying a superconductive powder to a wide variety of substrates

NASA Astrophysics Data System (ADS)

Hooker, Matthew W.; Wise, Stephanie A.; Tran, Sang Q.

1992-12-01

A fine superconducting powder such as YBa2Cu3O(7-x), wherein x is less than one, is blended into a liquid mixture comprising an epoxy resin and a thinner. This liquid mixture with the blended superconducting powder is coated onto a substrate. Next, the thinner is evaporated and the remaining coating cured, resulting in a coating of cured epoxy resin having superconducting powder suspended therein. This coating exhibits the Meissner effect, i.e., it expels a magnetic flux which protects the substrate from external magnetic interference. Since the coated substrate need only be heated for evaporation and curing at relatively low temperatures compared to firing, the superconducting coating can be applied to a wide variety of different materials.

Arbitrary Multicolor Photodetection by Hetero-integrated Semiconductor Nanostructures

PubMed Central

Sang, Liwen; Hu, Junqing; Zou, Rujia; Koide, Yasuo; Liao, Meiyong

2013-01-01

The typical photodetectors can only detect one specific optical spectral band, such as InGaAs and graphene-PbS quantum dots for near-infrared (NIR) light detection, CdS and Si for visible light detection, and ZnO and III-nitrides for UV light detection. So far, none of the developed photodetector can achieve the multicolor detection with arbitrary spectral selectivity, high sensitivity, high speed, high signal-to-noise ratio, high stability, and simplicity (called 6S requirements). Here, we propose a universal strategy to develop multicolor photodetectors with arbitrary spectral selectivity by integrating various semiconductor nanostructures on a wide-bandgap semiconductor or an insulator substrate. Because the photoresponse of each spectral band is determined by each semiconductor nanostructure or the semiconductor substrate, multicolor detection satisfying 6S requirements can be readily satisfied by selecting the right semiconductors. PMID:23917790

Regulatory role of calpain in neuronal death

PubMed Central

Cheng, Si-ying; Wang, Shu-chao; Lei, Ming; Wang, Zhen; Xiong, Kun

2018-01-01

Calpains are a group of calcium-dependent proteases that are over activated by increased intracellular calcium levels under pathological conditions. A wide range of substrates that regulate necrotic, apoptotic and autophagic pathways are affected by calpain. Calpain plays a very important role in neuronal death and various neurological disorders. This review introduces recent research progress related to the regulatory mechanisms of calpain in neuronal death. Various neuronal programmed death pathways including apoptosis, autophagy and regulated necrosis can be divided into receptor interacting protein-dependent necroptosis, mitochondrial permeability transition-dependent necrosis, pyroptosis and poly (ADP-ribose) polymerase 1-mediated parthanatos. Calpains cleave series of key substrates that may lead to cell death or participate in cell death. Regarding the investigation of calpain-mediated programed cell death, it is necessary to identify specific inhibitors that inhibit calpain mediated neuronal death and nervous system diseases. PMID:29623944

A universal mammalian vaccine cell line substrate.

PubMed

Murray, Jackelyn; Todd, Kyle V; Bakre, Abhijeet; Orr-Burks, Nichole; Jones, Les; Wu, Weilin; Tripp, Ralph A

2017-01-01

Using genome-wide small interfering RNA (siRNA) screens for poliovirus, influenza A virus and rotavirus, we validated the top 6 gene hits PV, RV or IAV to search for host genes that when knocked-down (KD) enhanced virus permissiveness and replication over wild type Vero cells or HEp-2 cells. The enhanced virus replication was tested for 12 viruses and ranged from 2-fold to >1000-fold. There were variations in virus-specific replication (strain differences) across the cell lines examined. Some host genes (CNTD2, COQ9, GCGR, NDUFA9, NEU2, PYCR1, SEC16G, SVOPL, ZFYVE9, and ZNF205) showed that KD resulted in enhanced virus replication. These findings advance platform-enabling vaccine technology, the creation of diagnostic cells substrates, and are informative about the host mechanisms that affect virus replication in mammalian cells.

Optically initiated silicon carbide high voltage switch

DOEpatents

Caporaso, George J [Livermore, CA; Sampayan, Stephen E [Manteca, CA; Sullivan, James S [Livermore, CA; Sanders,; David, M [Livermore, CA

2011-02-22

An improved photoconductive switch having a SiC or other wide band gap substrate material, such as GaAs and field-grading liners composed of preferably SiN formed on the substrate adjacent the electrode perimeters or adjacent the substrate perimeters for grading the electric fields.

Metabolism of Cryptic Peptides Derived from Neuropeptide FF Precursors: The Involvement of Insulin-Degrading Enzyme

PubMed Central

Grasso, Giuseppe; Mielczarek, Przemyslaw; Niedziolka, Magdalena; Silberring, Jerzy

2014-01-01

The term “cryptome” refers to the subset of cryptic peptides with bioactivities that are often unpredictable and very different from the parent protein. These cryptic peptides are generated by proteolytic cleavage of proteases, whose identification in vivo can be very challenging. In this work, we show that insulin-degrading enzyme (IDE) is able to degrade specific amino acid sequences present in the neuropeptide pro-NPFFA (NPFF precursor), generating some cryptic peptides that are also observed after incubation with rat brain cortex homogenate. The reported experimental findings support the increasingly accredited hypothesis, according to which, due to its wide substrate selectivity, IDE is involved in a wide variety of physiopathological processes. PMID:25247577

Photolithographic Synthesis of High-Density DNA and RNA Arrays on Flexible, Transparent, and Easily Subdivided Plastic Substrates.

PubMed

Holden, Matthew T; Carter, Matthew C D; Wu, Cheng-Hsien; Wolfer, Jamison; Codner, Eric; Sussman, Michael R; Lynn, David M; Smith, Lloyd M

2015-11-17

The photolithographic fabrication of high-density DNA and RNA arrays on flexible and transparent plastic substrates is reported. The substrates are thin sheets of poly(ethylene terephthalate) (PET) coated with cross-linked polymer multilayers that present hydroxyl groups suitable for conventional phosphoramidite-based nucleic acid synthesis. We demonstrate that by modifying array synthesis procedures to accommodate the physical and chemical properties of these materials, it is possible to synthesize plastic-backed oligonucleotide arrays with feature sizes as small as 14 μm × 14 μm and feature densities in excess of 125 000/cm(2), similar to specifications attainable using rigid substrates such as glass or glassy carbon. These plastic-backed arrays are tolerant to a wide range of hybridization temperatures, and improved synthetic procedures are described that enable the fabrication of arrays with sequences up to 50 nucleotides in length. These arrays hybridize with S/N ratios comparable to those fabricated on otherwise identical arrays prepared on glass or glassy carbon. This platform supports the enzymatic synthesis of RNA arrays and proof-of-concept experiments are presented showing that the arrays can be readily subdivided into smaller arrays (or "millichips") using common laboratory-scale laser cutting tools. These results expand the utility of oligonucleotide arrays fabricated on plastic substrates and open the door to new applications for these important bioanalytical tools.

Cloning and characterization of the rat HIF-1 alpha prolyl-4-hydroxylase-1 gene.

PubMed

Cobb, Ronald R; McClary, John; Manzana, Warren; Finster, Silke; Larsen, Brent; Blasko, Eric; Pearson, Jennifer; Biancalana, Sara; Kauser, Katalin; Bringmann, Peter; Light, David R; Schirm, Sabine

2005-08-01

Prolyl-4-hydroxylase domain-containing enzymes (PHDs) mediate the oxygen-dependent regulation of the heterodimeric transcription factor hypoxia-inducible factor-1 (HIF-1). Under normoxic conditions, one of the subunits of HIF-1, HIF-1alpha, is hydroxylated on specific proline residues to target HIF-1alpha for degradation by the ubiquitin-proteasome pathway. Under hypoxic conditions, the hydroxylation by the PHDs is attenuated by lack of the oxygen substrate, allowing HIF-1 to accumulate, translocate to the nucleus, and mediate HIF-mediated gene transcription. In several mammalian species including humans, three PHDs have been identified. We report here the cloning of a full-length rat cDNA that is highly homologous to the human and murine PHD-1 enzymes and encodes a protein that is 416 amino acids long. Both cDNA and protein are widely expressed in rat tissues and cell types. We demonstrate that purified and crude baculovirus-expressed rat PHD-1 exhibits HIF-1alpha specific prolyl hydroxylase activity with similar substrate affinities and is comparable to human PHD-1 protein.

Species specific inhibition of viral replication using dicer substrate siRNAs (DsiRNAs) targeting the viral nucleoprotein of the fish pathogenic rhabdovirus viral hemorrhagic septicemia virus (VHSV).

PubMed

Bohle, Harry; Lorenzen, Niels; Schyth, Brian Dall

2011-06-01

Gene knock down by the use of small interfering RNAs (siRNAs) is widely used as a method for reducing the expression of specific genes in eukaryotic cells via the RNA interference pathway. But, the effectivity of siRNA induced gene knock down in cells from fish has in several studies been questioned and the specificity seems to be a general problem in cells originating from both lower and higher vertebrates. Here we show that we are able to reduce the level of viral gene expression and replication specifically in fish cells in vitro. We do so by using 27/25-mer DsiRNAs acting as substrates for dicer for the generation of siRNAs targeting the nucleoprotein N gene of viral hemorrhagic septicemia virus (VHSV). This rhabdovirus infects salmonid fish and is responsible for large yearly losses in aquaculture production. Specificity of the DsiRNA is assured in two ways: first, by using the conventional method of testing a control DsiRNA which should not target the gene of interest. Second, by assuring that replication of a heterologous virus of the same genus as the target virus was not inhibited by the DsiRNA. Target controls are, as we have previously highlighted, essential for verification of the specificity of siRNA-induced interference with virus multiplication, but they are still not in general use. Copyright © 2011 Elsevier B.V. All rights reserved.

Genetics of the First Seven Proprotein Convertase Enzymes in Health and Disease

PubMed Central

Turpeinen, Hannu; Ortutay, Zsuzsanna; Pesu, Marko

2013-01-01

Members of the substilisin/kexin like proprotein convertase (PCSK) protease family cleave and convert immature pro-proteins into their biologically active forms. By cleaving for example prohormones, cytokines and cell membrane proteins, PCSKs participate in maintaining the homeostasis in a healthy human body. Conversely, erratic enzymatic function is thought to contribute to the pathogenesis of a wide variety of diseases, including obesity and hypercholestrolemia. The first characterized seven PCSK enzymes (PCSK1-2, FURIN, PCSK4-7) process their substrates at a motif made up of paired basic amino acid residues. This feature results in a variable degree of biochemical redundancy in vitro, and consequently, shared substrate molecules between the different PCSK enzymes. This redundancy has confounded our understanding of the specific biological functions of PCSKs. The physiological roles of these enzymes have been best illustrated by the phenotypes of genetically engineered mice and patients that carry mutations in the PCSK genes. Recent developments in genome-wide methodology have generated a large amount of novel information on the genetics of the first seven proprotein convertases. In this review we summarize the reported genetic alterations and their associated phenotypes. PMID:24396277

Flexible and conformable broadband metamaterial absorber with wide-angle and polarization stability for radar application

NASA Astrophysics Data System (ADS)

Chen, Huijie; Yang, Xiaoqing; Wu, Shiyue; Zhang, Di; Xiao, Hui; Huang, Kama; Zhu, Zhanxia; Yuan, Jianping

2018-01-01

In this work, a type of flexible, broadband electromagnetic microwave absorber is designed, fabricated and experimentally characterized. The absorber is composed of lumped resistors loaded frequency selective surface which is mounted on flexible substrate using silicone rubber and in turn backed by copper film. The simulated results show that an effective absorption (over 90%) bandwidth spans from 7.6 to 18.3 GHz, which covers both X (8-12 GHz) and Ku (12-18 GHz) bands, namely a 82.6% fraction bandwidth. And the bandwidth performs a good absorption response by varying the incident angle up to 60° for both TE and TM polarization. Moreover, the flexibility of the substrate enables the absorber conformably to bend and attach to cylinders of various radius without breakdown of the absorber. The designed structure has been fabricated and measured for both planar and conformable cases, and absorption responses show a good agreement of the broadband absorption feature with the simulated ones. This work has demonstrated specifically that proposed structure provides polarization-insensitive, wide-angle, flexible and conformable wideband absorption, which extends the absorber’s application to practical radar cross section reductions for radars and warships.

Microbial Breakdown of Organic Carbon in the Diverse Sediments of Guaymas Basin

NASA Astrophysics Data System (ADS)

Hoarfrost, A.; Snider, R.; Arnosti, C.

2015-12-01

Guaymas Basin is characterized by sediments under conditions ranging from hemipelagic to hydrothermal. This wide range in geochemical contexts results in diverse microbial communities that may have varying abilities to access organic matter. We can address these functional differences by comparing enzyme activities initializing the breakdown of organic matter across these sediment types; however, previous direct measurements of the extracellular hydrolysis of complex organic carbon in sediments are sparse. We measured this first step of heterotrophic processing of organic matter in sediments at 5-10cm and 55-60cm depth from a wide range of environmental settings in Guaymas Basin. Sediment sources included sulfidic seeps on the Sonora Margin, hemipelagic ridge flank sediments, and hydrothermically altered Sonora Margin sediments bordering a methane seep site. Hydrolysis of organic substrates varied by depth and by sediment source, but despite high energy potential and organic carbon load in sulfidic sediments, activity was not highest where hydrothermal influence was highest. These results suggest that heterotrophic breakdown of organic carbon in Guaymas Basin sediments may be sensitive to factors including varying composition of organic carbon available in different sediment types, or differences in microbial community capacities to access specific organic substrates.

High-intensity pulse light sintering of silver nanowire transparent films on polymer substrates: the effect of the thermal properties of substrates on the performance of silver films.

PubMed

Jiu, Jinting; Sugahara, Tohru; Nogi, Masaya; Araki, Teppei; Suganuma, Katsuaki; Uchida, Hiroshi; Shinozaki, Kenji

2013-12-07

Silver nanowire (AgNW) films with a random mesh structure have attracted considerable attention as high-performance flexible transparent electrodes that can replace the expensive and brittle ITO-sputtered films widely used in displays, touch screens, and solar cells. Methods such as heating, pressure treatment, and light treatment are usually used to obtain an optically transparent and electrically conductive film comparable to those of commercial ITO. However, the adhesion between the AgNW film and the substrate is so weak that other overcoatings or extra treatments are necessary. Here, a high-intensity pulsed light (HIPL) sintering technique was developed to rapidly and simply sinter the AgNW film and thus achieve strong adhesion and even high conductivity on these flexible polymer substrates which will be widely applied to the printing of electronic devices. The conductivity of the AgNW film closely depended on the thermal performance of substrates, and the adhesion was determined by the soft state of the substrate surface originating from the glass transition or melting of substrates with light intensity. The rapid sintering technique can be popularized to fabricate new devices on these polymer substrates by considering the thermal properties of the substrate to improve the performance of devices.

Surface derivatization strategy for combinatorial analysis of cell response to mixtures of protein domains.

PubMed

Chiang, Chunyi; Karuri, Stella W; Kshatriya, Pradnya P; Schwartz, Jeffrey; Schwarzbauer, Jean E; Karuri, Nancy W

2012-01-10

We report a robust strategy for conjugating mixtures of two or more protein domains to nonfouling polyurethane surfaces. In our strategy, the carbamate groups of polyurethane are reacted with zirconium alkoxide from the vapor phase to give a surface-bound oxide that serves as a chemical layer that can be used to bond organics to the polymer substrate. A hydroxyalkylphosphonate monolayer was synthesized on this layer, which was then used to covalently bind primary amine groups in protein domains using chloroformate-derived cross-linking. The effectiveness of this synthesis strategy was gauged by using an ELISA to measure competitive, covalent bonding of cell-binding (III(9-10)) and fibronectin-binding (III(1-2)) domains of the cell adhesion protein fibronectin. Cell adhesion, spreading, and fibronectin matrix assembly were examined on surfaces conjugated with single domains, a 1:1 surface mixture of III(1-2) and III(9-10), and a recombinant protein "duplex" containing both domains in one fusion protein. The mixture performed as well as or better than the other surfaces in these assays. Our surface activation strategy is amenable to a wide range of polymer substrates and free amino group-containing protein fragments. As such, this technique may be used to create biologically specific materials through the immobilization of specific protein groups or mixtures thereof on a substrate surface.

Chemical ubiquitination for decrypting a cellular code.

PubMed

Stanley, Mathew; Virdee, Satpal

2016-05-15

The modification of proteins with ubiquitin (Ub) is an important regulator of eukaryotic biology and deleterious perturbation of this process is widely linked to the onset of various diseases. The regulatory capacity of the Ub signal is high and, in part, arises from the capability of Ub to be enzymatically polymerised to form polyubiquitin (polyUb) chains of eight different linkage types. These distinct polyUb topologies can then be site-specifically conjugated to substrate proteins to elicit a number of cellular outcomes. Therefore, to further elucidate the biological significance of substrate ubiquitination, methodologies that allow the production of defined polyUb species, and substrate proteins that are site-specifically modified with them, are essential to progress our understanding. Many chemically inspired methods have recently emerged which fulfil many of the criteria necessary for achieving deeper insight into Ub biology. With a view to providing immediate impact in traditional biology research labs, the aim of this review is to provide an overview of the techniques that are available for preparing Ub conjugates and polyUb chains with focus on approaches that use recombinant protein building blocks. These approaches either produce a native isopeptide, or analogue thereof, that can be hydrolysable or non-hydrolysable by deubiquitinases. The most significant biological insights that have already been garnered using such approaches will also be summarized. © 2016 Authors; published by Portland Press Limited.

Quantitative Förster resonance energy transfer analysis for kinetic determinations of SUMO-specific protease.

PubMed

Liu, Yan; Song, Yang; Madahar, Vipul; Liao, Jiayu

2012-03-01

Förster resonance energy transfer (FRET) technology has been widely used in biological and biomedical research, and it is a very powerful tool for elucidating protein interactions in either dynamic or steady state. SUMOylation (the process of SUMO [small ubiquitin-like modifier] conjugation to substrates) is an important posttranslational protein modification with critical roles in multiple biological processes. Conjugating SUMO to substrates requires an enzymatic cascade. Sentrin/SUMO-specific proteases (SENPs) act as an endopeptidase to process the pre-SUMO or as an isopeptidase to deconjugate SUMO from its substrate. To fully understand the roles of SENPs in the SUMOylation cycle, it is critical to understand their kinetics. Here, we report a novel development of a quantitative FRET-based protease assay for SENP1 kinetic parameter determination. The assay is based on the quantitative analysis of the FRET signal from the total fluorescent signal at acceptor emission wavelength, which consists of three components: donor (CyPet-SUMO1) emission, acceptor (YPet) emission, and FRET signal during the digestion process. Subsequently, we developed novel theoretical and experimental procedures to determine the kinetic parameters, k(cat), K(M), and catalytic efficiency (k(cat)/K(M)) of catalytic domain SENP1 toward pre-SUMO1. Importantly, the general principles of this quantitative FRET-based protease kinetic determination can be applied to other proteases. Copyright © 2011 Elsevier Inc. All rights reserved.

Van der Waals interactions between planar substrate and tubular lipid membranes undergoing pearling instability

NASA Astrophysics Data System (ADS)

Valchev, G. S.; Djondjorov, P. A.; Vassilev, V. M.; Dantchev, D. M.

2017-10-01

In the current article we study the behavior of the van der Waals force between a planar substrate and an axisymmetric bilayer lipid membrane undergoing pearling instability, caused by uniform hydrostatic pressure difference. To do so, the recently suggested "surface integration approach" is used, which can be considered a generalization of the well known and widely used Derjaguin approximation. The static equilibrium shape after the occurrence of the instability is described in the framework of Helfrich's spontaneous curvature model. Some specific classes of exact analytical solutions to the corresponding shape equation are considered, and the components of the respective position vectors given in terms of elliptic integrals and Jacobi elliptic functions. The mutual orientation between the interacting objects is chosen such that the axis of revolution of the distorted cylinder be parallel to the plane bounding the substrate. Based on the discussed models and approaches we made some estimations for the studied force in real experimentally realizable systems, thus showing the possibility of pearling as an useful technique for reduction of the adhesion in variety of industrial processes using lipid membranes as carriers.

A novel robust quantitative Förster resonance energy transfer assay for protease SENP2 kinetics determination against its all natural substrates.

PubMed

Liu, Yan; Shen, Yali; Zheng, Shasha; Liao, Jiayu

2015-12-01

SUMOylation (the process of adding the SUMO [small ubiquitin-like modifier] to substrates) is an important post-translational modification of critical proteins in multiple processes. Sentrin/SUMO-specific proteases (SENPs) act as endopeptidases to process the pre-SUMO or as isopeptidases to deconjugate the SUMO from its substrate. Determining the kinetics of SENPs is important for understanding their activities. Förster resonance energy transfer (FRET) technology has been widely used in biomedical research and is a powerful tool for elucidating protein interactions. In this paper we report a novel quantitative FRET-based protease assay for SENP2 endopeptidase activity that accounts for the self-fluorescent emissions of the donor (CyPet) and the acceptor (YPet). The kinetic parameters, k(cat), K(M), and catalytic efficiency (k(cat)/K(M)) of catalytic domain SENP2 toward pre-SUMO1/2/3, were obtained by this novel design. Although we use SENP2 to demonstrate our method, the general principles of this quantitative FRET-based protease kinetic determination can be readily applied to other proteases.

High Carbon Use Efficiency is Not Explained by Production of Storage Compounds

NASA Astrophysics Data System (ADS)

Dijkstra, Paul; van Groenigen, Kees-Jan

2015-04-01

The efficiency with which microbes use substrate to make new microbial biomass (Carbon Use Efficiency or CUE; mol C / mol C) is an important variable in soil and ecosystem C cycling models. Estimates of CUE in soil microbial communities vary widely. It has been hypothesized that high values of CUE are associated with production of storage compounds following a sudden increases in substrate availability during CUE measurements. In that case, these high CUE values would not be representative for balanced microbial growth (i.e. the production of all compounds needed to make new microbial cells). To test this hypothesis, we added position-specific 13C-labeled glucose isotopomers in parallel incubations of a ponderosa pine and piñon-juniper soil. We compared the measured pattern of CO2 release for the six glucose C atoms with patterns of CO2 production expected for balanced growth with a low, medium, or high CUE, and with CO2 production patterns associated with production of storage compounds (glycogen, lipids, or polyhydroxybutyrate). The measured position-specific CO2 production did not match that for production of glycogen, lipids, or polyhydroxybutyrate, but agreed closely with that expected for balanced growth at high CUE and high pentose phosphate pathway activity. We conclude that soil microbial communities utilize glucose substrate for biomass growth with high CUE, and that addition of small amounts of 13C-labeled glucose tracers do not affect CUE or induce storage compounds production. We submit that the measurement of position-specific CO2 production offers a quick and easy way to test biochemically explicit hypotheses concerning microbial growth metabolism.

Cushing's syndrome mutant PKA L205R exhibits altered substrate specificity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lubner, Joshua M.; Dodge-Kafka, Kimberly L.; Carlson, Cathrine R.

The PKA L205R hotspot mutation has been implicated in Cushing's syndrome through hyperactive gain-of-function PKA signaling; however, its influence on substrate specificity has not been investigated. Here, we employ the Proteomic Peptide Library (ProPeL) approach to create high-resolution models for PKA WT and PKA L205R substrate specificity. We reveal that the L205R mutation reduces canonical hydrophobic preference at the substrate P + 1 position, and increases acidic preference in downstream positions. Using these models, we designed peptide substrates that exhibit altered selectivity for specific PKA variants, and demonstrate the feasibility of selective PKA L205R loss-of-function signaling. Through these results, wemore » suggest that substrate rewiring may contribute to Cushing's syndrome disease etiology, and introduce a powerful new paradigm for investigating mutation-induced kinase substrate rewiring in human disease.« less

Cushing's syndrome mutant PKA L205R exhibits altered substrate specificity

DOE PAGES

Lubner, Joshua M.; Dodge-Kafka, Kimberly L.; Carlson, Cathrine R.; ...

2017-02-01

The PKA L205R hotspot mutation has been implicated in Cushing's syndrome through hyperactive gain-of-function PKA signaling; however, its influence on substrate specificity has not been investigated. Here, we employ the Proteomic Peptide Library (ProPeL) approach to create high-resolution models for PKA WT and PKA L205R substrate specificity. We reveal that the L205R mutation reduces canonical hydrophobic preference at the substrate P + 1 position, and increases acidic preference in downstream positions. Using these models, we designed peptide substrates that exhibit altered selectivity for specific PKA variants, and demonstrate the feasibility of selective PKA L205R loss-of-function signaling. Through these results, wemore » suggest that substrate rewiring may contribute to Cushing's syndrome disease etiology, and introduce a powerful new paradigm for investigating mutation-induced kinase substrate rewiring in human disease.« less

Structural basis for the substrate specificity of PepA from Streptococcus pneumoniae, a dodecameric tetrahedral protease.

PubMed

Kim, Doyoun; San, Boi Hoa; Moh, Sang Hyun; Park, Hyejin; Kim, Dong Young; Lee, Sangho; Kim, Kyeong Kyu

2010-01-01

Regulated cytosolic proteolysis is one of the key cellular processes ensuring proper functioning of a cell. M42 family proteases show a broad spectrum of substrate specificities, but the structural basis for such diversity of the substrate specificities is lagging behind biochemical data. Here we report the crystal structure of PepA from Streptococcus pneumoniae, a glutamyl aminopeptidase belonging to M42 family (SpPepA). We found that Arg-257 in the substrate binding pocket is strategically positioned so that Arg-257 can make electrostatic interactions with the acidic residue of a substrate at its N-terminus. Structural comparison of the substrate binding pocket of the M42 family proteases, along with the structure-based multiple sequence alignment, argues that the appropriate electrostatic interactions contribute to the selective substrate specificity of SpPepA. Copyright 2009 Elsevier Inc. All rights reserved.

Understanding the Specificity and Random Collision of Enzyme-Substrate Interaction

ERIC Educational Resources Information Center

Kin, Ng Hong; Ling, Tan Aik

2016-01-01

The concept of specificity of enzyme action can potentially be abstract for some students as they fail to appreciate how the three-dimensional configuration of enzymes and the active sites confer perfect fit for specific substrates. In science text books, the specificity of enzyme-substrate binding is typically likened to the action of a lock and…

Structural, kinetic, and thermodynamic studies of specificity designed HIV-1 protease.

PubMed

Alvizo, Oscar; Mittal, Seema; Mayo, Stephen L; Schiffer, Celia A

2012-07-01

HIV-1 protease recognizes and cleaves more than 12 different substrates leading to viral maturation. While these substrates share no conserved motif, they are specifically selected for and cleaved by protease during viral life cycle. Drug resistant mutations evolve within the protease that compromise inhibitor binding but allow the continued recognition of all these substrates. While the substrate envelope defines a general shape for substrate recognition, successfully predicting the determinants of substrate binding specificity would provide additional insights into the mechanism of altered molecular recognition in resistant proteases. We designed a variant of HIV protease with altered specificity using positive computational design methods and validated the design using X-ray crystallography and enzyme biochemistry. The engineered variant, Pr3 (A28S/D30F/G48R), was designed to preferentially bind to one out of three of HIV protease's natural substrates; RT-RH over p2-NC and CA-p2. In kinetic assays, RT-RH binding specificity for Pr3 increased threefold compared to the wild-type (WT), which was further confirmed by isothermal titration calorimetry. Crystal structures of WT protease and the designed variant in complex with RT-RH, CA-p2, and p2-NC were determined. Structural analysis of the designed complexes revealed that one of the engineered substitutions (G48R) potentially stabilized heterogeneous flap conformations, thereby facilitating alternate modes of substrate binding. Our results demonstrate that while substrate specificity could be engineered in HIV protease, the structural pliability of protease restricted the propagation of interactions as predicted. These results offer new insights into the plasticity and structural determinants of substrate binding specificity of the HIV-1 protease. Copyright © 2012 The Protein Society.

Choline and N,N-dimethylethanolamine as direct substrates for methanogens.

PubMed

Watkins, Andrew J; Roussel, Erwan G; Webster, Gordon; Parkes, R John; Sass, Henrik

2012-12-01

Choline (N,N,N-trimethylethanolamine), which is widely distributed in membrane lipids and is a component of sediment biota, has been shown to be utilized anaerobically by mixed prokaryote cultures to produce methane but not by pure cultures of methanogens. Here, we show that five recently isolated Methanococcoides strains from a range of sediments (Aarhus Bay, Denmark; Severn Estuary mudflats at Portishead, United Kingdom; Darwin Mud Volcano, Gulf of Cadiz; Napoli mud volcano, eastern Mediterranean) can directly utilize choline for methanogenesis producing ethanolamine, which is not further metabolized. Di- and monomethylethanolamine are metabolic intermediates that temporarily accumulate. Consistent with this, dimethylethanolamine was shown to be another new growth substrate, but monomethylethanolamine was not. The specific methanogen inhibitor 2-bromoethanesulfonate (BES) inhibited methane production from choline. When choline and trimethylamine are provided together, diauxic growth occurs, with trimethylamine being utilized first, and then after a lag (∼7 days) choline is metabolized. Three type strains of Methanococcoides (M. methylutens, M. burtonii, and M. alaskense), in contrast, did not utilize choline. However, two of them (M. methylutens and M. burtonii) did metabolize dimethylethanolamine. These results extend the known substrates that can be directly utilized by some methanogens, giving them the advantage that they would not be reliant on bacterial syntrophs for their substrate supply.

Choline and N,N-Dimethylethanolamine as Direct Substrates for Methanogens

PubMed Central

Watkins, Andrew J.; Roussel, Erwan G.; Webster, Gordon; Parkes, R. John

2012-01-01

Choline (N,N,N-trimethylethanolamine), which is widely distributed in membrane lipids and is a component of sediment biota, has been shown to be utilized anaerobically by mixed prokaryote cultures to produce methane but not by pure cultures of methanogens. Here, we show that five recently isolated Methanococcoides strains from a range of sediments (Aarhus Bay, Denmark; Severn Estuary mudflats at Portishead, United Kingdom; Darwin Mud Volcano, Gulf of Cadiz; Napoli mud volcano, eastern Mediterranean) can directly utilize choline for methanogenesis producing ethanolamine, which is not further metabolized. Di- and monomethylethanolamine are metabolic intermediates that temporarily accumulate. Consistent with this, dimethylethanolamine was shown to be another new growth substrate, but monomethylethanolamine was not. The specific methanogen inhibitor 2-bromoethanesulfonate (BES) inhibited methane production from choline. When choline and trimethylamine are provided together, diauxic growth occurs, with trimethylamine being utilized first, and then after a lag (∼7 days) choline is metabolized. Three type strains of Methanococcoides (M. methylutens, M. burtonii, and M. alaskense), in contrast, did not utilize choline. However, two of them (M. methylutens and M. burtonii) did metabolize dimethylethanolamine. These results extend the known substrates that can be directly utilized by some methanogens, giving them the advantage that they would not be reliant on bacterial syntrophs for their substrate supply. PMID:23001649

Highly selective anti-Prelog synthesis of optically active aryl alcohols by recombinant Escherichia coli expressing stereospecific alcohol dehydrogenase.

PubMed

Li, Ming; Nie, Yao; Mu, Xiao Qing; Zhang, Rongzhen; Xu, Yan

2016-07-03

Biocatalytic asymmetric synthesis has been widely used for preparation of optically active chiral alcohols as the important intermediates and precursors of active pharmaceutical ingredients. However, the available whole-cell system involving anti-Prelog specific alcohol dehydrogenase is yet limited. A recombinant Escherichia coli system expressing anti-Prelog stereospecific alcohol dehydrogenase from Candida parapsilosis was established as a whole-cell system for catalyzing asymmetric reduction of aryl ketones to anti-Prelog configured alcohols. Using 2-hydroxyacetophenone as the substrate, reaction factors including pH, cell status, and substrate concentration had obvious impacts on the outcome of whole-cell biocatalysis, and xylose was found to be an available auxiliary substrate for intracellular cofactor regeneration, by which (S)-1-phenyl-1,2-ethanediol was achieved with an optical purity of 97%e.e. and yield of 89% under the substrate concentration of 5 g/L. Additionally, the feasibility of the recombinant cells toward different aryl ketones was investigated, and most of the corresponding chiral alcohol products were obtained with an optical purity over 95%e.e. Therefore, the whole-cell system involving recombinant stereospecific alcohol dehydrogenase was constructed as an efficient biocatalyst for highly enantioselective anti-Prelog synthesis of optically active aryl alcohols and would be promising in the pharmaceutical industry.

Foreign compounds and intermediary metabolism: sulfoxidation bridges the divide.

PubMed

Mitchell, S C; Steventon, G B

2009-03-01

It is widely appreciated that as a xenobiotic travels through an organism and interacts with the biochemical machinery of a living system, it most probably will undergo a number of metabolic alterations usually leading to a cluster of differing chemical species. Indeed, the modern 'metabonomic' approach, where earlier studied drug metabolism profiles have been reassessed, has indicated that there are normally many more previously unrecognised minor metabolites, and when all such biotransformation products are considered, then their total number is legion. It is now being recognised also that the same metabolic alteration of a substrate, especially a xenobiotic substrate, may be catalysed by more than one enzyme and that the previously sacrosanct notion of an enzyme's 'substrate specificity' may well be inverted to read a substrate's 'enzyme preference'. The following brief article attempts to highlight another aspect where our general acceptance of the 'status quo' needs to be reconsidered. The conventionally acknowledged division between the collection of enzymes that undertake intermediary metabolism and the group of enzymes responsible for xenobiotic metabolism may be becoming blurred. It may well be a prudent time to reassess the current dichotomous view. Overcoming inertia, with a realignment of ideas or alteration of perception, may permit new concepts to emerge leading to a more profound understanding and hopefully eventual benefits for mankind.

Wzx flippases exhibiting complex O-unit preferences require a new model for Wzx-substrate interactions.

PubMed

Liu, Michael A; Morris, Paraskevi; Reeves, Peter R

2018-06-10

The Wzx flippase is a critical component of the O-antigen biosynthesis pathway, being responsible for the translocation of oligosaccharide O units across the inner membrane in Gram-negative bacteria. Recent studies have shown that Wzx has a strong preference for its cognate O unit, but the types of O-unit structural variance that a given Wzx can accommodate are poorly understood. In this study, we identified two Yersinia pseudotuberculosis Wzx that can distinguish between different terminal dideoxyhexose sugars on a common O-unit main-chain, despite both being able to translocate several other structurally-divergent O units. We also identified other Y. pseudotuberculosis Wzx that can translocate a structurally divergent foreign O unit with high efficiency, and thus exhibit an apparently relaxed substrate preference. It now appears that Wzx substrate preference is more complex than previously suggested, and that not all O-unit residues are equally important determinants of translocation efficiency. We propose a new "Structure-Specific Triggering" model in which Wzx translocation proceeds at a low level for a wide variety of substrates, with high-frequency translocation only being triggered by Wzx interacting with one or more preferred O-unit structural elements found on its cognate O unit(s). © 2018 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

Specificity of a protein-protein interface: local dynamics direct substrate recognition of effector caspases.

PubMed

Fuchs, Julian E; von Grafenstein, Susanne; Huber, Roland G; Wallnoefer, Hannes G; Liedl, Klaus R

2014-04-01

Proteases are prototypes of multispecific protein-protein interfaces. Proteases recognize and cleave protein and peptide substrates at a well-defined position in a substrate binding groove and a plethora of experimental techniques provide insights into their substrate recognition. We investigate the caspase family of cysteine proteases playing a key role in programmed cell death and inflammation, turning caspases into interesting drug targets. Specific ligand binding to one particular caspase is difficult to achieve, as substrate specificities of caspase isoforms are highly similar. In an effort to rationalize substrate specificity of two closely related caspases, we investigate the substrate promiscuity of the effector Caspases 3 and 7 by data mining (cleavage entropy) and by molecular dynamics simulations. We find a strong correlation between binding site rigidity and substrate readout for individual caspase subpockets explaining more stringent substrate readout of Caspase 7 via its narrower conformational space. Caspase 3 subpockets S3 and S4 show elevated local flexibility explaining the more unspecific substrate readout of that isoform in comparison to Caspase 7. We show by in silico exchange mutations in the S3 pocket of the proteases that a proline residue in Caspase 7 contributes to the narrowed conformational space of the binding site. These findings explain the substrate specificities of caspases via a mechanism of conformational selection and highlight the crucial importance of binding site local dynamics in substrate recognition of proteases. Proteins 2014; 82:546-555. © 2013 Wiley Periodicals, Inc. Copyright © 2013 The Authors Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.

Probing the molecular determinants of aniline dioxygenase substrate specificity by saturation mutagenesis.

PubMed

Ang, Ee L; Obbard, Jeffrey P; Zhao, Huimin

2007-02-01

Aniline dioxygenase is a multicomponent Rieske nonheme-iron dioxygenase enzyme isolated from Acinetobacter sp. strain YAA. Saturation mutagenesis of the substrate-binding pocket residues, which were identified using a homology model of the alpha subunit of the terminal dioxygenase (AtdA3), was used to probe the molecular determinants of AtdA substrate specificity. The V205A mutation widened the substrate specificity of aniline dioxygenase to include 2-isopropylaniline, for which the wild-type enzyme has no activity. The V205A mutation also made 2-isopropylaniline a better substrate for the enzyme than 2,4-dimethylaniline, a native substrate of the wild-type enzyme. The I248L mutation improved the activity of aniline dioxygenase against aniline and 2,4-dimethylaniline approximately 1.7-fold and 2.1-fold, respectively. Thus, it is shown that the alpha subunit of the terminal dioxygenase indeed plays a part in the substrate specificity as well as the activity of aniline dioxygenase. Interestingly, the equivalent residues of V205 and I248 have not been previously reported to influence the substrate specificity of other Rieske dioxygenases. These results should facilitate future engineering of the enzyme for bioremediation and industrial applications.

Wafer bonded virtual substrate and method for forming the same

DOEpatents

Atwater, Jr., Harry A.; Zahler, James M [Pasadena, CA; Morral, Anna Fontcuberta i [Paris, FR

2007-07-03

A method of forming a virtual substrate comprised of an optoelectronic device substrate and handle substrate comprises the steps of initiating bonding of the device substrate to the handle substrate, improving or increasing the mechanical strength of the device and handle substrates, and thinning the device substrate to leave a single-crystal film on the virtual substrate such as by exfoliation of a device film from the device substrate. The handle substrate is typically Si or other inexpensive common substrate material, while the optoelectronic device substrate is formed of more expensive and specialized electro-optic material. Using the methodology of the invention a wide variety of thin film electro-optic materials of high quality can be bonded to inexpensive substrates which serve as the mechanical support for an optoelectronic device layer fabricated in the thin film electro-optic material.

Wafer bonded virtual substrate and method for forming the same

NASA Technical Reports Server (NTRS)

Atwater, Jr., Harry A. (Inventor); Zahler, James M. (Inventor); Morral, Anna Fontcuberta i (Inventor)

2007-01-01

A method of forming a virtual substrate comprised of an optoelectronic device substrate and handle substrate comprises the steps of initiating bonding of the device substrate to the handle substrate, improving or increasing the mechanical strength of the device and handle substrates, and thinning the device substrate to leave a single-crystal film on the virtual substrate such as by exfoliation of a device film from the device substrate. The handle substrate is typically Si or other inexpensive common substrate material, while the optoelectronic device substrate is formed of more expensive and specialized electro-optic material. Using the methodology of the invention a wide variety of thin film electro-optic materials of high quality can be bonded to inexpensive substrates which serve as the mechanical support for an optoelectronic device layer fabricated in the thin film electro-optic material.

Size-matched alkyne-conjugated cyanine fluorophores to identify differences in protein glycosylation.

PubMed

Burnham-Marusich, Amanda R; Plechaty, Anna M; Berninsone, Patricia M

2014-09-01

Currently, there are few methods to detect differences in posttranslational modifications (PTMs) in a specific manner from complex mixtures. Thus, we developed an approach that combines the sensitivity and specificity of click chemistry with the resolution capabilities of 2D-DIGE. In "Click-DIGE", posttranslationally modified proteins are metabolically labeled with azido-substrate analogs, then size- and charge-matched alkyne-Cy3 or alkyne-Cy5 dyes are covalently attached to the azide of the PTM by click chemistry. The fluorescently-tagged protein samples are then multiplexed for 2DE analysis. Whereas standard DIGE labels all proteins, Click-DIGE focuses the analysis of protein differences to a targeted subset of posttranslationally modified proteins within a complex sample (i.e. specific labeling and analysis of azido glycoproteins within a cell lysate). Our data indicate that (i) Click-DIGE specifically labels azido proteins, (ii) the resulting Cy-protein conjugates are spectrally distinct, and (iii) the conjugates are size- and charge-matched at the level of 2DE. We demonstrate the utility of this approach by detecting multiple differentially expressed glycoproteins between a mutant cell line defective in UDP-galactose transport and the parental cell line. We anticipate that the diversity of azido substrates already available will enable Click-DIGE to be compatible with analysis of a wide range of PTMs. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Extensive peptide and natural protein substrate screens reveal that mouse caspase-11 has much narrower substrate specificity than caspase-1

PubMed Central

Ramirez, Monica L. Gonzalez; Poreba, Marcin; Snipas, Scott J.; Groborz, Katarzyna; Drag, Marcin; Salvesen, Guy S.

2018-01-01

Inflammatory cell death, or pyroptosis, is triggered by pathogenic infections or events. It is executed by caspase-1 (in the canonical pyroptosis pathway) or caspase-11 (noncanonical pathway), each via production of a cell-lytic domain from the pyroptosis effector protein gasdermin D through specific and limited proteolysis. Pyroptosis is accompanied by the release of inflammatory mediators, including the proteolytically processed forms of interleukin-1β (IL-1β) and IL-18. Given the similar inflammatory outcomes of the canonical and noncanonical pyroptosis pathways, we hypothesized that caspase-1 and -11 should have very similar activities and substrate specificities. To test this hypothesis, we purified recombinant murine caspases and analyzed their primary specificities by massive hybrid combinatorial substrate library (HyCoSuL) screens. We correlated the substrate preferences of each caspase with their activities on the recombinant natural substrates IL-1β, IL-18, and gasdermin D. Although we identified highly selective and robust peptidyl substrates for caspase-1, we were unable to do so for caspase-11, because caspase-1 cleaved even the best caspase-11 substrates equally well. Caspase-1 rapidly processed pro-IL-1β and -18, but caspase-11 processed these two pro-ILs extremely poorly. However, both caspase-1 and -11 efficiently produced the cell-lytic domain from the gasdermin D precursor. We hypothesize that caspase-11 may have evolved a specific exosite to selectively engage pyroptosis without directly activating pro-IL-1β or -18. In summary, comparing the activities of caspase-1 and -11 in HyCoSuL screens and with three endogenous protein substrates, we conclude that caspase-11 has highly restricted substrate specificity, preferring gasdermin D over all other substrates examined. PMID:29414788

Fiber sensors for molecular detection

NASA Astrophysics Data System (ADS)

Gu, Claire; Yang, Xuan; Zhang, Jin; Newhouse, Rebecca; Cao, Liangcai

2010-11-01

The demand on sensors for detecting chemical and biological agents is greater than ever before, including medical, environmental, food safety, military, and security applications. At present, most detection or sensing techniques tend to be either non-molecular specific, bulky, expensive, relatively inaccurate, or unable to provide real time data. Clearly, alternative sensing technologies are urgently needed. Recently, we have been working to develop a compact fiber optic surface enhanced Raman scattering (SERS) sensor system that integrates various novel ideas to achieve compactness, high sensitivity and consistency, molecular specificity, and automatic preliminary identification capabilities. The unique sensor architecture is expected to bring SERS sensors to practical applications due to a combination of 1) novel SERS substrates that provide the high sensitivity and consistency, molecular specificity, and applicability to a wide range of compounds; 2) a unique hollow core optical fiber probe with double SERS substrate structure that provides the compactness, reliability, low cost, and ease of sampling; and 3) an innovative matched spectral filter set that provides automatic preliminary molecule identification. In this paper, we will review the principle of operation and some of the important milestones of fiber SERS sensor development with emphasis on our recent work to integrate photonic crystal fiber SERS probes with a portable Raman spectrometer and to demonstrate a matched spectral filter for molecule identification.

Substrate specificity of pyrimidine nucleoside phosphorylases of NP-II family probed by X-ray crystallography and molecular modeling

NASA Astrophysics Data System (ADS)

Balaev, V. V.; Lashkov, A. A.; Prokofev, I. I.; Gabdulkhakov, A. G.; Seregina, T. A.; Mironov, A. S.; Betzel, C.; Mikhailov, A. M.

2016-09-01

Pyrimidine nucleoside phosphorylases, which are widely used in the biotechnological production of nucleosides, have different substrate specificity for pyrimidine nucleosides. An interesting feature of these enzymes is that the three-dimensional structure of thymidine-specific nucleoside phosphorylase is similar to the structure of nonspecific pyrimidine nucleoside phosphorylase. The three-dimensional structures of thymidine phosphorylase from Salmonella typhimurium and nonspecific pyrimidine nucleoside phosphorylase from Bacillus subtilis in complexes with a sulfate anion were determined for the first time by X-ray crystallography. An analysis of the structural differences between these enzymes demonstrated that Lys108, which is involved in the phosphate binding in pyrimidine nucleoside phosphorylase, corresponds to Met111 in thymidine phosphorylases. This difference results in a decrease in the charge on one of the hydroxyl oxygens of the phosphate anion in thymidine phosphorylase and facilitates the catalysis through SN2 nucleophilic substitution. Based on the results of X-ray crystallography, the virtual screening was performed for identifying a potent inhibitor (anticancer agent) of nonspecific pyrimidine nucleoside phosphorylase, which does not bind to thymidine phosphorylase. The molecular dynamics simulation revealed the stable binding of the discovered compound—2-pyrimidin-2-yl-1H-imidazole-4-carboxylic acid—to the active site of pyrimidine nucleoside phosphorylase.

Substrate specificity of the ubiquitin and Ubl proteases

PubMed Central

Ronau, Judith A; Beckmann, John F; Hochstrasser, Mark

2016-01-01

Conjugation and deconjugation of ubiquitin and ubiquitin-like proteins (Ubls) to cellular proteins are highly regulated processes integral to cellular homeostasis. Most often, the C-termini of these small polypeptides are attached to lysine side chains of target proteins by an amide (isopeptide) linkage. Deubiquitinating enzymes (DUBs) and Ubl-specific proteases (ULPs) comprise a diverse group of proteases that recognize and remove ubiquitin and Ubls from their substrates. How DUBs and ULPs distinguish among different modifiers, or different polymeric forms of these modifiers, remains poorly understood. The specificity of ubiquitin/Ubl-deconjugating enzymes for particular substrates depends on multiple factors, ranging from the topography of specific substrate features, as in different polyubiquitin chain types, to structural elements unique to each enzyme. Here we summarize recent structural and biochemical studies that provide insights into mechanisms of substrate specificity among various DUBs and ULPs. We also discuss the unexpected specificities of non-eukaryotic proteases in these families. PMID:27012468

Demixing of polymers under nanoimprinting process

NASA Astrophysics Data System (ADS)

Wang, Zhen

Polymer blend has been an important area in polymer science for decades. The knowledge of polymer blend in bulk is well established and technologies based on it have created products ubiquitous in our daily life. More intriguing problem arises when the phase separation of a polymer blend occurs under physical confinement. In this thesis, we investigated the effect of interfacial interactions between constituent polymers and confinement environment on phase evolution. Specifically, morphologies of thin films of binary polymer blends were examined on chemically homogenous substrates (preferential surface, neutral surface), on chemical pattern, between two parallel rigid substrates, and under thermal embossing/step-and-flash nanoimprint lithography conditions. We found that preferential wetting of selective component dominates the phase evolution, which can be suppressed by the use of neutral surfaces or external pressure. By manipulating these factors, a wide range of unique non-equilibrium micro or nanostructures can thus be achieved.

A simple, multidimensional approach to high-throughput discovery of catalytic reactions.

PubMed

Robbins, Daniel W; Hartwig, John F

2011-09-09

Transition metal complexes catalyze many important reactions that are employed in medicine, materials science, and energy production. Although high-throughput methods for the discovery of catalysts that would mirror related approaches for the discovery of medicinally active compounds have been the focus of much attention, these methods have not been sufficiently general or accessible to typical synthetic laboratories to be adopted widely. We report a method to evaluate a broad range of catalysts for potential coupling reactions with the use of simple laboratory equipment. Specifically, we screen an array of catalysts and ligands with a diverse mixture of substrates and then use mass spectrometry to identify reaction products that, by design, exceed the mass of any single substrate. With this method, we discovered a copper-catalyzed alkyne hydroamination and two nickel-catalyzed hydroarylation reactions, each of which displays excellent functional-group tolerance.

Functional evolution and structural conservation in chimeric cytochromes p450: calibrating a structure-guided approach.

PubMed

Otey, Christopher R; Silberg, Jonathan J; Voigt, Christopher A; Endelman, Jeffrey B; Bandara, Geethani; Arnold, Frances H

2004-03-01

Recombination generates chimeric proteins whose ability to fold depends on minimizing structural perturbations that result when portions of the sequence are inherited from different parents. These chimeric sequences can display functional properties characteristic of the parents or acquire entirely new functions. Seventeen chimeras were generated from two CYP102 members of the functionally diverse cytochrome p450 family. Chimeras predicted to have limited structural disruption, as defined by the SCHEMA algorithm, displayed CO binding spectra characteristic of folded p450s. Even this small population exhibited significant functional diversity: chimeras displayed altered substrate specificities, a wide range in thermostabilities, up to a 40-fold increase in peroxidase activity, and ability to hydroxylate a substrate toward which neither parent heme domain shows detectable activity. These results suggest that SCHEMA-guided recombination can be used to generate diverse p450s for exploring function evolution within the p450 structural framework.

GaN-on-silicon high-electron-mobility transistor technology with ultra-low leakage up to 3000 V using local substrate removal and AlN ultra-wide bandgap

NASA Astrophysics Data System (ADS)

Dogmus, Ezgi; Zegaoui, Malek; Medjdoub, Farid

2018-03-01

We report on extremely low off-state leakage current in AlGaN/GaN-on-silicon metal–insulator–semiconductor high-electron-mobility transistors (MISHEMTs) up to a high blocking voltage. Remarkably low off-state gate and drain leakage currents below 1 µA/mm up to 3 kV have been achieved owing to the use of a thick in situ SiN gate dielectric under the gate, and a local Si substrate removal technique combined with a cost effective 15-µm-thick AlN dielectric layer followed by a Cu deposition. This result establishes a manufacturable state-of-the-art high-voltage GaN-on-silicon power transistors while maintaining a low specific on-resistance of approximately 10 mΩ·cm2.

Tritium target manufacturing for use in accelerators

NASA Astrophysics Data System (ADS)

Bach, P.; Monnin, C.; Van Rompay, M.; Ballanger, A.

2001-07-01

As a neutron tube manufacturer, SODERN is now in charge of manufacturing tritium targets for accelerators, in cooperation with CEA/DAM/DTMN in Valduc. Specific deuterium and tritium targets are manufactured on request, according to the requirements of the users, starting from titanium target on copper substrate, and going to more sophisticated devices. A wide range of possible uses is covered, including thin targets for neutron calibration, thick targets with controlled loading of deuterium and tritium, rotating targets for higher lifetimes, or large size rotating targets for accelerators used in boron neutron therapy. Activity of targets lies in the 1 to 1000 Curie, diameter of targets being up to 30 cm. Special targets are also considered, including surface layer targets for lowering tritium desorption under irradiation, or those made from different kinds of occluders such as titanium, zirconium, erbium, scandium, with different substrates. It is then possible to optimize either neutron output, or lifetime and stability, or thermal behavior.

Altered Substrate Specificity of Drug-Resistant Human Immunodeficiency Virus Type 1 Protease

PubMed Central

Dauber, Deborah S.; Ziermann, Rainer; Parkin, Neil; Maly, Dustin J.; Mahrus, Sami; Harris, Jennifer L.; Ellman, Jon A.; Petropoulos, Christos; Craik, Charles S.

2002-01-01

Resistance to human immunodeficiency virus type 1 protease (HIV PR) inhibitors results primarily from the selection of multiple mutations in the protease region. Because many of these mutations are selected for the ability to decrease inhibitor binding in the active site, they also affect substrate binding and potentially substrate specificity. This work investigates the substrate specificity of a panel of clinically derived protease inhibitor-resistant HIV PR variants. To compare protease specificity, we have used positional-scanning, synthetic combinatorial peptide libraries as well as a select number of individual substrates. The subsite preferences of wild-type HIV PR determined by using the substrate libraries are consistent with prior reports, validating the use of these libraries to compare specificity among a panel of HIV PR variants. Five out of seven protease variants demonstrated subtle differences in specificity that may have significant impacts on their abilities to function in viral maturation. Of these, four variants demonstrated up to fourfold changes in the preference for valine relative to alanine at position P2 when tested on individual peptide substrates. This change correlated with a common mutation in the viral NC/p1 cleavage site. These mutations may represent a mechanism by which severely compromised, drug-resistant viral strains can increase fitness levels. Understanding the altered substrate specificity of drug-resistant HIV PR should be valuable in the design of future generations of protease inhibitors as well as in elucidating the molecular basis of regulation of proteolysis in HIV. PMID:11773410

A Single Glycan at the 99-Loop of Human Kallikrein-related Peptidase 2 Regulates Activation and Enzymatic Activity*

PubMed Central

Guo, Shihui; Skala, Wolfgang; Magdolen, Viktor; Briza, Peter; Biniossek, Martin L.; Schilling, Oliver; Kellermann, Josef; Brandstetter, Hans; Goettig, Peter

2016-01-01

Human kallikrein-related peptidase 2 (KLK2) is a key serine protease in semen liquefaction and prostate cancer together with KLK3/prostate-specific antigen. In order to decipher the function of its potential N-glycosylation site, we produced pro-KLK2 in Leishmania tarentolae cells and compared it with its non-glycosylated counterpart from Escherichia coli expression. Mass spectrometry revealed that Asn-95 carries a core glycan, consisting of two GlcNAc and three hexoses. Autocatalytic activation was retarded in glyco-pro-KLK2, whereas the activated glyco-form exhibited an increased proteolytic resistance. The specificity patterns obtained by the PICS (proteomic identification of protease cleavage sites) method are similar for both KLK2 variants, with a major preference for P1-Arg. However, glycosylation changes the enzymatic activity of KLK2 in a drastically substrate-dependent manner. Although glyco-KLK2 has a considerably lower catalytic efficiency than glycan-free KLK2 toward peptidic substrates with P2-Phe, the situation was reverted toward protein substrates, such as glyco-pro-KLK2 itself. These findings can be rationalized by the glycan-carrying 99-loop that prefers to cover the active site like a lid. By contrast, the non-glycosylated 99-loop seems to favor a wide open conformation, which mostly increases the apparent affinity for the substrates (i.e. by a reduction of Km). Also, the cleavage pattern and kinetics in autolytic inactivation of both KLK2 variants can be explained by a shift of the target sites due to the presence of the glycan. These striking effects of glycosylation pave the way to a deeper understanding of kallikrein-related peptidase biology and pathology. PMID:26582203

Substrate specificity of sheep liver sorbitol dehydrogenase.

PubMed Central

Lindstad, R I; Köll, P; McKinley-McKee, J S

1998-01-01

The substrate specificity of sheep liver sorbitol dehydrogenase has been studied by steady-state kinetics over the range pH 7-10. Sorbitol dehydrogenase stereo-selectively catalyses the reversible NAD-linked oxidation of various polyols and other secondary alcohols into their corresponding ketones. The kinetic constants are given for various novel polyol substrates, including L-glucitol, L-mannitol, L-altritol, D-altritol, D-iditol and eight heptitols, as well as for many aliphatic and aromatic alcohols. The maximum velocities (kcat) and the substrate specificity-constants (kcat/Km) are positively correlated with increasing pH. The enzyme-catalysed reactions occur by a compulsory ordered kinetic mechanism with the coenzyme as the first, or leading, substrate. With many substrates, the rate-limiting step for the overall reaction is the enzyme-NADH product dissociation. However, with several substrates there is a transition to a mechanism with partial rate-limitation at the ternary complex level, especially at low pH. The kinetic data enable the elucidation of new empirical rules for the substrate specificity of sorbitol dehydrogenase. The specificity-constants for polyol oxidation vary as a function of substrate configuration with D-xylo> D-ribo > L-xylo > D-lyxo approximately L-arabino > D-arabino > L-lyxo. Catalytic activity with a polyol or an aromatic substrate and various 1-deoxy derivatives thereof varies with -CH2OH > -CH2NH2 > -CH2OCH3 approximately -CH3. The presence of a hydroxyl group at each of the remaining chiral centres of a polyol, apart from the reactive C2, is also nonessential for productive ternary complex formation and catalysis. A predominantly nonpolar enzymic epitope appears to constitute an important structural determinant for the substrate specificity of sorbitol dehydrogenase. The existence of two distinct substrate binding regions in the enzyme active site, along with that of the catalytic zinc, is suggested to account for the lack of stereospecificity at C2 in some polyols. PMID:9461546

Kinetics of Mixed Microbial Assemblages Enhance Removal of Highly Dilute Organic Substrates

PubMed Central

Lewis, David L.; Hodson, Robert E.; Hwang, Huey-Min

1988-01-01

Our experiments with selected organic substrates reveal that the rate-limiting process governing microbial degradation rates changes with substrate concentration, S, in such a manner that substrate removal is enhanced at lower values of S. This enhancement is the result of the dominance of very efficient systems for substrate removal at low substrate concentrations. The variability of dominant kinetic parameters over a range of S causes the kinetics of complex assemblages to be profoundly dissimilar to those of systems possessing a single set of kinetic parameters; these findings necessitate taking a new approach to predicting substrate removal rates over wide ranges of S. PMID:16347715

Kinetics of reactions of the Actinomadura R39 DD-peptidase with specific substrates.

PubMed

Adediran, S A; Kumar, Ish; Nagarajan, Rajesh; Sauvage, Eric; Pratt, R F

2011-01-25

The Actinomadura R39 DD-peptidase catalyzes the hydrolysis and aminolysis of a number of small peptides and depsipeptides. Details of its substrate specificity and the nature of its in vivo substrate are not, however, well understood. This paper describes the interactions of the R39 enzyme with two peptidoglycan-mimetic substrates 3-(D-cysteinyl)propanoyl-D-alanyl-D-alanine and 3-(D-cysteinyl)propanoyl-D-alanyl-D-thiolactate. A detailed study of the reactions of the former substrate, catalyzed by the enzyme, showed DD-carboxypeptidase, DD-transpeptidase, and DD-endopeptidase activities. These results confirm the specificity of the enzyme for a free D-amino acid at the N-terminus of good substrates and indicated a preference for extended D-amino acid leaving groups. The latter was supported by determination of the structural specificity of amine nucleophiles for the acyl-enzyme generated by reaction of the enzyme with the thiolactate substrate. It was concluded that a specific substrate for this enzyme, and possibly the in vivo substrate, may consist of a partly cross-linked peptidoglycan polymer where a free side chain N-terminal un-cross-linked amino acid serves as the specific acyl group in an endopeptidase reaction. The enzyme is most likely a DD-endopeptidase in vivo. pH-rate profiles for reactions of the enzyme with peptides, the thiolactate named above, and β-lactams indicated the presence of complex proton dissociation pathways with sticky substrates and/or protons. The local structure of the active site may differ significantly for reactions of peptides and β-lactams. Solvent kinetic deuterium isotope effects indicate the presence of classical general acid/base catalysis in both acylation and deacylation; there is no evidence of the low fractionation factor active site hydrogen found previously in class A and C β-lactamases.

E3Net: a system for exploring E3-mediated regulatory networks of cellular functions.

PubMed

Han, Youngwoong; Lee, Hodong; Park, Jong C; Yi, Gwan-Su

2012-04-01

Ubiquitin-protein ligase (E3) is a key enzyme targeting specific substrates in diverse cellular processes for ubiquitination and degradation. The existing findings of substrate specificity of E3 are, however, scattered over a number of resources, making it difficult to study them together with an integrative view. Here we present E3Net, a web-based system that provides a comprehensive collection of available E3-substrate specificities and a systematic framework for the analysis of E3-mediated regulatory networks of diverse cellular functions. Currently, E3Net contains 2201 E3s and 4896 substrates in 427 organisms and 1671 E3-substrate specific relations between 493 E3s and 1277 substrates in 42 organisms, extracted mainly from MEDLINE abstracts and UniProt comments with an automatic text mining method and additional manual inspection and partly from high throughput experiment data and public ubiquitination databases. The significant functions and pathways of the extracted E3-specific substrate groups were identified from a functional enrichment analysis with 12 functional category resources for molecular functions, protein families, protein complexes, pathways, cellular processes, cellular localization, and diseases. E3Net includes interactive analysis and navigation tools that make it possible to build an integrative view of E3-substrate networks and their correlated functions with graphical illustrations and summarized descriptions. As a result, E3Net provides a comprehensive resource of E3s, substrates, and their functional implications summarized from the regulatory network structures of E3-specific substrate groups and their correlated functions. This resource will facilitate further in-depth investigation of ubiquitination-dependent regulatory mechanisms. E3Net is freely available online at http://pnet.kaist.ac.kr/e3net.

Relationships between substrate, surface characteristics, and vegetation in an initial ecosystem

NASA Astrophysics Data System (ADS)

Biber, P.; Seifert, S.; Zaplata, M. K.; Schaaf, W.; Pretzsch, H.; Fischer, A.

2013-12-01

We investigated surface and vegetation dynamics in the artificial initial ecosystem "Chicken Creek" (Lusatia, Germany) in the years 2006-2011 across a wide spectrum of empirical data. We scrutinized three overarching hypotheses concerning (1) the relations between initial geomorphological and substrate characteristics with surface structure and terrain properties, (2) the effects of the latter on the occurrence of grouped plant species, and (3) vegetation density effects on terrain surface change. Our data comprise and conflate annual vegetation monitoring results, biennial terrestrial laser scans (starting in 2008), annual groundwater levels, and initially measured soil characteristics. The empirical evidence mostly confirms the hypotheses, revealing statistically significant relations for several goal variables: (1) the surface structure properties, local rill density, local relief energy and terrain surface height change; (2) the cover of different plant groups (annual, herbaceous, grass-like, woody, Fabaceae), and local vegetation height; and (3) terrain surface height change showed significant time-dependent relations with a variable that proxies local plant biomass. Additionally, period specific effects (like a calendar-year optimum effect for the occurrence of Fabaceae) were proven. Further and beyond the hypotheses, our findings on the spatiotemporal dynamics during the system's early development grasp processes which generally mark the transition from a geo-hydro-system towards a bio-geo-hydro system (weakening geomorphology effects on substrate surface dynamics, while vegetation effects intensify with time), where pure geomorphology or substrate feedbacks are changing into vegetation-substrate feedback processes.

Glycan microarray screening assay for glycosyltransferase specificities.

PubMed

Peng, Wenjie; Nycholat, Corwin M; Razi, Nahid

2013-01-01

Glycan microarrays represent a high-throughput approach to determining the specificity of glycan-binding proteins against a large set of glycans in a single format. This chapter describes the use of a glycan microarray platform for evaluating the activity and substrate specificity of glycosyltransferases (GTs). The methodology allows simultaneous screening of hundreds of immobilized glycan acceptor substrates by in situ incubation of a GT and its appropriate donor substrate on the microarray surface. Using biotin-conjugated donor substrate enables direct detection of the incorporated sugar residues on acceptor substrates on the array. In addition, the feasibility of the method has been validated using label-free donor substrate combined with lectin-based detection of product to assess enzyme activity. Here, we describe the application of both procedures to assess the specificity of a recombinant human α2-6 sialyltransferase. This technique is readily adaptable to studying other glycosyltransferases.

Characterizing Protease Specificity: How Many Substrates Do We Need?

PubMed Central

Schauperl, Michael; Fuchs, Julian E.; Waldner, Birgit J.; Huber, Roland G.; Kramer, Christian; Liedl, Klaus R.

2015-01-01

Calculation of cleavage entropies allows to quantify, map and compare protease substrate specificity by an information entropy based approach. The metric intrinsically depends on the number of experimentally determined substrates (data points). Thus a statistical analysis of its numerical stability is crucial to estimate the systematic error made by estimating specificity based on a limited number of substrates. In this contribution, we show the mathematical basis for estimating the uncertainty in cleavage entropies. Sets of cleavage entropies are calculated using experimental cleavage data and modeled extreme cases. By analyzing the underlying mathematics and applying statistical tools, a linear dependence of the metric in respect to 1/n was found. This allows us to extrapolate the values to an infinite number of samples and to estimate the errors. Analyzing the errors, a minimum number of 30 substrates was found to be necessary to characterize substrate specificity, in terms of amino acid variability, for a protease (S4-S4’) with an uncertainty of 5 percent. Therefore, we encourage experimental researchers in the protease field to record specificity profiles of novel proteases aiming to identify at least 30 peptide substrates of maximum sequence diversity. We expect a full characterization of protease specificity helpful to rationalize biological functions of proteases and to assist rational drug design. PMID:26559682

The pathology and pathophysiology of vascular dementia.

PubMed

Kalaria, Raj N

2017-12-19

Vascular dementia (VaD) is widely recognised as the second most common type of dementia. Consensus and accurate diagnosis of clinically suspected VaD relies on wide-ranging clinical, neuropsychological and neuroimaging measures in life but more importantly pathological confirmation. Factors defining subtypes of VaD include the nature and extent of vascular pathologies, degree of involvement of extra and intracranial vessels and the anatomical location of tissue changes as well as time after the initial vascular event. Atherosclerotic and cardioembolic diseases combined appear the most common subtypes of vascular brain injury. In recent years, cerebral small vessel disease (SVD) has gained prominence worldwide as an important substrate of cognitive impairment. SVD is characterised by arteriolosclerosis, lacunar infarcts and cortical and subcortical microinfarcts and diffuse white matter changes, which involve myelin loss and axonal abnormalities. Global brain atrophy and focal degeneration of the cerebrum including medial temporal lobe atrophy are also features of VaD similar to Alzheimer's disease. Hereditary arteriopathies have provided insights into the mechanisms of dementia particularly how arteriolosclerosis, a major contributor of SVD promotes cognitive impairment. Recently developed and validated neuropathology guidelines indicated that the best predictors of vascular cognitive impairment were small or lacunar infarcts, microinfarcts, perivascular space dilation, myelin loss, arteriolosclerosis and leptomeningeal cerebral amyloid angiopathy. While these substrates do not suggest high specificity, VaD is likely defined by key neuronal and dendro-synaptic changes resulting in executive dysfunction and related cognitive deficits. Greater understanding of the molecular pathology is needed to clearly define microvascular disease and vascular substrates of dementia. Copyright © 2017 Elsevier Ltd. All rights reserved.

Chaperone activity of human small heat shock protein-GST fusion proteins.

PubMed

Arbach, Hannah; Butler, Caley; McMenimen, Kathryn A

2017-07-01

Small heat shock proteins (sHsps) are a ubiquitous part of the machinery that maintains cellular protein homeostasis by acting as molecular chaperones. sHsps bind to and prevent the aggregation of partially folded substrate proteins in an ATP-independent manner. sHsps are dynamic, forming an ensemble of structures from dimers to large oligomers through concentration-dependent equilibrium dissociation. Based on structural studies and mutagenesis experiments, it is proposed that the dimer is the smallest active chaperone unit, while larger oligomers may act as storage depots for sHsps or play additional roles in chaperone function. The complexity and dynamic nature of their structural organization has made elucidation of their chaperone function challenging. HspB1 and HspB5 are two canonical human sHsps that vary in sequence and are expressed in a wide variety of tissues. In order to determine the role of the dimer in chaperone activity, glutathione-S-transferase (GST) was genetically linked as a fusion protein to the N-terminus regions of both HspB1 and HspB5 (also known as Hsp27 and αB-crystallin, respectively) proteins in order to constrain oligomer formation of HspB1 and HspB5, by using GST, since it readily forms a dimeric structure. We monitored the chaperone activity of these fusion proteins, which suggest they primarily form dimers and monomers and function as active molecular chaperones. Furthermore, the two different fusion proteins exhibit different chaperone activity for two model substrate proteins, citrate synthase (CS) and malate dehydrogenase (MDH). GST-HspB1 prevents more aggregation of MDH compared to GST-HspB5 and wild type HspB1. However, when CS is the substrate, both GST-HspB1 and GST-HspB5 are equally effective chaperones. Furthermore, wild type proteins do not display equal activity toward the substrates, suggesting that each sHsp exhibits different substrate specificity. Thus, substrate specificity, as described here for full-length GST fusion proteins with MDH and CS, is modulated by both sHsp oligomeric conformation and by variations of sHsp sequences.

An Alternative Wearable Tracking System Based on a Low-Power Wide-Area Network

PubMed Central

Fernández-Garcia, Raul; Gil, Ignacio

2017-01-01

This work presents an alternative wearable tracking system based on a low-power wide area network. A complete GPS receiver was integrated with a textile substrate, and the latitude and longitude coordinates were sent to the cloud by means of the SIM-less SIGFOX network. To send the coordinates over SIGFOX protocol, a specific codification algorithm was used and a customized UHF antenna on jeans fabric was designed, simulated and tested. Moreover, to guarantee the compliance to international regulations for human body exposure to electromagnetic radiation, the electromagnetic specific absorption rate of this antenna was analyzed. A specific remote server was developed to decode the latitude and longitude coordinates. Once the coordinates have been decoded, the remote server sends this information to the open source data viewer SENTILO to show the location of the sensor node in a map. The functionality of this system has been demonstrated experimentally. The results guarantee the utility and wearability of the proposed tracking system for the development of sensor nodes and point out that it can be a low cost alternative to other commercial products based on GSM networks. PMID:28335424

Salt-Driven Deposition of Thermoresponsive Polymer-Coated Metal Nanoparticles on Solid Substrates.

PubMed

Zhang, Zhiyue; Maji, Samarendra; da Fonseca Antunes, André B; De Rycke, Riet; Hoogenboom, Richard; De Geest, Bruno G

2016-06-13

Here we report on a simple, generally applicable method for depositing metal nanoparticles on a wide variety of solid surfaces under all aqueous conditions. Noble-metal nanoparticles obtained by citrate reduction followed by coating with thermoresponsive polymers spontaneously form a monolayer-like structure on a wide variety of substrates in presence of sodium chloride whereas this phenomenon does not occur in salt-free medium. Interestingly, this phenomenon occurs below the cloud point temperature of the polymers and we hypothesize that salt ion-induced screening of electrostatic charges on the nanoparticle surface entropically favors hydrophobic association between the polymer-coated nanoparticles and a hydrophobic substrate. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Effect of mechanical and electrical stimuli in conductive atomic force microscopy with noble metal-coated tips

NASA Astrophysics Data System (ADS)

Zade, Vishal; Kang, Hung-Sen; Lee, Min Hwan

2018-01-01

Conductive atomic force microscopy has been widely employed to study the localized electrical properties of a wide range of substrates in non-vacuum conditions by the use of noble metal-coated tips. However, quantitative characterization of the electrical properties was often precluded by unpredictable changes in the tip apex morphology, and/or electronic transport characteristics of undesired oxide overcoats on the tip. In this paper, the impact of mechanical and electrical stimuli on the apex geometry of gold coated tips and electrical conduction properties at the tip-substrate contact is discussed by choosing gold and highly ordered pyrolytic graphite as the representative tip and substrate materials, respectively.

The core microprocessor component DiGeorge syndrome critical region 8 (DGCR8) is a nonspecific RNA-binding protein.

PubMed

Roth, Braden M; Ishimaru, Daniella; Hennig, Mirko

2013-09-13

MicroRNA (miRNA) biogenesis follows a conserved succession of processing steps, beginning with the recognition and liberation of an miRNA-containing precursor miRNA hairpin from a large primary miRNA transcript (pri-miRNA) by the Microprocessor, which consists of the nuclear RNase III Drosha and the double-stranded RNA-binding domain protein DGCR8 (DiGeorge syndrome critical region protein 8). Current models suggest that specific recognition is driven by DGCR8 detection of single-stranded elements of the pri-miRNA stem-loop followed by Drosha recruitment and pri-miRNA cleavage. Because countless RNA transcripts feature single-stranded-dsRNA junctions and DGCR8 can bind hundreds of mRNAs, we explored correlations between RNA binding properties of DGCR8 and specific pri-miRNA substrate processing. We found that DGCR8 bound single-stranded, double-stranded, and random hairpin transcripts with similar affinity. Further investigation of DGCR8/pri-mir-16 interactions by NMR detected intermediate exchange regimes over a wide range of stoichiometric ratios. Diffusion analysis of DGCR8/pri-mir-16 interactions by pulsed field gradient NMR lent further support to dynamic complex formation involving free components in exchange with complexes of varying stoichiometry, although in vitro processing assays showed exclusive cleavage of pri-mir-16 variants bearing single-stranded flanking regions. Our results indicate that DGCR8 binds RNA nonspecifically. Therefore, a sequential model of DGCR8 recognition followed by Drosha recruitment is unlikely. Known RNA substrate requirements are broad and include 70-nucleotide hairpins with unpaired flanking regions. Thus, specific RNA processing is likely facilitated by preformed DGCR8-Drosha heterodimers that can discriminate between authentic substrates and other hairpins.

The Core Microprocessor Component DiGeorge Syndrome Critical Region 8 (DGCR8) Is a Nonspecific RNA-binding Protein*

PubMed Central

Roth, Braden M.; Ishimaru, Daniella; Hennig, Mirko

2013-01-01

MicroRNA (miRNA) biogenesis follows a conserved succession of processing steps, beginning with the recognition and liberation of an miRNA-containing precursor miRNA hairpin from a large primary miRNA transcript (pri-miRNA) by the Microprocessor, which consists of the nuclear RNase III Drosha and the double-stranded RNA-binding domain protein DGCR8 (DiGeorge syndrome critical region protein 8). Current models suggest that specific recognition is driven by DGCR8 detection of single-stranded elements of the pri-miRNA stem-loop followed by Drosha recruitment and pri-miRNA cleavage. Because countless RNA transcripts feature single-stranded-dsRNA junctions and DGCR8 can bind hundreds of mRNAs, we explored correlations between RNA binding properties of DGCR8 and specific pri-miRNA substrate processing. We found that DGCR8 bound single-stranded, double-stranded, and random hairpin transcripts with similar affinity. Further investigation of DGCR8/pri-mir-16 interactions by NMR detected intermediate exchange regimes over a wide range of stoichiometric ratios. Diffusion analysis of DGCR8/pri-mir-16 interactions by pulsed field gradient NMR lent further support to dynamic complex formation involving free components in exchange with complexes of varying stoichiometry, although in vitro processing assays showed exclusive cleavage of pri-mir-16 variants bearing single-stranded flanking regions. Our results indicate that DGCR8 binds RNA nonspecifically. Therefore, a sequential model of DGCR8 recognition followed by Drosha recruitment is unlikely. Known RNA substrate requirements are broad and include 70-nucleotide hairpins with unpaired flanking regions. Thus, specific RNA processing is likely facilitated by preformed DGCR8-Drosha heterodimers that can discriminate between authentic substrates and other hairpins. PMID:23893406

Calorie restriction hysteretically primes aging Saccharomyces cerevisiae toward more effective oxidative metabolism.

PubMed

Tahara, Erich B; Cunha, Fernanda M; Basso, Thiago O; Della Bianca, Bianca E; Gombert, Andreas K; Kowaltowski, Alicia J

2013-01-01

Calorie restriction (CR) is an intervention known to extend the lifespan of a wide variety of organisms. In S. cerevisiae, chronological lifespan is prolonged by decreasing glucose availability in the culture media, a model for CR. The mechanism has been proposed to involve an increase in the oxidative (versus fermentative) metabolism of glucose. Here, we measured wild-type and respiratory incompetent (ρ(0)) S. cerevisiae biomass formation, pH, oxygen and glucose consumption, and the evolution of ethanol, glycerol, acetate, pyruvate and succinate levels during the course of 28 days of chronological aging, aiming to identify metabolic changes responsible for the effects of CR. The concomitant and quantitative measurements allowed for calculations of conversion factors between different pairs of substrates and products, maximum specific substrate consumption and product formation rates and maximum specific growth rates. Interestingly, we found that the limitation of glucose availability in CR S. cerevisiae cultures hysteretically increases oxygen consumption rates many hours after the complete exhaustion of glucose from the media. Surprisingly, glucose-to-ethanol conversion and cellular growth supported by glucose were not quantitatively altered by CR. Instead, we found that CR primed the cells for earlier, faster and more efficient metabolism of respiratory substrates, especially ethanol. Since lifespan-enhancing effects of CR are absent in respiratory incompetent ρ(0) cells, we propose that the hysteretic effect of glucose limitation on oxidative metabolism is central toward chronological lifespan extension by CR in this yeast.

Extensive peptide and natural protein substrate screens reveal that mouse caspase-11 has much narrower substrate specificity than caspase-1.

PubMed

Ramirez, Monica L Gonzalez; Poreba, Marcin; Snipas, Scott J; Groborz, Katarzyna; Drag, Marcin; Salvesen, Guy S

2018-05-04

Inflammatory cell death, or pyroptosis, is triggered by pathogenic infections or events. It is executed by caspase-1 (in the canonical pyroptosis pathway) or caspase-11 (noncanonical pathway), each via production of a cell-lytic domain from the pyroptosis effector protein gasdermin D through specific and limited proteolysis. Pyroptosis is accompanied by the release of inflammatory mediators, including the proteolytically processed forms of interleukin-1β (IL-1β) and IL-18. Given the similar inflammatory outcomes of the canonical and noncanonical pyroptosis pathways, we hypothesized that caspase-1 and -11 should have very similar activities and substrate specificities. To test this hypothesis, we purified recombinant murine caspases and analyzed their primary specificities by massive hybrid combinatorial substrate library (HyCoSuL) screens. We correlated the substrate preferences of each caspase with their activities on the recombinant natural substrates IL-1β, IL-18, and gasdermin D. Although we identified highly selective and robust peptidyl substrates for caspase-1, we were unable to do so for caspase-11, because caspase-1 cleaved even the best caspase-11 substrates equally well. Caspase-1 rapidly processed pro-IL-1β and -18, but caspase-11 processed these two pro-ILs extremely poorly. However, both caspase-1 and -11 efficiently produced the cell-lytic domain from the gasdermin D precursor. We hypothesize that caspase-11 may have evolved a specific exosite to selectively engage pyroptosis without directly activating pro-IL-1β or -18. In summary, comparing the activities of caspase-1 and -11 in HyCoSuL screens and with three endogenous protein substrates, we conclude that caspase-11 has highly restricted substrate specificity, preferring gasdermin D over all other substrates examined. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

A Xylenol Orange-Based Screening Assay for the Substrate Specificity of Flavin-Dependent para-Phenol Oxidases.

PubMed

Ewing, Tom A; van Noord, Aster; Paul, Caroline E; van Berkel, Willem J H

2018-01-14

Vanillyl alcohol oxidase (VAO) and eugenol oxidase (EUGO) are flavin-dependent enzymes that catalyse the oxidation of para -substituted phenols. This makes them potentially interesting biocatalysts for the conversion of lignin-derived aromatic monomers to value-added compounds. To facilitate their biocatalytic exploitation, it is important to develop methods by which variants of the enzymes can be rapidly screened for increased activity towards substrates of interest. Here, we present the development of a screening assay for the substrate specificity of para -phenol oxidases based on the detection of hydrogen peroxide using the ferric-xylenol orange complex method. The assay was used to screen the activity of VAO and EUGO towards a set of twenty-four potential substrates. This led to the identification of 4-cyclopentylphenol as a new substrate of VAO and EUGO and 4-cyclohexylphenol as a new substrate of VAO. Screening of a small library of VAO and EUGO active-site variants for alterations in their substrate specificity led to the identification of a VAO variant (T457Q) with increased activity towards vanillyl alcohol (4-hydroxy-3-methoxybenzyl alcohol) and a EUGO variant (V436I) with increased activity towards chavicol (4-allylphenol) and 4-cyclopentylphenol. This assay provides a quick and efficient method to screen the substrate specificity of para -phenol oxidases, facilitating the enzyme engineering of known para- phenol oxidases and the evaluation of the substrate specificity of novel para -phenol oxidases.

Highly sensitive and adaptable fluorescence-quenched pair discloses the substrate specificity profiles in diverse protease families

PubMed Central

Poreba, Marcin; Szalek, Aleksandra; Rut, Wioletta; Kasperkiewicz, Paulina; Rutkowska-Wlodarczyk, Izabela; Snipas, Scott J.; Itoh, Yoshifumi; Turk, Dusan; Turk, Boris; Overall, Christopher M.; Kaczmarek, Leszek; Salvesen, Guy S.; Drag, Marcin

2017-01-01

Internally quenched fluorescent (IQF) peptide substrates originating from FRET (Förster Resonance Energy Transfer) are powerful tool for examining the activity and specificity of proteases, and a variety of donor/acceptor pairs are extensively used to design individual substrates and combinatorial libraries. We developed a highly sensitive and adaptable donor/acceptor pair that can be used to investigate the substrate specificity of cysteine proteases, serine proteases and metalloproteinases. This novel pair comprises 7-amino-4-carbamoylmethylcoumarin (ACC) as the fluorophore and 2,4-dinitrophenyl-lysine (Lys(DNP)) as the quencher. Using caspase-3, caspase-7, caspase-8, neutrophil elastase, legumain, and two matrix metalloproteinases (MMP2 and MMP9), we demonstrated that substrates containing ACC/Lys(DNP) exhibit 7 to 10 times higher sensitivity than conventional 7-methoxy-coumarin-4-yl acetic acid (MCA)/Lys(DNP) substrates; thus, substantially lower amounts of substrate and enzyme can be used for each assay. We therefore propose that the ACC/Lys(DNP) pair can be considered a novel and sensitive scaffold for designing substrates for any group of endopeptidases. We further demonstrate that IQF substrates containing unnatural amino acids can be used to investigate protease activities/specificities for peptides containing post-translationally modified amino acids. Finally, we used IQF substrates to re-investigate the P1-Asp characteristic of caspases, thus demonstrating that some human caspases can also hydrolyze substrates after glutamic acid. PMID:28230157

Gallium phosphide nanowires as a substrate for cultured neurons.

PubMed

Hällström, Waldemar; Mårtensson, Thomas; Prinz, Christelle; Gustavsson, Per; Montelius, Lars; Samuelson, Lars; Kanje, Martin

2007-10-01

Dissociated sensory neurons were cultured on epitaxial gallium phosphide (GaP) nanowires grown vertically from a gallium phosphide surface. Substrates covered by 2.5 microm long, 50 nm wide nanowires supported cell adhesion and axonal outgrowth. Cell survival was better on nanowire substrates than on planar control substrates. The cells interacted closely with the nanostructures, and cells penetrated by hundreds of wires were observed as well as wire bending due to forces exerted by the cells.

High voltage photoconductive switch package

DOE Office of Scientific and Technical Information (OSTI.GOV)

Caporaso, George J.

2016-11-22

A photoconductive switch having a wide bandgap material substrate between opposing electrodes, and a doped dielectric filler that is in contact with both the electrodes and the substrate at the triple point. The dielectric filler material is doped with a conductive material to make it partially or completely conducting, to minimize the field enhancement near the triple point both when the substrate is not conducting in the "off" state and when the substrate is rendered conducting by radiation in the "on" state.

N-mustard analogs of S-adenosyl-L-methionine as biochemical probes of protein arginine methylation.

PubMed

Hymbaugh Bergman, Sarah J; Comstock, Lindsay R

2015-08-01

Nucleosomes, the fundamental building blocks of eukaryotic chromatin, undergo post-synthetic modifications and play a major role in the regulation of transcriptional processes. Combinations of these modifications, including methylation, regulate chromatin structure, determining its different functional states and playing a central role in differentiation. The biological significance of cellular methylation, particularly on chromatin, is widely recognized, yet we know little about the mechanisms that link biological methylation events. To characterize and fully understand protein methylation, we describe here novel N-mustard analogs of S-adenosyl-l-methionine (SAM) as biochemical tools to better understand protein arginine methylation events using protein arginine methyltransferase 1 (PRMT1). Specifically, azide- and alkyne-functionalized N-mustard analogs serve as cofactor mimics of SAM and are enzymatically transferred to a model peptide substrate in a PRMT1-dependent fashion. Once incorporated, the resulting alkynes and azides can be modified through chemoselective ligations, including click chemistry and the Staudinger ligation. These results readily demonstrate the feasibility of utilizing N-mustard analogs as biochemical tools to site-specifically label substrates of PRMT1 and serve as an alternative approach to study protein methylation events. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Subdigital setae of chameleon feet: friction-enhancing microstructures for a wide range of substrate roughness.

PubMed

Spinner, Marlene; Westhoff, Guido; Gorb, Stanislav N

2014-06-27

Hairy adhesive systems of microscopic setae with triangular flattened tips have evolved convergently in spiders, insects and arboreal lizards. The ventral sides of the feet and tails in chameleons are also covered with setae. However, chameleon setae feature strongly elongated narrow spatulae or fibrous tips. The friction enhancing function of these microstructures has so far only been demonstrated in contact with glass spheres. In the present study, the frictional properties of subdigital setae of Chamaeleo calyptratus were measured under normal forces in the physical range on plane substrates having different roughness. We showed that chameleon setae maximize friction on a wide range of substrate roughness. The highest friction was measured on asperities of 1 μm. However, our observations of the climbing ability of Ch. calyptratus on rods of different diameters revealed that also claws and grasping feet are additionally responsible for the force generation on various substrates during locomotion.

Subdigital setae of chameleon feet: Friction-enhancing microstructures for a wide range of substrate roughness

NASA Astrophysics Data System (ADS)

Spinner, Marlene; Westhoff, Guido; Gorb, Stanislav N.

2014-06-01

Hairy adhesive systems of microscopic setae with triangular flattened tips have evolved convergently in spiders, insects and arboreal lizards. The ventral sides of the feet and tails in chameleons are also covered with setae. However, chameleon setae feature strongly elongated narrow spatulae or fibrous tips. The friction enhancing function of these microstructures has so far only been demonstrated in contact with glass spheres. In the present study, the frictional properties of subdigital setae of Chamaeleo calyptratus were measured under normal forces in the physical range on plane substrates having different roughness. We showed that chameleon setae maximize friction on a wide range of substrate roughness. The highest friction was measured on asperities of 1 μm. However, our observations of the climbing ability of Ch. calyptratus on rods of different diameters revealed that also claws and grasping feet are additionally responsible for the force generation on various substrates during locomotion.

Surface Pre-treatment for Thermally Sprayed ZnAl15 Coatings

NASA Astrophysics Data System (ADS)

Bobzin, K.; Öte, M.; Knoch, M. A.

2017-02-01

Pre-treatment of substrates is an important step in thermal spraying. It is widely accepted that mechanical interlocking is the dominant adhesion mechanism for most substrate-coating combinations. To prevent premature failure, minimum coating adhesion strength, surface preparation grades, and roughness parameters are often specified. For corrosion-protection coatings for offshore wind turbines, an adhesion strength ≥ 5 MPa is commonly assumed to ensure adhesion over service lifetime. In order to fulfill this requirement, Rz > 80 µm and a preparation grade of Sa3 are common specifications. In this study, the necessity of these requirements is investigated using the widely used combination of twin-wire arc-sprayed ZnAl15 on S355J2 + N as a test case. By using different blasting media and parameters, the correlation between coating adhesion and roughness parameters is analyzed. The adhesion strength of these systems is measured using a test method allowing measurements on real parts. The results are compared to DIN EN 582:1993, the European equivalent of ASTM-C633. In another series of experiments, the influence of surface pre-treatment grades Sa2.5 and Sa3 is considered. By combining the results of these three sets of experiments, a guideline for surface pre-treatment and adhesion testing on real parts is proposed for the considered system.

Transversely-illuminated high current photoconductive switches with geometry-constrained conductivity path

DOEpatents

Nelson, Scott D.

2016-05-10

A photoconductive switch having a wide bandgap semiconductor material substrate between opposing electrodes, with one of the electrodes having an aperture or apertures at an electrode-substrate interface for transversely directing radiation therethrough from a radiation source into a triple junction region of the substrate, so as to geometrically constrain the conductivity path to within the triple junction region.

Specificity profiling of protein-binding domains using one-bead-one-compound Peptide libraries.

PubMed

Kunys, Andrew R; Lian, Wenlong; Pei, Dehua

2012-12-01

One-bead-one-compound (OBOC) libraries consist of structurally related compounds (e.g., peptides) covalently attached to a solid support, with each resin bead carrying a unique compound. OBOC libraries of high structural diversity can be rapidly synthesized and screened without the need for any special equipment, and therefore can be employed in any chemical or biochemical laboratory. OBOC peptide libraries have been widely used to map the ligand specificity of proteins, to determine the substrate specificity of enzymes, and to develop inhibitors against macromolecular targets. They have proven particularly useful in profiling the binding specificity of protein modular domains (e.g., SH2 domains, BIR domains, and PDZ domains); subsequently, the specificity information can be used to predict the protein targets of these domains. The protocols outlined in this article describe the methodologies for synthesizing and screening OBOC peptide libraries against SH2 and PDZ domains, and the related data analysis. Curr. Protoc. Chem. Biol. 4:331-355 © 2012 by John Wiley & Sons, Inc.

Understanding transporter specificity and the discrete appearance of channel-like gating domains in transporters

PubMed Central

Diallinas, George

2014-01-01

Transporters are ubiquitous proteins mediating the translocation of solutes across cell membranes, a biological process involved in nutrition, signaling, neurotransmission, cell communication and drug uptake or efflux. Similarly to enzymes, most transporters have a single substrate binding-site and thus their activity follows Michaelis-Menten kinetics. Substrate binding elicits a series of structural changes, which produce a transporter conformer open toward the side opposite to the one from where the substrate was originally bound. This mechanism, involving alternate outward- and inward-facing transporter conformers, has gained significant support from structural, genetic, biochemical and biophysical approaches. Most transporters are specific for a given substrate or a group of substrates with similar chemical structure, but substrate specificity and/or affinity can vary dramatically, even among members of a transporter family that show high overall amino acid sequence and structural similarity. The current view is that transporter substrate affinity or specificity is determined by a small number of interactions a given solute can make within a specific binding site. However, genetic, biochemical and in silico modeling studies with the purine transporter UapA of the filamentous ascomycete Aspergillus nidulans have challenged this dogma. This review highlights results leading to a novel concept, stating that substrate specificity, but also transport kinetics and transporter turnover, are determined by subtle intramolecular interactions between a major substrate binding site and independent outward- or cytoplasmically-facing gating domains, analogous to those present in channels. This concept is supported by recent structural evidence from several, phylogenetically and functionally distinct transporter families. The significance of this concept is discussed in relationship to the role and potential exploitation of transporters in drug action. PMID:25309439

Controlled Spalling in (100)-Oriented Germanium by Electroplating

NASA Astrophysics Data System (ADS)

Crouse, Dustin Ray

This work investigates controlled spalling as a method to exfoliate thin films of various thickness from rigid, crystalline germanium (Ge) substrates and to enable substrate reuse for III-V single junction photovoltaic devices. Technological limitations impeding wide-spread adoption of flexible electronics and high-material-cost photovoltaic devices have motivated significant interest in a method to remove devices from their substrates. DC magnetron sputtering has been previously utilized to remove semiconductor devices of various thicknesses from Ge substrates, but this method is expensive and time-consuming. Controlled spalling via high-speed electrodeposition is a fast, inexpensive exfoliation method that utilizes a tensile-stressed metal layer deposited on a (100)-oriented Ge substrate and an external force to mechanically propagate a crack parallel to the surface at a desired depth in the substrate material. Suo and Hutchinson's quantitative models describe critical combinations of film thickness and strain mismatch between a film and substrate at which a stressed bilayer system spontaneously spalls; however, fine control over a wide steady-state spall depth range has been limited by the ability to experimentally tailor strain mismatch caused by residual stress within deposited stressor layers. This work investigates the effect of tuning electroplating current density and electrolyte chemistry on the residual stress in a nickel stressor film and their impact on the achievable spall depth range. Steady-state spall depth is found to increase with increasing stressor layer thickness and decrease with increasing residual stress. By tailoring residual stress through adjusting plating conditions and the electrolyte's phosphorous concentration, wide control over spall depth within Ge substrates from sub-micron to 76microm-thicknesses were achieved. To assess the viability of utilizing controlled spalling for substrate reuse, this dissertation demonstrates the first III-V solar cells (GaInAsP, Eg 1.7 eV) grown directly on a spalled-Ge substrate without any additional surface preparation. Widespread adoption of high-efficiency III-V solar cells has been limited by expensive deposition processes and high material cost of substrates. Substrate reuse offers a promising route towards enabling III-V devices to become cost-competitive for one-sun terrestrial applications. In this study, the quality of spalled Ge surfaces is characterized to assess lattice matching capability between the device layer materials and the substrate. GaAs films grown on spalled Ge substrates by hydride vapor phase epitaxy were single-crystal in nature. III-V solar cells grown on spalled and pristine Ge substrates show nearly equivalent efficiency of 8%, despite the roughness of the spalled-Ge substrate. Principles of fractography were used to deduce that surface roughness originated from non-uniform crack propagation and mixed-mode loading during the spalling process.

Durable anti-fogging effect and adhesion improvement on polymer surfaces

NASA Astrophysics Data System (ADS)

Moser, E. M.; Gilliéron, D.; Henrion, G.

2010-01-01

The hydrophobic properties of polymeric surfaces may cause fogging in transparent packaging and poor adhesion to printing colours and coatings. Novel plasma processes for durable functionalization of polypropylene and polyethylene terephthalate substrates were developed and analysed using optical emission spectroscopy. A worm-like nano pattern was created on the polypropylene surface prior to the deposition of thin polar plasma polymerised layers. For both substrates, highly polar surfaces exhibiting a surface tension of up to 69 mN/m and a water contact angle of about 10° were produced - providing the anti-fogging effect. The deposition of thin plasma polymerised layers protects the increased surface areas and enables to tailoring the surface energy of the substrate in a wide range. Wetting characteristics were determined by dynamic contact angle measurements. Investigations of the chemical composition of several layers using X-ray photoelectron spectroscopy and FT-infrared spectroscopy were correlated with functional testing. The surface topography was investigated using atomic force microscopy. The weldability and peeling-off characteristics of the plasma treated polymer films could be adjusted by varying the process parameters. Global and specific migration analyses were undertaken in order to ensure the manufacturing of plasma treated polymer surfaces for direct food contact purposes.

Production and characterization of glucoamylase from fungus Aspergillus awamori expressed in yeast Saccharomyces cerevisiae using different carbon sources

PubMed Central

Pavezzi, Fabiana Carina; Gomes, Eleni; da Silva, Roberto

2008-01-01

Glucoamylase is widely used in the food industry to produce high glucose syrup, and also in fermentation processes for production beer and ethanol. In this work the productivity of the glucoamylase of Aspergillus awamori expressed by the yeast Saccharomyces cerevisiae, produced in submerged fermentation using different starches, was evaluated and characterized physico-chemically. The enzyme presented high specific activity, 13.8 U/mgprotein or 2.9 U/mgbiomass, after 48 h of fermentation using soluble starch as substrate. Glucoamylase presented optimum activity at temperature of 55°C, and, in the substratum absence, the thermostability was for 1h at 50°C. The optimum pH of activity was pH 3.5 - 4.0 and the pH stability between 5.0 and 7.0. The half life at 65°C was at 30.2 min, and the thermal energy of denaturation was 234.3 KJ mol-1. The hydrolysis of different substrate showed the enzyme’s preference for the substrate with a larger polymerization degree. The gelatinized corn starch was the substratum most susceptible to the enzymatic action. PMID:24031189

Is Oxidized Thioredoxin a Major Trigger for Cysteine Oxidation? Clues from a Redox Proteomics Approach

PubMed Central

García-Santamarina, Sarela; Boronat, Susanna; Calvo, Isabel A.; Rodríguez-Gabriel, Miguel; Ayté, José; Molina, Henrik

2013-01-01

Abstract Cysteine oxidation mediates oxidative stress toxicity and signaling. It has been long proposed that the thioredoxin (Trx) system, which consists of Trx and thioredoxin reductase (Trr), is not only involved in recycling classical Trx substrates, such as ribonucleotide reductase, but it also regulates general cytoplasmic thiol homeostasis. To investigate such a role, we have performed a proteome-wide analysis of cells expressing or not the two components of the Trx system. We have compared the reversibly oxidized thiol proteomes of wild-type Schizosaccharomyces pombe cells with mutants lacking Trx or Trr. Specific Trx substrates are reversibly-oxidized in both strain backgrounds; however, in the absence of Trr, Trx can weakly recycle its substrates at the expense of an alternative electron donor. A massive thiol oxidation occurs only in cells lacking Trr, with 30% of all cysteine-containing peptides being reversibly oxidized; this oxidized cysteine proteome depends on the presence of Trxs. Our observations lead to the hypothesis that, in the absence of its reductase, the natural electron donor Trx becomes a powerful oxidant and triggers general thiol oxidation. Antioxid. Redox Signal. 18, 1549–1556. PMID:23121505

High-throughput sequencing reveals circular substrates for an archaeal RNA ligase

PubMed Central

Becker, Hubert F.; Héliou, Alice; Djaout, Kamel; Lestini, Roxane; Regnier, Mireille; Myllykallio, Hannu

2017-01-01

ABSTRACT It is only recently that the abundant presence of circular RNAs (circRNAs) in all kingdoms of Life, including the hyperthermophilic archaeon Pyrococcus abyssi, has emerged. This led us to investigate the physiologic significance of a previously observed weak intramolecular ligation activity of Pab1020 RNA ligase. Here we demonstrate that this enzyme, despite sharing significant sequence similarity with DNA ligases, is indeed an RNA-specific polynucleotide ligase efficiently acting on physiologically significant substrates. Using a combination of RNA immunoprecipitation assays and RNA-seq, our genome-wide studies revealed 133 individual circRNA loci in P. abyssi. The large majority of these loci interacted with Pab1020 in cells and circularization of selected C/D Box and 5S rRNA transcripts was confirmed biochemically. Altogether these studies revealed that Pab1020 is required for RNA circularization. Our results further suggest the functional speciation of an ancestral NTase domain and/or DNA ligase toward RNA ligase activity and prompt for further characterization of the widespread functions of circular RNAs in prokaryotes. Detailed insight into the cellular substrates of Pab1020 may facilitate the development of new biotechnological applications e.g. in ligation of preadenylated adaptors to RNA molecules. PMID:28277897

The MAP kinase substrate MKS1 is a regulator of plant defense responses

PubMed Central

Andreasson, Erik; Jenkins, Thomas; Brodersen, Peter; Thorgrimsen, Stephan; Petersen, Nikolaj H T; Zhu, Shijiang; Qiu, Jin-Long; Micheelsen, Pernille; Rocher, Anne; Petersen, Morten; Newman, Mari-Anne; Bjørn Nielsen, Henrik; Hirt, Heribert; Somssich, Imre; Mattsson, Ole; Mundy, John

2005-01-01

Arabidopsis MAP kinase 4 (MPK4) functions as a regulator of pathogen defense responses, because it is required for both repression of salicylic acid (SA)-dependent resistance and for activation of jasmonate (JA)-dependent defense gene expression. To understand MPK4 signaling mechanisms, we used yeast two-hybrid screening to identify the MPK4 substrate MKS1. Analyses of transgenic plants and genome-wide transcript profiling indicated that MKS1 is required for full SA-dependent resistance in mpk4 mutants, and that overexpression of MKS1 in wild-type plants is sufficient to activate SA-dependent resistance, but does not interfere with induction of a defense gene by JA. Further yeast two-hybrid screening revealed that MKS1 interacts with the WRKY transcription factors WRKY25 and WRKY33. WRKY25 and WRKY33 were shown to be in vitro substrates of MPK4, and a wrky33 knockout mutant was found to exhibit increased expression of the SA-related defense gene PR1. MKS1 may therefore contribute to MPK4-regulated defense activation by coupling the kinase to specific WRKY transcription factors. PMID:15990873

High-resolution proton NMR studies of intracellular metabolites in yeast using 13C decoupling

NASA Astrophysics Data System (ADS)

Sillerud, Laurel O.; Alger, Jeffry R.; Shulman, Robert G.

The resolution and specificity of 1H NMR in studies of yeast cellular metabolism were increased by feeding a 13C-labeled substrate and observing 1H difference spectra in the presence and absence of 13C decoupling fields. [2- 13C]Acetate was utilized as a respiratory substrate in an aerobic suspension of Saccharomyces cerevisiae. The broad cellular background proton resonances are removed by the technique, leaving only signals from the protons of the substrate, or its metabolites, that are coupled to 13C. Spectra of the yeast suspension after acetate feeding show the disappearance of label from the acetate pool and the subsequent appearance of 13C in glutamate C 3 and C 4 and in aspartate C 3. These results are in accord with the known fluxes of metabolites. Selective single-frequency 13C decoupling was used to provide assignments for the difference signals. The limitations on single-frequency decoupling coming from finite decoupling fields are investigated. The technique shows a potential for application in a wide variety of systems where the resolution of the 13C spectrum may be combined with the sensitivity for proton detection to observe metabolites that have been previously unobservable.

A new buckwheat dihydroflavonol 4-reductase (DFR), with a unique substrate binding structure, has altered substrate specificity.

PubMed

Katsu, Kenjiro; Suzuki, Rintaro; Tsuchiya, Wataru; Inagaki, Noritoshi; Yamazaki, Toshimasa; Hisano, Tomomi; Yasui, Yasuo; Komori, Toshiyuki; Koshio, Motoyuki; Kubota, Seiji; Walker, Amanda R; Furukawa, Kiyoshi; Matsui, Katsuhiro

2017-12-11

Dihydroflavonol 4-reductase (DFR) is the key enzyme committed to anthocyanin and proanthocyanidin biosynthesis in the flavonoid biosynthetic pathway. DFR proteins can catalyse mainly the three substrates (dihydrokaempferol, dihydroquercetin, and dihydromyricetin), and show different substrate preferences. Although relationships between the substrate preference and amino acids in the region responsible for substrate specificity have been investigated in several plant species, the molecular basis of the substrate preference of DFR is not yet fully understood. By using degenerate primers in a PCR, we isolated two cDNA clones that encoded DFR in buckwheat (Fagopyrum esculentum). Based on sequence similarity, one cDNA clone (FeDFR1a) was identical to the FeDFR in DNA databases (DDBJ/Gen Bank/EMBL). The other cDNA clone, FeDFR2, had a similar sequence to FeDFR1a, but a different exon-intron structure. Linkage analysis in an F 2 segregating population showed that the two loci were linked. Unlike common DFR proteins in other plant species, FeDFR2 contained a valine instead of the typical asparagine at the third position and an extra glycine between sites 6 and 7 in the region that determines substrate specificity, and showed less activity against dihydrokaempferol than did FeDFR1a with an asparagine at the third position. Our 3D model suggested that the third residue and its neighbouring residues contribute to substrate specificity. FeDFR1a was expressed in all organs that we investigated, whereas FeDFR2 was preferentially expressed in roots and seeds. We isolated two buckwheat cDNA clones of DFR genes. FeDFR2 has unique structural and functional features that differ from those of previously reported DFRs in other plants. The 3D model suggested that not only the amino acid at the third position but also its neighbouring residues that are involved in the formation of the substrate-binding pocket play important roles in determining substrate preferences. The unique characteristics of FeDFR2 would provide a useful tool for future studies on the substrate specificity and organ-specific expression of DFRs.

Substrate- and isoform-specific proteome stability in normal and stressed cardiac mitochondria.

PubMed

Lau, Edward; Wang, Ding; Zhang, Jun; Yu, Hongxiu; Lam, Maggie P Y; Liang, Xiangbo; Zong, Nobel; Kim, Tae-Young; Ping, Peipei

2012-04-27

Mitochondrial protein homeostasis is an essential component of the functions and oxidative stress responses of the heart. To determine the specificity and efficiency of proteome turnover of the cardiac mitochondria by endogenous and exogenous proteolytic mechanisms. Proteolytic degradation of the murine cardiac mitochondria was assessed by 2-dimensional differential gel electrophoresis and liquid chromatography-tandem mass spectrometry. Mitochondrial proteases demonstrated a substrate preference for basic protein variants, which indicates a possible recognition mechanism based on protein modifications. Endogenous mitochondrial proteases and the cytosolic 20S proteasome exhibited different substrate specificities. The cardiac mitochondrial proteome contains low amounts of proteases and is remarkably stable in isolation. Oxidative damage lowers the proteolytic capacity of cardiac mitochondria and reduces substrate availability for mitochondrial proteases. The 20S proteasome preferentially degrades specific substrates in the mitochondria and may contribute to cardiac mitochondrial proteostasis.

Replacement of Antibodies in Pseudo-ELISAs: Molecularly Imprinted Nanoparticles for Vancomycin Detection.

PubMed

Canfarotta, Francesco; Smolinska-Kempisty, Katarzyna; Piletsky, Sergey

2017-01-01

The enzyme-linked immunosorbent assay (ELISA) is a widely employed analytical test used to quantify a given molecule. It relies on the use of specific antibodies, linked to an enzyme, to target the desired molecule. The reaction between the enzyme and its substrate gives rise to the analytical signal that can be quantified. Thanks to their robustness and low cost, molecularly imprinted polymer nanoparticles (nanoMIPs) are a viable alternative to antibodies. Herein, we describe the synthesis of nanoMIPs imprinted for vancomycin and their subsequent application in an ELISA-like format for direct replacement of antibodies.

Relative Expression Levels Rather Than Specific Activity Plays the Major Role in Determining In Vivo AKT Isoform Substrate Specificity

PubMed Central

Lee, Rachel S.; House, Colin M.; Cristiano, Briony E.; Hannan, Ross D.; Pearson, Richard B.; Hannan, Katherine M.

2011-01-01

The AKT protooncogene mediates many cellular processes involved in normal development and disease states such as cancer. The three structurally similar isoforms: AKT1, AKT2, and AKT3 exhibit both functional redundancy and isoform-specific functions; however the basis for their differential signalling remains unclear. Here we show that in vitro, purified AKT3 is ∼47-fold more active than AKT1 at phosphorylating peptide and protein substrates. Despite these marked variations in specific activity between the individual isoforms, a comprehensive analysis of phosphorylation of validated AKT substrates indicated only subtle differences in signalling via individual isoforms in vivo. Therefore, we hypothesise, at least in this model system, that relative tissue/cellular abundance, rather than specific activity, plays the dominant role in determining AKT substrate specificity in situ. PMID:21869924

Method of fabricating low-dislocation-density epitaxially-grown films with textured surfaces

DOEpatents

Li, Qiming; Wang, George T

2015-01-13

A method for forming a surface-textured single-crystal film layer by growing the film atop a layer of microparticles on a substrate and subsequently selectively etching away the microparticles to release the surface-textured single-crystal film layer from the substrate. This method is applicable to a very wide variety of substrates and films. In some embodiments, the film is an epitaxial film that has been grown in crystallographic alignment with respect to a crystalline substrate.

Methods of making monolayers

DOEpatents

Alford, Kentin L [Pasco, WA; Simmons, Kevin L [Kennewick, WA; Samuels, William D [Richland, WA; Zemanian, Thomas S [Richland, WA; Liu, Jun [Albuquerque, NM; Shin, Yongsoon [Richland, WA; Fryxell, Glen E [Kennewick, WA

2009-12-08

The invention pertains to methods of forming monolayers on various surfaces. The surfaces can be selected from a wide array of materials, including, for example, aluminum dioxide, silicon dioxide, carbon and SiC. The substrates can be planar or porous. The monolayer is formed under enhanced pressure conditions. The monolayer contains functionalized molecules, and accordingly functionalizes a surface of the substrate. The properties of the functionalized substrate can enhance the substrate's applicability for numerous purposes including, for example, utilization in extracting contaminants, or incorporation into a polymeric matrix.

Methods of making monolayers

DOEpatents

Alford, Kentin L [Pasco, WA; Simmons, Kevin L [Kennewick, WA; Samuels, William D [Richland, WA; Zemanian, Thomas S [Richland, WA; Liu, Jun [Albuquerque, NM; Shin, Yongsoon [Richland, WA; Fryxell, Glen E [Kennewick, WA

2009-09-15

The invention pertains to methods of forming monolayers on various surfaces. The surfaces can be selected from a wide array of materials, including, for example, aluminum dioxide, silicon dioxide, carbon and SiC. The substrates can be planar or porous. The monolayer is formed under enhanced pressure conditions. The monolayer contains functionalized molecules, and accordingly functionalizes a surface of the substrate. The properties of the functionalized substrate can enhance the substrate's applicability for numerous purposes including, for example, utilization in extracting contaminants, or incorporation into a polymeric matrix.

Involvement of arginine 878 together with Ca2+ in mouse aminopeptidase A substrate specificity for N-terminal acidic amino-acid residues

PubMed Central

Couvineau, Pierre; de Almeida, Hugo; Maigret, Bernard; Llorens-Cortes, Catherine

2017-01-01

Aminopeptidase A (APA) is a membrane-bound zinc metalloprotease cleaving, in the brain, the N-terminal aspartyl residue of angiotensin II to generate angiotensin III, which exerts a tonic stimulatory effect on the control of blood pressure in hypertensive animals. Using a refined APA structure derived from the human APA crystal structure, we docked the specific and selective APA inhibitor, EC33 in the presence of Ca2+. We report the presence in the S1 subsite of Arg-887 (Arg-878 in mouse APA), the guanidinium moiety of which established an interaction with the electronegative sulfonate group of EC33. Mutagenic replacement of Arg-878 with an alanine or a lysine residue decreased the affinity of the recombinant enzymes for the acidic substrate, α-L-glutamyl-β-naphthylamide, with a slight decrease in substrate hydrolysis velocity either with or without Ca2+. In the absence of Ca2+, the mutations modified the substrate specificity of APA for the acidic substrate, the mutated enzymes hydrolyzing more efficiently basic and neutral substrates, although the addition of Ca2+ partially restored the acidic substrate specificity. The analysis of the 3D models of the Arg-878 mutated APAs revealed a change in the volume of the S1 subsite, which may impair the binding and/or the optimal positioning of the substrate in the active site as well as its hydrolysis. These findings demonstrate the key role of Arg-878 together with Ca2 + in APA substrate specificity for N-terminal acidic amino acid residues by ensuring the optimal positioning of acidic substrates during catalysis. PMID:28877217

Wide-Field Fluorescence Microscopy of Real-Time Bioconjugation Sensing

PubMed Central

Szalkowski, Marcin; Sulowska, Karolina; Grzelak, Justyna; Niedziółka-Jönsson, Joanna; Roźniecka, Ewa

2018-01-01

We apply wide-field fluorescence microscopy to measure real-time attachment of photosynthetic proteins to plasmonically active silver nanowires. The observation of this effect is enabled, on the one hand, by sensitive detection of fluorescence and, on the other hand, by plasmonic enhancement of protein fluorescence. We examined two sample configurations with substrates being a bare glass coverslip and a coverslip functionalized with a monolayer of streptavidin. The different preparation of the substrate changes the observed behavior as far as attachment of the protein is concerned as well as its subsequent photobleaching. For the latter substrate the conjugation process is measurably slower. The described method can be universally applied in studying protein-nanostructure interactions for real-time fluorescence-based sensing. PMID:29351211

Wide-Field Fluorescence Microscopy of Real-Time Bioconjugation Sensing.

PubMed

Szalkowski, Marcin; Sulowska, Karolina; Grzelak, Justyna; Niedziółka-Jönsson, Joanna; Roźniecka, Ewa; Kowalska, Dorota; Maćkowski, Sebastian

2018-01-19

We apply wide-field fluorescence microscopy to measure real-time attachment of photosynthetic proteins to plasmonically active silver nanowires. The observation of this effect is enabled, on the one hand, by sensitive detection of fluorescence and, on the other hand, by plasmonic enhancement of protein fluorescence. We examined two sample configurations with substrates being a bare glass coverslip and a coverslip functionalized with a monolayer of streptavidin. The different preparation of the substrate changes the observed behavior as far as attachment of the protein is concerned as well as its subsequent photobleaching. For the latter substrate the conjugation process is measurably slower. The described method can be universally applied in studying protein-nanostructure interactions for real-time fluorescence-based sensing.

Evaluating Factor XIII Specificity for Glutamine-Containing Substrates Using a MALDI-TOF Mass Spectrometry Assay

PubMed Central

Doiphode, Prakash G.; Malovichko, Marina V.; Mouapi, Kelly Njine; Maurer, Muriel C.

2014-01-01

Activated Factor XIII (FXIIIa) catalyzes the formation of γ-glutamyl-ε-lysyl cross-links within the fibrin blood clot network. Although several cross-linking targets have been identified, the characteristic features that define FXIIIa substrate specificity are not well understood. To learn more about how FXIIIa selects its targets, a matrix-assisted laser desorption ionization – time of flight mass spectrometry (MALDI-TOF MS) based assay was developed that could directly follow the consumption of a glutamine-containing substrate and the formation of a cross-linked product with glycine ethylester. This FXIIIa kinetics assay is no longer reliant on a secondary coupled reaction, on substrate labeling, or on detecting the final deacylation portion of the transglutaminase reaction. With the MALDI-TOF MS assay, glutamine-containing peptides derived from α2-antiplasmin, S. Aureus fibronectin binding protein A, and thrombin activatable fibrinolysis inhibitor were examined directly. Results suggest that the FXIIIa active site surface responds to changes in substrate residues following the reactive glutamine. The P-1 substrate position is sensitive to charge character and the P-2 and P-3 to the broad FXIIIa substrate specificity pockets. The more distant P-8 to P-11 region serves as a secondary substrate anchoring point. New knowledge on FXIIIa specificity may be used to design better substrates or inhibitors of this transglutaminase. PMID:24751466

Structural Basis of Substrate Specificity and Regiochemistry in the MycF/TylF Family of Sugar O -Methyltransferases.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bernard, Steffen M.; Akey, David L.; Tripathi, Ashootosh

Sugar moieties in natural products are frequently modified by O-methylation. In the biosynthesis of the macrolide antibiotic mycinamicin, methylation of a 6'-deoxyallose substituent occurs in a stepwise manner first at the 2'- and then the 3'-hydroxyl groups to produce the mycinose moiety in the final product. The timing and placement of the O-methylations impact final stage C-H functionalization reactions mediated by the P450 monooxygenase MycG. The structural basis of pathway ordering and substrate specificity is unknown. A series of crystal structures of MycF, the 3'-O-methyltransferase, including the free enzyme and complexes with S-adenosyl homocysteine (SAH), substrate, product, and unnatural substrates,more » show that SAM binding induces substantial ordering that creates the binding site for the natural substrate, and a bound metal ion positions the substrate for catalysis. A single amino acid substitution relaxed the 2'-methoxy specificity but retained regiospecificity. The engineered variant produced a new mycinamicin analog, demonstrating the utility of structural information to facilitate bioengineering approaches for the chemoenzymatic synthesis of complex small molecules containing modified sugars. Using the MycF substrate complex and the modeled substrate complex of a 4'-specific homolog, active site residues were identified that correlate with the 3'- or 4'- specificity of MycF family members and define the protein and substrate features that direct the regiochemistry of methyltransfer. Lastly, this classification scheme will be useful in the annotation of new secondary metabolite pathways that utilize this family of enzymes.« less

Structural Basis of Substrate Specificity and Regiochemistry in the MycF/TylF Family of Sugar O -Methyltransferases.

DOE PAGES

Bernard, Steffen M.; Akey, David L.; Tripathi, Ashootosh; ...

2015-02-18

Sugar moieties in natural products are frequently modified by O-methylation. In the biosynthesis of the macrolide antibiotic mycinamicin, methylation of a 6'-deoxyallose substituent occurs in a stepwise manner first at the 2'- and then the 3'-hydroxyl groups to produce the mycinose moiety in the final product. The timing and placement of the O-methylations impact final stage C-H functionalization reactions mediated by the P450 monooxygenase MycG. The structural basis of pathway ordering and substrate specificity is unknown. A series of crystal structures of MycF, the 3'-O-methyltransferase, including the free enzyme and complexes with S-adenosyl homocysteine (SAH), substrate, product, and unnatural substrates,more » show that SAM binding induces substantial ordering that creates the binding site for the natural substrate, and a bound metal ion positions the substrate for catalysis. A single amino acid substitution relaxed the 2'-methoxy specificity but retained regiospecificity. The engineered variant produced a new mycinamicin analog, demonstrating the utility of structural information to facilitate bioengineering approaches for the chemoenzymatic synthesis of complex small molecules containing modified sugars. Using the MycF substrate complex and the modeled substrate complex of a 4'-specific homolog, active site residues were identified that correlate with the 3'- or 4'- specificity of MycF family members and define the protein and substrate features that direct the regiochemistry of methyltransfer. Lastly, this classification scheme will be useful in the annotation of new secondary metabolite pathways that utilize this family of enzymes.« less

Site-Specific Protein Labeling via Sortase-Mediated Transpeptidation

PubMed Central

Antos, John M.; Ingram, Jessica; Fang, Tao; Pishesha, Novalia; Truttmann, Matthias C.; Ploegh, Hidde L.

2017-01-01

Strategies for site-specific protein modification are highly desirable for the construction of conjugates containing non-genetically encoded functional groups. Ideally, these strategies should proceed under mild conditions, and be compatible with a wide range of protein targets and non-natural moieties. The transpeptidation reaction catalyzed by bacterial sortases is a prominent strategy for protein derivatization that possesses these features. Naturally occurring or engineered variants of sortase A from Staphylococcus aureus catalyze a ligation reaction between a five amino acid substrate motif (LPXTG) and oligoglycine nucleophiles. By pairing proteins and synthetic peptides that possess these ligation handles, it is possible to install modifications onto the protein N- or C-terminus in site-specific fashion. As described in this unit, the successful implementation of sortase-mediated labeling involves straightforward solid-phase synthesis and molecular biology techniques, and this method is compatible with proteins in solution or on the surface of live cells. PMID:19365788

Substrate-specific regulation of ubiquitination by the anaphase-promoting complex

PubMed Central

Song, Ling

2011-01-01

By orchestrating the sequential degradation of a large number of cell cycle regulators, the ubiquitin ligase anaphase-promoting complex (APC/C) is essential for proliferation in all eukaryotes. The correct timing of APC/C-dependent substrate degradation, a critical feature of progression through mitosis, was long known to be controlled by mechanisms targeting the core APC/C-machinery. Recent experiments, however have revealed an important contribution of substrate-specific regulation of the APC/C to achieve accurate cell division. In this perspective, we describe different mechanisms of substrate-specific APC/C-regulation and discuss their importance for cell division. PMID:21191176

Exploring the specific features of interfacial enzymology based on lipase studies.

PubMed

Aloulou, Ahmed; Rodriguez, Jorge A; Fernandez, Sylvie; van Oosterhout, Dirk; Puccinelli, Delphine; Carrière, Frédéric

2006-09-01

Many enzymes are active at interfaces in the living world (such as in the signaling processes at the surface of cell membranes, digestion of dietary lipids, starch and cellulose degradation, etc.), but fundamental enzymology remains largely focused on the interactions between enzymes and soluble substrates. The biochemical and kinetic characterization of lipolytic enzymes has opened up new paths of research in the field of interfacial enzymology. Lipases are water-soluble enzymes hydrolyzing insoluble triglyceride substrates, and studies on these enzymes have led to the development of specific interfacial kinetic models. Structure-function studies on lipases have thrown light on the interfacial recognition sites present in the molecular structure of these enzymes, the conformational changes occurring in the presence of lipids and amphiphiles, and the stability of the enzymes present at interfaces. The pH-dependent activity, substrate specificity and inhibition of these enzymes can all result from both "classical" interactions between a substrate or inhibitor and the active site, as well as from the adsorption of the enzymes at the surface of aggregated substrate particles such as oil drops, lipid bilayers or monomolecular lipid films. The adsorption step can provide an alternative target for improving substrate specificity and developing specific enzyme inhibitors. Several data obtained with gastric lipase, classical pancreatic lipase, pancreatic lipase-related protein 2 and phosphatidylserine-specific phospholipase A1 were chosen here to illustrate these specific features of interfacial enzymology.

Quantitative Comparison of Protein Adsorption and Conformational Changes on Dielectric-Coated Nanoplasmonic Sensing Arrays.

PubMed

Ferhan, Abdul Rahim; Jackman, Joshua A; Sut, Tun Naw; Cho, Nam-Joon

2018-04-22

Nanoplasmonic sensors are a popular, surface-sensitive measurement tool to investigate biomacromolecular interactions at solid-liquid interfaces, opening the door to a wide range of applications. In addition to high surface sensitivity, nanoplasmonic sensors have versatile surface chemistry options as plasmonic metal nanoparticles can be coated with thin dielectric layers. Within this scope, nanoplasmonic sensors have demonstrated promise for tracking protein adsorption and substrate-induced conformational changes on oxide film-coated arrays, although existing studies have been limited to single substrates. Herein, we investigated human serum albumin (HSA) adsorption onto silica- and titania-coated arrays of plasmonic gold nanodisks by localized surface plasmon resonance (LSPR) measurements and established an analytical framework to compare responses across multiple substrates with different sensitivities. While similar responses were recorded on the two substrates for HSA adsorption under physiologically-relevant ionic strength conditions, distinct substrate-specific behavior was observed at lower ionic strength conditions. With decreasing ionic strength, larger measurement responses occurred for HSA adsorption onto silica surfaces, whereas HSA adsorption onto titania surfaces occurred independently of ionic strength condition. Complementary quartz crystal microbalance-dissipation (QCM-D) measurements were also performed, and the trend in adsorption behavior was similar. Of note, the magnitudes of the ionic strength-dependent LSPR and QCM-D measurement responses varied, and are discussed with respect to the measurement principle and surface sensitivity of each technique. Taken together, our findings demonstrate how the high surface sensitivity of nanoplasmonic sensors can be applied to quantitatively characterize protein adsorption across multiple surfaces, and outline broadly-applicable measurement strategies for biointerfacial science applications.

Deciphering kinase-substrate relationships by analysis of domain-specific phosphorylation network.

PubMed

Damle, Nikhil Prakash; Mohanty, Debasisa

2014-06-15

In silico prediction of site-specific kinase-substrate relationships (ssKSRs) is crucial for deciphering phosphorylation networks by linking kinomes to phosphoproteomes. However, currently available predictors for ssKSRs give rise to a large number of false-positive results because they use only a short sequence stretch around phosphosite as determinants of kinase specificity and do not consider the biological context of kinase-substrate recognition. Based on the analysis of domain-specific kinase-substrate relationships, we have constructed a domain-level phosphorylation network that implicitly incorporates various contextual factors. It reveals preferential phosphorylation of specific domains by certain kinases. These novel correlations have been implemented in PhosNetConstruct, an automated program for predicting target kinases for a substrate protein. PhosNetConstruct distinguishes cognate kinase-substrate pairs from a large number of non-cognate combinations. Benchmarking on independent datasets using various statistical measures demonstrates the superior performance of PhosNetConstruct over ssKSR-based predictors. PhosNetConstruct is freely available at http://www.nii.ac.in/phosnetconstruct.html. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Substrate Specificity and Possible Heterologous Targets of Phytaspase, a Plant Cell Death Protease*

PubMed Central

Galiullina, Raisa A.; Kasperkiewicz, Paulina; Chichkova, Nina V.; Szalek, Aleksandra; Serebryakova, Marina V.; Poreba, Marcin; Drag, Marcin; Vartapetian, Andrey B.

2015-01-01

Plants lack aspartate-specific cell death proteases homologous to animal caspases. Instead, a subtilisin-like serine-dependent plant protease named phytaspase shown to be involved in the accomplishment of programmed death of plant cells is able to hydrolyze a number of peptide-based caspase substrates. Here, we determined the substrate specificity of rice (Oryza sativa) phytaspase by using the positional scanning substrate combinatorial library approach. Phytaspase was shown to display an absolute specificity of hydrolysis after an aspartic acid residue. The preceding amino acid residues, however, significantly influence the efficiency of hydrolysis. Efficient phytaspase substrates demonstrated a remarkable preference for an aromatic amino acid residue in the P3 position. The deduced optimum phytaspase recognition motif has the sequence IWLD and is strikingly hydrophobic. The established pattern was confirmed through synthesis and kinetic analysis of cleavage of a set of optimized peptide substrates. An amino acid motif similar to the phytaspase cleavage site is shared by the human gastrointestinal peptide hormones gastrin and cholecystokinin. In agreement with the established enzyme specificity, phytaspase was shown to hydrolyze gastrin-1 and cholecystokinin at the predicted sites in vitro, thus destroying the active moieties of the hormones. PMID:26283788

Substrate Sorting by a Supercharged Nanoreactor

PubMed Central

2017-01-01

Compartmentalization of proteases enables spatially and temporally controlled protein degradation in cells. Here we show that an engineered lumazine synthase protein cage, which possesses a negatively supercharged lumen, can exploit electrostatic effects to sort substrates for an encapsulated protease. This proteasome-like nanoreactor preferentially cleaves positively charged polypeptides over both anionic and zwitterionic substrates, inverting the inherent substrate specificity of the guest enzyme approximately 480 fold. Our results suggest that supercharged nanochambers could provide a simple and potentially general means of conferring substrate specificity to diverse encapsulated catalysts. PMID:29278496

Allosteric regulation of rhomboid intramembrane proteolysis.

PubMed

Arutyunova, Elena; Panwar, Pankaj; Skiba, Pauline M; Gale, Nicola; Mak, Michelle W; Lemieux, M Joanne

2014-09-01

Proteolysis within the lipid bilayer is poorly understood, in particular the regulation of substrate cleavage. Rhomboids are a family of ubiquitous intramembrane serine proteases that harbour a buried active site and are known to cleave transmembrane substrates with broad specificity. In vitro gel and Förster resonance energy transfer (FRET)-based kinetic assays were developed to analyse cleavage of the transmembrane substrate psTatA (TatA from Providencia stuartii). We demonstrate significant differences in catalytic efficiency (kcat/K0.5) values for transmembrane substrate psTatA (TatA from Providencia stuartii) cleavage for three rhomboids: AarA from P. stuartii, ecGlpG from Escherichia coli and hiGlpG from Haemophilus influenzae demonstrating that rhomboids specifically recognize this substrate. Furthermore, binding of psTatA occurs with positive cooperativity. Competitive binding studies reveal an exosite-mediated mode of substrate binding, indicating allostery plays a role in substrate catalysis. We reveal that exosite formation is dependent on the oligomeric state of rhomboids, and when dimers are dissociated, allosteric substrate activation is not observed. We present a novel mechanism for specific substrate cleavage involving several dynamic processes including positive cooperativity and homotropic allostery for this interesting class of intramembrane proteases. © 2014 The Authors.

Allosteric regulation of rhomboid intramembrane proteolysis

PubMed Central

Arutyunova, Elena; Panwar, Pankaj; Skiba, Pauline M; Gale, Nicola; Mak, Michelle W; Lemieux, M Joanne

2014-01-01

Proteolysis within the lipid bilayer is poorly understood, in particular the regulation of substrate cleavage. Rhomboids are a family of ubiquitous intramembrane serine proteases that harbour a buried active site and are known to cleave transmembrane substrates with broad specificity. In vitro gel and Förster resonance energy transfer (FRET)-based kinetic assays were developed to analyse cleavage of the transmembrane substrate psTatA (TatA from Providencia stuartii). We demonstrate significant differences in catalytic efficiency (kcat/K0.5) values for transmembrane substrate psTatA (TatA from Providencia stuartii) cleavage for three rhomboids: AarA from P. stuartii, ecGlpG from Escherichia coli and hiGlpG from Haemophilus influenzae demonstrating that rhomboids specifically recognize this substrate. Furthermore, binding of psTatA occurs with positive cooperativity. Competitive binding studies reveal an exosite-mediated mode of substrate binding, indicating allostery plays a role in substrate catalysis. We reveal that exosite formation is dependent on the oligomeric state of rhomboids, and when dimers are dissociated, allosteric substrate activation is not observed. We present a novel mechanism for specific substrate cleavage involving several dynamic processes including positive cooperativity and homotropic allostery for this interesting class of intramembrane proteases. PMID:25009246

Combined eye tracking and fMRI reveals neural basis of linguistic predictions during sentence comprehension.

PubMed

Bonhage, Corinna E; Mueller, Jutta L; Friederici, Angela D; Fiebach, Christian J

2015-07-01

It is widely agreed upon that linguistic predictions are an integral part of language comprehension. Yet, experimental proof of their existence remains challenging. Here, we introduce a new predictive eye gaze reading task combining eye tracking and functional magnetic resonance imaging (fMRI) that allows us to infer the existence and timing of linguistic predictions via anticipatory eye-movements. Participants read different types of word sequences (i.e., regular sentences, meaningless jabberwocky sentences, non-word lists) up to the pre-final word. The final target word was displayed with a temporal delay and its screen position was dependent on the syntactic word category (nouns vs verbs). During the delay, anticipatory eye-movements into the correct target word area were indicative of linguistic predictions. For fMRI analysis, the predictive sentence conditions were contrasted to the non-word condition, with the anticipatory eye-movements specifying differences in timing across conditions. A conjunction analysis of both sentence conditions revealed the neural substrate of word category prediction, namely a distributed network of cortical and subcortical brain regions including language systems, basal ganglia, thalamus, and hippocampus. Direct contrasts between the regular sentence condition and the jabberwocky condition indicate that prediction of word category in meaningless jabberwocky sentences relies on classical left-hemispheric language systems involving Brodman's area 44/45 in the left inferior frontal gyrus, left superior temporal areas, and the dorsal caudate nucleus. Regular sentences, in contrast, allowed for the prediction of specific words. Word-specific predictions were specifically associated with more widely distributed temporal and parietal cortical systems, most prominently in the right hemisphere. Our results support the presence of linguistic predictions during sentence processing and demonstrate the validity of the predictive eye gaze paradigm for measuring syntactic and semantic aspects of linguistic predictions, as well as for investigating their neural substrates. Copyright © 2015 Elsevier Ltd. All rights reserved.

LAND USE AND NATURAL HYDRAULIC CONTROLS ON STREAM SUBSTRATE AND MACROINVERTEBRATE ASSEMBLAGES IN REGIONAL SURVEYS

EPA Science Inventory

In large regions, human land uses typically overlay wide ranges of natural geomorphic factors that control stream habitat characteristics and benthic macroinvertebrate assemblages. Many macroinvertebrate measures of stream "health" show strong association with substrate size, a ...

Evolutionary dynamics of enzymes.

PubMed

Demetrius, L

1995-08-01

This paper codifies and rationalizes the large diversity in reaction rates and substrate specificity of enzymes in terms of a model which postulates that the kinetic properties of present-day enzymes are the consequence of the evolutionary force of mutation and selection acting on a class of primordial enzymes with poor catalytic activity and broad substrate specificity. Enzymes are classified in terms of their thermodynamic parameters, activation enthalpy delta H* and activation entropy delta S*, in their kinetically significant transition states as follows: type 1, delta H* > 0, delta S* < 0; type 2, delta H* < or = 0, delta S* < or = 0; type 3, delta H* > 0, delta S* > 0. We study the evolutionary dynamics of these three classes of enzymes subject to mutation, which acts at the level of the gene which codes for the enzyme and selection, which acts on the organism that contains the enzyme. Our model predicts the following evolutionary trends in the reaction rate and binding specificity for the three classes of molecules. In type 1 enzymes, evolution results in random, non-directional changes in the reaction rate and binding specificity. In type 2 and 3 enzymes, evolution results in a unidirectional increase in both the reaction rate and binding specificity. We exploit these results in order to codify the diversity in functional properties of present-day enzymes. Type 1 molecules will be described by intermediate reaction rates and broad substrate specificity. Type 2 enzymes will be characterized by diffusion-controlled rates and absolute substrate specificity. The type 3 catalysts can be further subdivided in terms of their activation enthalpy into two classes: type 3a (delta H* small) and type 3b (delta H* large). We show that type 3a will be represented by the same functional properties that identify type 2, namely, diffusion-controlled rates and absolute substrate specificity, whereas type 3b will be characterized by non-diffusion-controlled rates and absolute substrate specificity. We infer from this depiction of the three classes of enzymes, a general relation between the two functional properties, reaction rate and substrate specificity, namely, enzymes with diffusion-controlled rates have absolute substrate specificity. By appealing to energetic considerations, we furthermore show that enzymes with diffusion-controlled rates (types 2 and 3a) form a small subset of the class of all enzymes. This codification of present-day enzymes derived from an evolutionary model, essentially relates the structural properties of enzymes, as described by their thermodynamic parameters, to their functional properties, as represented by the reaction rate and substrate specificity.

Engineering the Substrate Specificity of the DhbE Adenylation Domain by Yeast Cell Surface Display

PubMed Central

Zhang, Keya; Nelson, Kathryn M.; Bhuripanyo, Karan; Grimes, Kimberly D.; Zhao, Bo; Aldrich, Courtney C.; Yin, Jun

2013-01-01

SUMMARY The adenylation (A) domains of nonribosomal peptide synthetases (NRPSs) activate aryl acids or amino acids to launch their transfer through the NRPS assembly line for the biosynthesis of many medicinally important natural products. In order to expand the substrate pool of NRPSs, we developed a method based on yeast cell surface display to engineer the substrate specificities of the A-domains. We acquired A-domain mutants of DhbE that have 11- and 6-fold increases in kcat/Km with nonnative substrates 3-hydroxybenzoic acid and 2-aminobenzoic acid, respectively and corresponding 3- and 33-fold decreases in kcat/Km values with the native substrate 2,3-dihydroxybenzoic acid, resulting in a dramatic switch in substrate specificity of up to 200-fold. Our study demonstrates that yeast display can be used as a high throughput selection platform to reprogram the “nonribosomal code” of A-domains. PMID:23352143

Simulation of an integrated system for the production of methane and single cell protein from biomass

DOE Office of Scientific and Technical Information (OSTI.GOV)

Thomas, M.V.

1989-01-01

A numerical model was developed to simulate the operation of an integrated system for the production of methane and single-cell algal protein from a variety of biomass energy crops or waste streams. Economic analysis was performed at the end of each simulation. The model was capable of assisting in the determination of design parameters by providing relative economic information for various strategies. Three configurations of anaerobic reactors were simulated. These included fed-bed reactors, conventional stirred tank reactors, and continuously expanding reactors. A generic anaerobic digestion process model, using lumped substrate parameters, was developed for use by type-specific reactor models. Themore » generic anaerobic digestion model provided a tool for the testing of conversion efficiencies and kinetic parameters for a wide range of substrate types and reactor designs. Dynamic growth models were used to model the growth of algae and Eichornia crassipes was modeled as a function of daily incident radiation and temperature. The growth of Eichornia crassipes was modeled for the production of biomass as a substrate for digestion. Computer simulations with the system model indicated that tropical or subtropical locations offered the most promise for a viable system. The availability of large quantities of digestible waste and low land prices were found to be desirable in order to take advantage of the economies of scale. Other simulations indicated that poultry and swine manure produced larger biogas yields than cattle manure. The model was created in a modular fashion to allow for testing of a wide variety of unit operations. Coding was performed in the Pascal language for use on personal computers.« less

Structural features of Aspergillus niger β-galactosidase define its activity against glycoside linkages.

PubMed

Rico-Díaz, Agustín; Ramírez-Escudero, Mercedes; Vizoso-Vázquez, Ángel; Cerdán, M Esperanza; Becerra, Manuel; Sanz-Aparicio, Julia

2017-06-01

β-Galactosidases are biotechnologically interesting enzymes that catalyze the hydrolysis or transgalactosylation of β-galactosides. Among them, the Aspergillus niger β-galactosidase (AnβGal) belongs to the glycoside hydrolase family 35 (GH35) and is widely used in the industry due to its high hydrolytic activity which is used to degrade lactose. We present here its three-dimensional structure in complex with different oligosaccharides, to illustrate the structural determinants of the broad specificity of the enzyme against different glycoside linkages. Remarkably, the residues Phe264, Tyr304, and Trp806 make a dynamic hydrophobic platform that accommodates the sugar at subsite +1 suggesting a main role on the recognition of structurally different substrates. Moreover, complexes with the trisaccharides show two potential subsites +2 depending on the substrate type. This feature and the peculiar shape of its wide cavity suggest that AnβGal might accommodate branched substrates from the complex net of polysaccharides composing the plant material in its natural environment. Relevant residues were selected and mutagenesis analyses were performed to evaluate their role in the catalytic performance and the hydrolase/transferase ratio of AnβGal. Thus, we generated mutants with improved transgalactosylation activity. In particular, the variant Y304F/Y355H/N357G/W806F displays a higher level of galacto-oligosaccharides production than the Aspergillus oryzae β-galactosidase, which is the preferred enzyme in the industry owing to its high transferase activity. Our results provide new knowledge on the determinants modulating specificity and the catalytic performance of fungal GH35 β-galactosidases. In turn, this fundamental background gives novel tools for the future improvement of these enzymes, which represent an interesting target for rational design. Structural data are available in PDB database under the accession numbers 5IFP (native form), 5IHR (in complex with 6GalGlu), 5IFT (in complex with 3GalGlu), 5JUV (in complex with 6GalGal), 5MGC (in complex with 4GalLac), and 5MGD (in complex with 6GalLac). © 2017 Federation of European Biochemical Societies.

Calcium-dependent protein kinases from Arabidopsis show substrate specificity differences in an analysis of 103 substrates.

PubMed

Curran, Amy; Chang, Ing-Feng; Chang, Chia-Lun; Garg, Shilpi; Miguel, Rodriguez Milla; Barron, Yoshimi D; Li, Ying; Romanowsky, Shawn; Cushman, John C; Gribskov, Michael; Harmon, Alice C; Harper, Jeffrey F

2011-01-01

The identification of substrates represents a critical challenge for understanding any protein kinase-based signal transduction pathway. In Arabidopsis, there are more than 1000 different protein kinases, 34 of which belong to a family of Ca(2+)-dependent protein kinases (CPKs). While CPKs are implicated in regulating diverse aspects of plant biology, from ion transport to transcription, relatively little is known about isoform-specific differences in substrate specificity, or the number of phosphorylation targets. Here, in vitro kinase assays were used to compare phosphorylation targets of four CPKs from Arabidopsis (CPK1, 10, 16, and 34). Significant differences in substrate specificity for each kinase were revealed by assays using 103 different substrates. For example CPK16 phosphorylated Serine 109 in a peptide from the stress-regulated protein, Di19-2 with K(M) ∼70 μM, but this site was not phosphorylated significantly by CPKs 1, 10, or 34. In contrast, CPKs 1, 10, and 34 phosphorylated 93 other peptide substrates not recognized by CPK16. Examples of substrate specificity differences among all four CPKs were verified by kinetic analyses. To test the correlation between in vivo phosphorylation events and in vitro kinase activities, assays were performed with 274 synthetic peptides that contained phosphorylation sites previously mapped in proteins isolated from plants (in vivo-mapped sites). Of these, 74 (27%) were found to be phosphorylated by at least one of the four CPKs tested. This 27% success rate validates a robust strategy for linking the activities of specific kinases, such as CPKs, to the thousands of in planta phosphorylation sites that are being uncovered by emerging technologies.

Calcium-Dependent Protein Kinases from Arabidopsis Show Substrate Specificity Differences in an Analysis of 103 Substrates

PubMed Central

Curran, Amy; Chang, Ing-Feng; Chang, Chia-Lun; Garg, Shilpi; Miguel, Rodriguez Milla; Barron, Yoshimi D.; Li, Ying; Romanowsky, Shawn; Cushman, John C.; Gribskov, Michael; Harmon, Alice C.; Harper, Jeffrey F.

2011-01-01

The identification of substrates represents a critical challenge for understanding any protein kinase-based signal transduction pathway. In Arabidopsis, there are more than 1000 different protein kinases, 34 of which belong to a family of Ca2+-dependent protein kinases (CPKs). While CPKs are implicated in regulating diverse aspects of plant biology, from ion transport to transcription, relatively little is known about isoform-specific differences in substrate specificity, or the number of phosphorylation targets. Here, in vitro kinase assays were used to compare phosphorylation targets of four CPKs from Arabidopsis (CPK1, 10, 16, and 34). Significant differences in substrate specificity for each kinase were revealed by assays using 103 different substrates. For example CPK16 phosphorylated Serine 109 in a peptide from the stress-regulated protein, Di19-2 with KM ∼70 μM, but this site was not phosphorylated significantly by CPKs 1, 10, or 34. In contrast, CPKs 1, 10, and 34 phosphorylated 93 other peptide substrates not recognized by CPK16. Examples of substrate specificity differences among all four CPKs were verified by kinetic analyses. To test the correlation between in vivo phosphorylation events and in vitro kinase activities, assays were performed with 274 synthetic peptides that contained phosphorylation sites previously mapped in proteins isolated from plants (in vivo-mapped sites). Of these, 74 (27%) were found to be phosphorylated by at least one of the four CPKs tested. This 27% success rate validates a robust strategy for linking the activities of specific kinases, such as CPKs, to the thousands of in planta phosphorylation sites that are being uncovered by emerging technologies. PMID:22645532

Sugar microarray via click chemistry: molecular recognition with lectins and amyloid β (1-42)

NASA Astrophysics Data System (ADS)

Matsumoto, Erino; Yamauchi, Takahiro; Fukuda, Tomohiro; Miura, Yoshiko

2009-06-01

Sugar microarrays were fabricated on various substrates via click chemistry. Acetylene-terminated substrates were prepared by forming self-assembled monolayers (SAMs) on a gold substrate with alkyl-disulfide and on silicon, quartz and glass substrates with a silane-coupling reagent. The gold substrates were subjected to surface plasmon resonance measurements, and the quartz and glass substrates were subjected to spectroscopy measurements and optical microscopy observation. The saccharide-immobilized substrate on the gold substrate showed specific interaction with the corresponding lectin, and the saccharides showed inert surface properties to other proteins with a high signal-to-noise ratio. We also focused on the saccharide-protein interaction on protein amyloidosis of Alzheimer amyloid β. Amyloid β peptide showed conformation transition on the saccharide-immobilization substrate into a β-sheet, and fibril formation and amyloid aggregates were found on the specific saccharides.

Genome-wide identification and characterization of aquaporin gene family in common bean (Phaseolus vulgaris L.).

PubMed

Ariani, Andrea; Gepts, Paul

2015-10-01

Plant aquaporins are a large and diverse family of water channel proteins that are essential for several physiological processes in living organisms. Numerous studies have linked plant aquaporins with a plethora of processes, such as nutrient acquisition, CO2 transport, plant growth and development, and response to abiotic stresses. However, little is known about this protein family in common bean. Here, we present a genome-wide identification of the aquaporin gene family in common bean (Phaseolus vulgaris L.), a legume crop essential for human nutrition. We identified 41 full-length coding aquaporin sequences in the common bean genome, divided by phylogenetic analysis into five sub-families (PIPs, TIPs, NIPs, SIPs and XIPs). Residues determining substrate specificity of aquaporins (i.e., NPA motifs and ar/R selectivity filter) seem conserved between common bean and other plant species, allowing inference of substrate specificity for these proteins. Thanks to the availability of RNA-sequencing datasets, expression levels in different organs and in leaves of wild and domesticated bean accessions were evaluated. Three aquaporins (PvTIP1;1, PvPIP2;4 and PvPIP1;2) have the overall highest mean expressions, with PvTIP1;1 having the highest expression among all aquaporins. We performed an EST database mining to identify drought-responsive aquaporins in common bean. This analysis showed a significant increase in expression for PvTIP1;1 in drought stress conditions compared to well-watered environments. The pivotal role suggested for PvTIP1;1 in regulating water homeostasis and drought stress response in the common bean should be verified by further field experimentation under drought stress.

Human XPA and RPA DNA repair proteins participate in specific recognition of triplex-induced helical distortions

NASA Astrophysics Data System (ADS)

Vasquez, Karen M.; Christensen, Jesper; Li, Lei; Finch, Rick A.; Glazer, Peter M.

2002-04-01

Nucleotide excision repair (NER) plays a central role in maintaining genomic integrity by detecting and repairing a wide variety of DNA lesions. Xeroderma pigmentosum complementation group A protein (XPA) is an essential component of the repair machinery, and it is thought to be involved in the initial step as a DNA damage recognition and/or confirmation factor. Human replication protein A (RPA) and XPA have been reported to interact to form a DNA damage recognition complex with greater specificity for damaged DNA than XPA alone. The mechanism by which these two proteins recognize such a wide array of structures resulting from different types of DNA damage is not known. One possibility is that they recognize a common feature of the lesions, such as distortions of the helical backbone. We have tested this idea by determining whether human XPA and RPA proteins can recognize the helical distortions induced by a DNA triple helix, a noncanonical DNA structure that has been shown to induce DNA repair, mutagenesis, and recombination. We measured binding of XPA and RPA, together or separately, to substrates containing triplexes with three, two, or no strands covalently linked by psoralen conjugation and photoaddition. We found that RPA alone recognizes all covalent triplex structures, but also forms multivalent nonspecific DNA aggregates at higher concentrations. XPA by itself does not recognize the substrates, but it binds them in the presence of RPA. Addition of XPA decreases the nonspecific DNA aggregate formation. These results support the hypothesis that the NER machinery is targeted to helical distortions and demonstrate that RPA can recognize damaged DNA even without XPA.

Quinone-based stable isotope probing for assessment of 13C substrate-utilizing bacteria

NASA Astrophysics Data System (ADS)

Kunihiro, Tadao; Katayama, Arata; Demachi, Toyoko; Veuger, Bart; Boschker, Henricus T. S.; van Oevelen, Dick

2015-04-01

In this study, we attempted to establish quinone-stable-isotope probing (SIP) technique to link substrate-utilizing bacterial group to chemotaxonomic group in bacterial community. To identify metabolically active bacterial group in various environments, SIP techniques combined with biomarkers have been widely utilized as an attractive method for environmental study. Quantitative approaches of the SIP technique have unique advantage to assess substrate-incorporation into bacteria. As a most major quantitative approach, SIP technique based on phospholipid-derived fatty acids (PLFA) have been applied to simultaneously assess substrate-incorporation rate into bacteria and microbial community structure. This approach is powerful to estimate the incorporation rate because of the high sensitivity due to the detection by a gas chromatograph-combustion interface-isotope ratio mass spectrometer (GC-c-IRMS). However, its phylogenetic resolution is limited by specificity of a compound-specific marker. We focused on respiratory quinone as a biomarker. Our previous study found a good correlation between concentrations of bacteria-specific PLFAs and quinones over several orders of magnitude in various marine sediments, and the quinone method has a higher resolution (bacterial phylum level) for resolving differences in bacterial community composition more than that of bacterial PLFA. Therefore, respiratory quinones are potentially good biomarkers for quantitative approaches of the SIP technique. The LC-APCI-MS method as molecular-mass based detection method for quinone was developed and provides useful structural information for identifying quinone molecular species in environmental samples. LC-MS/MS on hybrid triple quadrupole/linear ion trap, which enables to simultaneously identify and quantify compounds in a single analysis, can detect high molecular compounds with their isotope ions. Use of LC-MS/MS allows us to develop quinone-SIP based on molecular mass differences due to 13C abundance in the quinone. In this study, we verified carbon stable isotope of quinone compared with bulk carbon stable isotope of bacterial culture. Results indicated a good correlation between carbon stable isotope of quinone compared with bulk carbon stable isotope. However, our measurement conditions for detection of quinone isotope-ions incurred underestimation of 13C abundance in the quinone. The quinone-SIP technique needs further optimization for measurement conditions of LC-MS/MS.

Structure of the carboxypeptidase B complex with N-sulfamoyl-L-phenylalanine - a transition state analog of non-specific substrate.

PubMed

Akparov, Valery; Timofeev, Vladimir; Khaliullin, Ilyas; Švedas, Vytas; Kuranova, Inna

2018-03-01

Carboxypeptidase B (EC 3.4.17.2) (CPB) is commonly used in the industrial insulin production and as a template for drug design. However, its ability to discriminate substrates with hydrophobic, hydrophilic, and charged side chains is not well understood. We report structure of CPB complex with a transition state analog N-sulfamoyl-L-phenylalanine solved at 1.74Å. The study provided an insight into structural basis of CPB substrate specificity. Ligand binding is affected by structure-depended conformational changes of Asp255 in S1'-subsite, interactions with Asn144 and Arg145 in C-terminal binding subsite, and Glu270 in the catalytic center. Side chain of the non-specific substrate analog SPhe in comparison with that of specific substrate analog SArg (reported earlier) not only loses favorable electrostatic interactions and two hydrogen bonds with Asp255 and three fixed water molecules, but is forced to be in the unfavorable hydrophilic environment. Thus, Ser207, Gly253, Tyr248, and Asp255 residues play major role in the substrate recognition by S1'-subsite.

Novel α-L-arabinofuranosidase from Cellulomonas fimi ATCC 484 and its substrate-specificity analysis with the aid of computer.

PubMed

Yang, Ying; Zhang, Lujia; Guo, Mingrong; Sun, Jiaqi; Matsukawa, Shingo; Xie, Jingli; Wei, Dongzhi

2015-04-15

In the process of gene mining for novel α-L-arabinofuranosidases (AFs), the gene Celf_3321 from Cellulomonas fimi ATCC 484 encodes an AF, termed as AbfCelf, with potent activity, 19.4 U/mg under the optimum condition, pH 6.0 and 40 °C. AbfCelf can hydrolyze α-1,5-linked oligosaccharides, sugar beet arabinan, linear 1,5-α-arabinan, and wheat flour arabinoxylan, which is partly different from some previously well-characterized GH 51 AFs. The traditional substrate-specificity analysis for AFs is labor-consuming and money costing, because the substrates include over 30 kinds of various 4-nitrophenol (PNP)-glycosides, oligosaccharides, and polysaccharides. Hence, a preliminary structure and mechanism based method was applied for substrate-specificity analysis. The binding energy (ΔG, kcal/mol) obtained by docking suggested the reaction possibility and coincided with the experimental results. AbfA crystal 1QW9 was used to test the rationality of docking method in simulating the interaction between enzyme and substrate, as well the credibility of the substrate-specificity analysis method in silico.

Reliability Prediction Modeling of New Devices.

DTIC Science & Technology

1980-07-01

film of magnetic material on a nonmagnetic garnet crystal substrate with a surround- ing magnetic bias field mechanism to complete the basic hybrid...semiconductor processes. The magnetic domain centers are formed in a magnetic epitaxial film of garnet crystal on a nonmagnetic garnet substrate, and...polarity. The most widely used basic substrate is a high-purity gadolinium garnet . The thin- film magnetic layer is of the same crystal class with a

CVD growth of large-area and high-quality HfS2 nanoforest on diverse substrates

NASA Astrophysics Data System (ADS)

Zheng, Binjie; Wang, Zegao; Qi, Fei; Wang, Xinqiang; Yu, Bo; Zhang, Wanli; Chen, Yuanfu

2018-03-01

Two-dimensional layered transition metal dichalcogenides (TMDs) have attracted burgeoning attention due to their various properties and wide potential applications. As a new TMD, hafnium disulfide (HfS2) is theoretically predicted to have better electrical performance than widely studied MoS2. The experimental researches also confirmed the extraordinary feature in electronics and optoelectronics. However, the maximal device performance may not be achieved due to its own limitation of planar structure and challenge of transfer without contamination. Here, through the chemical vapor deposition (CVD) technique, inch-size HfS2 nanoforest has been directly grown on diverse objective substrates covering insulating, semiconducting and conducting substrates. This direct CVD growth without conventional transfer process avoids contamination and degradation in quality, suggesting its promising and wide applications in high-quality and multifarious devices. It is noted that all the HfS2 nanoforests grown on diverse substrates are constructed with vertically aligned few-layered HfS2 nanosheets with high crystalline quality and edge orientation. Moreover, due to its unique structure, the HfS2 nanoforest owns abundant exposed edge sites and large active surface area, which is essential to apply in high-performance catalyst, sensor, and energy storage or field emitter.

Toward a mechanistic understanding of patterns in biomineralization and new insights for old dogmas in geological settings (Invited)

NASA Astrophysics Data System (ADS)

Dove, P. M.; Hamm, L.; Giuffre, A. J.; Han, N.; De Yoreo, J. J.

2013-12-01

The ability of organisms to mineralize tissues into skeletons and other functional structures is a remarkable achievement of biology. Yet, the physical basis for how macromolecules regulate the placement and onset of mineral formation is not well established. Efforts to understand nucleation onto organic substrates have produced two, seemingly contradictory, lines of thought: The biomineralization community widely assumes the organic matrix promotes nucleation through stereochemical matching to guide the organization of solute ions, while materials synthesis groups use simple binding assays to correlate high binding strength with good promoters of nucleation. This study reconciles the two views and provides a mechanistic explanation for template-directed nucleation by correlating heterogeneous nucleation barriers with crystal-substrate binding free energies. Using surface assembled monolayers (SAM) as simple model systems, we first measure the kinetics of calcite nucleation onto model substrates that present different functional group chemistries (carboxyl, thiol, phosphate, hydroxyl) and conformations (C11, C16 chain lengths). We find rates are substrate-specific and obey predictions of classical nucleation theory at supersaturations that extend above the solubility of amorphous calcium carbonate (ACC). Analysis of the kinetic data shows the thermodynamic barrier to nucleation is reduced by minimizing the interfacial free energy of the system, γ. We then use dynamic force spectroscopy to independently measure calcite-substrate binding free energies, ΔGb. Moreover, we show that within the classical theory of nucleation, γ and ΔGb should be linearly related. The results bear out this prediction and demonstrate that low energy barriers to nucleation correlate with strong crystal-substrate binding. This relationship is general to all functional group chemistries and conformations. These findings reconcile the long-standing concept of templated nucleation through stereochemical matching with the conventional wisdom that ';good binders are good nucleators'. Alternative perspectives become internally consistent when viewed through the lens of crystal-substrate binding and provide a physical basis for how organic chemistry can direct temporal and spatial patterns of carbonate nucleation.

Active site specificity profiling datasets of matrix metalloproteinases (MMPs) 1, 2, 3, 7, 8, 9, 12, 13 and 14.

PubMed

Eckhard, Ulrich; Huesgen, Pitter F; Schilling, Oliver; Bellac, Caroline L; Butler, Georgina S; Cox, Jennifer H; Dufour, Antoine; Goebeler, Verena; Kappelhoff, Reinhild; Auf dem Keller, Ulrich; Klein, Theo; Lange, Philipp F; Marino, Giada; Morrison, Charlotte J; Prudova, Anna; Rodriguez, David; Starr, Amanda E; Wang, Yili; Overall, Christopher M

2016-06-01

The data described provide a comprehensive resource for the family-wide active site specificity portrayal of the human matrix metalloproteinase family. We used the high-throughput proteomic technique PICS (Proteomic Identification of protease Cleavage Sites) to comprehensively assay 9 different MMPs. We identified more than 4300 peptide cleavage sites, spanning both the prime and non-prime sides of the scissile peptide bond allowing detailed subsite cooperativity analysis. The proteomic cleavage data were expanded by kinetic analysis using a set of 6 quenched-fluorescent peptide substrates designed using these results. These datasets represent one of the largest specificity profiling efforts with subsequent structural follow up for any protease family and put the spotlight on the specificity similarities and differences of the MMP family. A detailed analysis of this data may be found in Eckhard et al. (2015) [1]. The raw mass spectrometry data and the corresponding metadata have been deposited in PRIDE/ProteomeXchange with the accession number PXD002265.

Matrix metalloproteinase proteomics: substrates, targets, and therapy.

PubMed

Morrison, Charlotte J; Butler, Georgina S; Rodríguez, David; Overall, Christopher M

2009-10-01

Proteomics encompasses powerful techniques termed 'degradomics' for unbiased high-throughput protease substrate discovery screens that have been applied to an important family of extracellular proteases, the matrix metalloproteinases (MMPs). Together with the data generated from genetic deletion and transgenic mouse models and genomic profiling, these screens can uncover the diverse range of MMP functions, reveal which MMPs and MMP-mediated pathways exacerbate pathology, and which are involved in protection and the resolution of disease. This information can be used to identify and validate candidate drug targets and antitargets, and is critical for the development of new inhibitors of MMP function. Such inhibitors may target either the MMP directly in a specific manner or pathways upstream and downstream of MMP activity that are mediating deleterious effects in disease. Since MMPs do not operate alone but are part of the 'protease web', it is necessary to use system-wide approaches to understand MMP proteolysis in vivo, to discover new biological roles and their potential for therapeutic modification.

High-speed atomic force microscopy reveals strongly polarized movement of clostridial collagenase along collagen fibrils

PubMed Central

Watanabe-Nakayama, Takahiro; Itami, Masahiro; Kodera, Noriyuki; Ando, Toshio; Konno, Hiroki

2016-01-01

Bacterial collagenases involved in donor infection are widely applied in many fields due to their high activity and specificity; however, little is known regarding the mechanisms by which bacterial collagenases degrade insoluble collagen in host tissues. Using high-speed atomic force microscopy, we simultaneously visualized the hierarchical structure of collagen fibrils and the movement of a representative bacterial collagenase, Clostridium histolyticum type I collagenase (ColG), to determine the relationship between collagen structure and collagenase movement. Notably, ColG moved ~14.5 nm toward the collagen N terminus in ~3.8 s in a manner dependent on a catalytic zinc ion. While ColG was engaged, collagen molecules were not only degraded but also occasionally rearranged to thicken neighboring collagen fibrils. Importantly, we found a similarity of relationship between the enzyme-substrate interface structure and enzyme migration in collagen-collagenase and DNA-nuclease systems, which share a helical substrate structure, suggesting a common strategy in enzyme evolution. PMID:27373458

Release of MEMS devices with hard-baked polyimide sacrificial layer

NASA Astrophysics Data System (ADS)

Boroumand Azad, Javaneh; Rezadad, Imen; Nath, Janardan; Smith, Evan; Peale, Robert E.

2013-03-01

Removal of polyimides used as sacrificial layer in fabricating MEMS devices can be challenging after hardbaking, which may easily result by the end of multiple-step processing. We consider the specific commercial co-developable polyimide ProLift 100 (Brewer Science). Excessive heat hardens this material, so that during wet release in TMAH based solvents, intact sheets break free from the substrate, move around in the solution, and break delicate structures. On the other hand, dry reactive-ion etching of hard-baked ProLift is so slow, that MEMS structures are damaged from undesirably-prolonged physical bombardment by plasma ions. We found that blanket exposure to ultraviolet light allows rapid dry etch of the ProLift surrounding the desired structures without damaging them. Subsequent removal of ProLift from under the devices can then be safely performed using wet or dry etch. We demonstrate the approach on PECVD-grown silicon-oxide cantilevers of 100 micron × 100 micron area supported 2 microns above the substrate by ~100-micron-long 8-micron-wide oxide arms.

Analysis and characterization of Cu2CdSnS4 quaternary alloy nanostructures deposited on GaN

NASA Astrophysics Data System (ADS)

Odeh, Ali Abu; Al-Douri, Y.; Ameri, M.; Bouhemadou, A.

2018-06-01

Through using spin coating technique, Cu2CdSnS4 (CCTS) quaternary alloy nanostructures were successfully deposited on GaN substrate using a wide range of spin coating speeds; 1500, 2000, 2500, 3000 and 3500 RPM at annealing temperature 300 °C. The optical properties were investigated through UV-vis which revealed the changing of energy band gap as the spin coating speed increases, in addition, to verify specific models of refractive index and optical dielectric constant. The structural properties were studied by X-ray diffraction which indicated that the number and intensity of the peaks were changed as the spin coating speed changes. The morphological and topographical studies of CCTS were elaborated by field emission-scanning electron microscopy and atomic force microscopy. The obtained results suggest that CCTS nanostructures deposited on GaN substrate are very suitable for optoelectronic applications, that are in accordance with the available theoretical and experimental data.

LUSH-based SPR sensor for the detection of alcohols and pheromone

NASA Astrophysics Data System (ADS)

Lau, Hui-Chong; Lee, Yeon-Kyung; Kwon, Jae-Young; Sohn, Young-Soo; Lim, Jeong Ok

2013-05-01

Protein is a widely used sensing substrate in the biosensing technology. In the study conducted here, we used odorant binding protein, LUSH from Drosophila as a biosensing substrate in a miniaturized surface plasmon resonance (SPR) sensor. LUSH contains the specific alcohols binding sites, which mediates the detection of alcohols and pheromone. We first modified the surface of the gold sensor chip using the self assembled monolayer in the chloroform solution. The saturated concentration was determined prior to the detection of alcohols and pheromone at various concentrations. The results showed that the LUSH was saturated at 1000 μg/ml on the gold sensor chip. The detection response of LUSH was significant at higher concentration of alcohols. LUSH detected ethanol at concentration >=50% propanol was detected at >=25% whereas pheromone was detected at >=1.25 μg/μl. The results provide some fundamental information on the potential use of LUSH-based SPR as a simple and easy protein-based sensor in the near future.

Preferential orientation of NV defects in CVD diamond films grown on (113)-oriented substrates

NASA Astrophysics Data System (ADS)

Lesik, M.; Plays, T.; Tallaire, A.; Achard, J.; Brinza, O.; William, L.; Chipaux, M.; Toraille, L.; Debuisschert, T.; Gicquel, A.; Roch, J. F.; Jacques, V.

2015-06-01

Thick CVD diamond layers were successfully grown on (113)-oriented substrates. They exhibited smooth surface morphologies and a crystalline quality comparable to (100) electronic grade material, and much better than (111)-grown layers. High growth rates (15-50 {\\mu}m/h) were obtained while nitrogen doping could be achieved in a fairly wide range without seriously imparting crystalline quality. Electron spin resonance measurements were carried out to determine NV centers orientation and concluded that one specific orientation has an occurrence probability of 73 % when (100)-grown layers show an equal distribution in the 4 possible directions. A spin coherence time of around 270 {\\mu}s was measured which is equivalent to that reported for material with similar isotopic purity. Although a higher degree of preferential orientation was achieved with (111)-grown layers (almost 100 %), the ease of growth and post-processing of the (113) orientation make it a potentially useful material for magnetometry or other quantum mechanical applications.

Subdigital setae of chameleon feet: Friction-enhancing microstructures for a wide range of substrate roughness

PubMed Central

Spinner, Marlene; Westhoff, Guido; Gorb, Stanislav N.

2014-01-01

Hairy adhesive systems of microscopic setae with triangular flattened tips have evolved convergently in spiders, insects and arboreal lizards. The ventral sides of the feet and tails in chameleons are also covered with setae. However, chameleon setae feature strongly elongated narrow spatulae or fibrous tips. The friction enhancing function of these microstructures has so far only been demonstrated in contact with glass spheres. In the present study, the frictional properties of subdigital setae of Chamaeleo calyptratus were measured under normal forces in the physical range on plane substrates having different roughness. We showed that chameleon setae maximize friction on a wide range of substrate roughness. The highest friction was measured on asperities of 1 μm. However, our observations of the climbing ability of Ch. calyptratus on rods of different diameters revealed that also claws and grasping feet are additionally responsible for the force generation on various substrates during locomotion. PMID:24970387

Gold/silver coated nanoporous ceramic membranes: a new substrate for SERS studies

NASA Astrophysics Data System (ADS)

Kassu, A.; Robinson, P.; Sharma, A.; Ruffin, P. B.; Brantley, C.; Edwards, E.

2010-08-01

Surface Enhanced Raman Scattering (SERS) is a recently discovered powerful technique which has demonstrated sensitivity and selectivity for detecting single molecules of certain chemical species. This is due to an enhancement of Raman scattered light by factors as large as 1015. Gold and Silver-coated substrates fabricated by electron-beam lithography on Silicon are widely used in SERS technique. In this paper, we report the use of nanoporous ceramic membranes for SERS studies. Nanoporous membranes are widely used as a separation membrane in medical devices, fuel cells and other studies. Three different pore diameter sizes of commercially available nanoporous ceramic membranes: 35 nm, 55nm and 80nm are used in the study. To make the membranes SERS active, they are coated with gold/silver using sputtering techniques. We have seen that the membranes coated with gold layer remain unaffected even when immersed in water for several days. The results show that gold coated nanoporous membranes have sensitivity comparable to substrates fabricated by electron-beam lithography on Silicon substrates.

Substrate Specificities and Conformational Flexibility of 3-Ketosteroid 9α-Hydroxylases*

PubMed Central

Penfield, Jonathan S.; Worrall, Liam J.; Strynadka, Natalie C.; Eltis, Lindsay D.

2014-01-01

KshA is the oxygenase component of 3-ketosteroid 9α-hydroxylase, a Rieske oxygenase involved in the bacterial degradation of steroids. Consistent with its role in bile acid catabolism, KshA1 from Rhodococcus rhodochrous DSM43269 had the highest apparent specificity (kcat/Km) for steroids with an isopropyl side chain at C17, such as 3-oxo-23,24-bisnorcholesta-1,4-diene-22-oate (1,4-BNC). By contrast, the KshA5 homolog had the highest apparent specificity for substrates with no C17 side chain (kcat/Km >105 s−1 m−1 for 4-estrendione, 5α-androstandione, and testosterone). Unexpectedly, substrates such as 4-androstene-3,17-dione (ADD) and 4-BNC displayed strong substrate inhibition (KiS ∼100 μm). By comparison, the cholesterol-degrading KshAMtb from Mycobacterium tuberculosis had the highest specificity for CoA-thioesterified substrates. These specificities are consistent with differences in the catabolism of cholesterol and bile acids, respectively, in actinobacteria. X-ray crystallographic structures of the KshAMtb·ADD, KshA1·1,4-BNC-CoA, KshA5·ADD, and KshA5·1,4-BNC-CoA complexes revealed that the enzymes have very similar steroid-binding pockets with the substrate's C17 oriented toward the active site opening. Comparisons suggest Tyr-245 and Phe-297 are determinants of KshA1 specificity. All enzymes have a flexible 16-residue “mouth loop,” which in some structures completely occluded the substrate-binding pocket from the bulk solvent. Remarkably, the catalytic iron and α-helices harboring its ligands were displaced up to 4.4 Å in the KshA5·substrate complexes as compared with substrate-free KshA, suggesting that Rieske oxygenases may have a dynamic nature similar to cytochrome P450. PMID:25049233

Rapid and general profiling of protease specificity by using combinatorial fluorogenic substrate libraries

PubMed Central

Harris, Jennifer L.; Backes, Bradley J.; Leonetti, Francesco; Mahrus, Sami; Ellman, Jonathan A.; Craik, Charles S.

2000-01-01

A method is presented for the preparation and use of fluorogenic peptide substrates that allows for the configuration of general substrate libraries to rapidly identify the primary and extended specificity of proteases. The substrates contain the fluorogenic leaving group 7-amino-4-carbamoylmethylcoumarin (ACC). Substrates incorporating the ACC leaving group show kinetic profiles comparable to those with the traditionally used 7-amino-4-methylcoumarin (AMC) leaving group. The bifunctional nature of ACC allows for the efficient production of single substrates and substrate libraries by using 9-fluorenylmethoxycarbonyl (Fmoc)-based solid-phase synthesis techniques. The approximately 3-fold-increased quantum yield of ACC over AMC permits reduction in enzyme and substrate concentrations. As a consequence, a greater number of substrates can be tolerated in a single assay, thus enabling an increase in the diversity space of the library. Soluble positional protease substrate libraries of 137,180 and 6,859 members, possessing amino acid diversity at the P4-P3-P2-P1 and P4-P3-P2 positions, respectively, were constructed. Employing this screening method, we profiled the substrate specificities of a diverse array of proteases, including the serine proteases thrombin, plasmin, factor Xa, urokinase-type plasminogen activator, tissue plasminogen activator, granzyme B, trypsin, chymotrypsin, human neutrophil elastase, and the cysteine proteases papain and cruzain. The resulting profiles create a pharmacophoric portrayal of the proteases to aid in the design of selective substrates and potent inhibitors. PMID:10869434

Characterisation of phenol oxidase and peroxidase from maize silk.

PubMed

Sukalović, V Hadzi-Tasković; Veljović-Jovanović, S; Maksimović, J Dragisić; Maksimović, V; Pajić, Z

2010-05-01

Silk of some maize genotypes contains a high level of phenolics that undergo enzymatic oxidation to form quinones, which condense among themselves or with proteins to form brown pigments. Two phenolic oxidizing enzymes, peroxidase (POD; EC 1.11.1.7) and polyphenol oxidase (PPO; EC 1.10.3.1), from maize (Zea mays L.) silk were characterised with respect to their preferred substrate, different isoforms and specific effectors. One browning silk sample with high, and two non-browning samples with low phenolic content were investigated. Although POD oxidizes a wide range of phenolic substrates in vitro, its activity rate was independent of silk phenolic content. PPO activity, detected with o-diphenolic substrates, was abundant only in browning silk, and low or absent in non-browning silk. Pollination increased POD but not PPO activity. Isoelectric-focusing (IEF) and specific staining for POD and PPO showed a high degree of polymorphism that varied with silk origin. The IEF pattern of POD revealed a number of anionic and several cationic isoenzymes, with the most pronounced having neutral pI 7 and a basic isoform with pI 10. Detected isoforms of PPO were anionic, except for one neutral form found only in browning silk, and occupied positions different from those of POD. Different inhibitory effects of NaN(3), EDTA, KCN, and L-cysteine, as well as different impacts of a variety of cations on the oxidation of chlorogenic acid, mediated by PPO or POD, were detected. The findings are discussed in terms of a possible roles of these enzymes in defence and pollination.

The prospects of cellulase-producing bacteria for the bioconversion of lignocellulosic biomass.

PubMed

Maki, Miranda; Leung, Kam Tin; Qin, Wensheng

2009-07-29

Lignocellulosic biomass is a renewable and abundant resource with great potential for bioconversion to value-added bioproducts. However, the biorefining process remains economically unfeasible due to a lack of biocatalysts that can overcome costly hurdles such as cooling from high temperature, pumping of oxygen/stirring, and, neutralization from acidic or basic pH. The extreme environmental resistance of bacteria permits screening and isolation of novel cellulases to help overcome these challenges. Rapid, efficient cellulase screening techniques, using cellulase assays and metagenomic libraries, are a must. Rare cellulases with activities on soluble and crystalline cellulose have been isolated from strains of Paenibacillus and Bacillus and shown to have high thermostability and/or activity over a wide pH spectrum. While novel cellulases from strains like Cellulomonas flavigena and Terendinibacter turnerae, produce multifunctional cellulases with broader substrate utilization. These enzymes offer a framework for enhancement of cellulases including: specific activity, thermalstability, or end-product inhibition. In addition, anaerobic bacteria like the clostridia offer potential due to species capable of producing compound multienzyme complexes called cellulosomes. Cellulosomes provide synergy and close proximity of enzymes to substrate, increasing activity towards crystalline cellulose. This has lead to the construction of designer cellulosomes enhanced for specific substrate activity. Furthermore, cellulosome-producing Clostridium thermocellum and its ability to ferment sugars to ethanol; its amenability to co-culture and, recent advances in genetic engineering, offer a promising future in biofuels. The exploitation of bacteria in the search for improved enzymes or strategies provides a means to upgrade feasibility for lignocellulosic biomass conversion, ultimately providing means to a 'greener' technology.

The prospects of cellulase-producing bacteria for the bioconversion of lignocellulosic biomass

PubMed Central

Maki, Miranda; Leung, Kam Tin; Qin, Wensheng

2009-01-01

Lignocellulosic biomass is a renewable and abundant resource with great potential for bioconversion to value-added bioproducts. However, the biorefining process remains economically unfeasible due to a lack of biocatalysts that can overcome costly hurdles such as cooling from high temperature, pumping of oxygen/stirring, and, neutralization from acidic or basic pH. The extreme environmental resistance of bacteria permits screening and isolation of novel cellulases to help overcome these challenges. Rapid, efficient cellulase screening techniques, using cellulase assays and metagenomic libraries, are a must. Rare cellulases with activities on soluble and crystalline cellulose have been isolated from strains of Paenibacillus and Bacillus and shown to have high thermostability and/or activity over a wide pH spectrum. While novel cellulases from strains like Cellulomonas flavigena and Terendinibacter turnerae, produce multifunctional cellulases with broader substrate utilization. These enzymes offer a framework for enhancement of cellulases including: specific activity, thermalstability, or end-product inhibition. In addition, anaerobic bacteria like the clostridia offer potential due to species capable of producing compound multienzyme complexes called cellulosomes. Cellulosomes provide synergy and close proximity of enzymes to substrate, increasing activity towards crystalline cellulose. This has lead to the construction of designer cellulosomes enhanced for specific substrate activity. Furthermore, cellulosome-producing Clostridium thermocellum and its ability to ferment sugars to ethanol; its amenability to co-culture and, recent advances in genetic engineering, offer a promising future in biofuels. The exploitation of bacteria in the search for improved enzymes or strategies provides a means to upgrade feasibility for lignocellulosic biomass conversion, ultimately providing means to a 'greener' technology. PMID:19680472

A thermophone on porous polymeric substrate

NASA Astrophysics Data System (ADS)

Chitnis, G.; Kim, A.; Song, S. H.; Jessop, A. M.; Bolton, J. S.; Ziaie, B.

2012-07-01

In this Letter, we present a simple, low-temperature method for fabricating a wide-band (>80 kHz) thermo-acoustic sound generator on a porous polymeric substrate. We were able to achieve up to 80 dB of sound pressure level with an input power of 0.511 W. No significant surface temperature increase was observed in the device even at an input power level of 2.5 W. Wide-band ultrasonic performance, simplicity of structure, and scalability of the fabrication process make this device suitable for many ranging and imaging applications.

Cleavage-site specificity of prolyl endopeptidase FAP investigated with a full-length protein substrate.

PubMed

Huang, Chih-Hsiang; Suen, Ching-Shu; Lin, Ching-Ting; Chien, Chia-Hui; Lee, Hsin-Ying; Chung, Kuei-Min; Tsai, Ting-Yueh; Jiaang, Weir-Tong; Hwang, Ming-Jing; Chen, Xin

2011-06-01

Fibroblast activation protein (FAP) is a prolyl-cleaving endopeptidase proposed as an anti-cancer drug target. It is necessary to define its cleavage-site specificity to facilitate the identification of its in vivo substrates and to understand its biological functions. We found that the previously identified substrate of FAP, α(2)-anti-plasmin, is not a robust substrate in vitro. Instead, an intracellular protein, SPRY2, is cleavable by FAP and more suitable for investigation of its substrate specificity in the context of the full-length globular protein. FAP prefers uncharged residues, including small or bulky hydrophobic amino acids, but not charged amino acids, especially acidic residue at P1', P3 and P4 sites. Molecular modelling analysis shows that the substrate-binding site of FAP is surrounded by multiple tyrosine residues and some negatively charged residues, which may exert least preference for substrates with acidic residues. This provides an explanation why FAP cannot cleave interleukins, which have a glutamate at either P4 or P2', despite their P3-P2-P1 sites being identical to SPRY2 or α-AP. Our study provided new information on FAP cleavage-site specificity, which differs from the data obtained by profiling with a peptide library or with the denatured protein, gelatin, as the substrate. Furthermore, our study suggests that negatively charged residues should be avoided when designing FAP inhibitors.

Bauhinia proteinase inhibitor-based synthetic fluorogenic substrates for enzymes isolated from insect midgut and caterpillar bristles.

PubMed

Andrade, Sonia A; Santomauro-Vaz, Eugênio M; Lopes, Adriana R; Chudzinski-Tavassi, Ana M; Juliano, Maria A; Terra, Walter R; Sampaio, Misako U; Sampaio, Claudio A M; Oliva, Maria Luiza V

2003-03-01

Bauhinia ungulata factor Xa inhibitor (BuXI) inactivates factor Xa and LOPAP, a prothrombin activator proteinase isolated from the venom of Lonomia obliqua caterpillar bristles. The reactive site of the enzyme-inhibitor interaction was explored to design specific substrates for both enzymes. Methionine is crucial for LOPAP and factor Xa substrate interaction, since the change of both Met residues in the substrates abolished the hydrolysis. Synthetic substrates containing the sequence around the reactive site of BbKI, a plasma kallikrein inhibitor, were shown to be specific for trypsin hydrolysis. Therefore, these substrates may be an alternative in studies aiming at a characterization of trypsin-like enzyme activities, especially non-mammalian enzymes.

Live single-cell laser tag.

PubMed

Binan, Loïc; Mazzaferri, Javier; Choquet, Karine; Lorenzo, Louis-Etienne; Wang, Yu Chang; Affar, El Bachir; De Koninck, Yves; Ragoussis, Jiannis; Kleinman, Claudia L; Costantino, Santiago

2016-05-20

The ability to conduct image-based, non-invasive cell tagging, independent of genetic engineering, is key to cell biology applications. Here we introduce cell labelling via photobleaching (CLaP), a method that enables instant, specific tagging of individual cells based on a wide array of criteria such as shape, behaviour or positional information. CLaP uses laser illumination to crosslink biotin onto the plasma membrane, coupled with streptavidin conjugates to label individual cells for genomic, cell-tracking, flow cytometry or ultra-microscopy applications. We show that the incorporated mark is stable, non-toxic, retained for several days, and transferred by cell division but not to adjacent cells in culture. To demonstrate the potential of CLaP for genomic applications, we combine CLaP with microfluidics-based single-cell capture followed by transcriptome-wide next-generation sequencing. Finally, we show that CLaP can also be exploited for inducing transient cell adhesion to substrates for microengineering cultures with spatially patterned cell types.

Discovery of a widely distributed toxin biosynthetic gene cluster

PubMed Central

Lee, Shaun W.; Mitchell, Douglas A.; Markley, Andrew L.; Hensler, Mary E.; Gonzalez, David; Wohlrab, Aaron; Dorrestein, Pieter C.; Nizet, Victor; Dixon, Jack E.

2008-01-01

Bacteriocins represent a large family of ribosomally produced peptide antibiotics. Here we describe the discovery of a widely conserved biosynthetic gene cluster for the synthesis of thiazole and oxazole heterocycles on ribosomally produced peptides. These clusters encode a toxin precursor and all necessary proteins for toxin maturation and export. Using the toxin precursor peptide and heterocycle-forming synthetase proteins from the human pathogen Streptococcus pyogenes, we demonstrate the in vitro reconstitution of streptolysin S activity. We provide evidence that the synthetase enzymes, as predicted from our bioinformatics analysis, introduce heterocycles onto precursor peptides, thereby providing molecular insight into the chemical structure of streptolysin S. Furthermore, our studies reveal that the synthetase exhibits relaxed substrate specificity and modifies toxin precursors from both related and distant species. Given our findings, it is likely that the discovery of similar peptidic toxins will rapidly expand to existing and emerging genomes. PMID:18375757

Long-range electrostatic complementarity governs substrate recognition by human chymotrypsin C, a key regulator of digestive enzyme activation.

PubMed

Batra, Jyotica; Szabó, András; Caulfield, Thomas R; Soares, Alexei S; Sahin-Tóth, Miklós; Radisky, Evette S

2013-04-05

Human chymotrypsin C (CTRC) is a pancreatic serine protease that regulates activation and degradation of trypsinogens and procarboxypeptidases by targeting specific cleavage sites within their zymogen precursors. In cleaving these regulatory sites, which are characterized by multiple flanking acidic residues, CTRC shows substrate specificity that is distinct from that of other isoforms of chymotrypsin and elastase. Here, we report the first crystal structure of active CTRC, determined at 1.9-Å resolution, revealing the structural basis for binding specificity. The structure shows human CTRC bound to the small protein protease inhibitor eglin c, which binds in a substrate-like manner filling the S6-S5' subsites of the substrate binding cleft. Significant binding affinity derives from burial of preferred hydrophobic residues at the P1, P4, and P2' positions of CTRC, although acidic P2' residues can also be accommodated by formation of an interfacial salt bridge. Acidic residues may also be specifically accommodated in the P6 position. The most unique structural feature of CTRC is a ring of intense positive electrostatic surface potential surrounding the primarily hydrophobic substrate binding site. Our results indicate that long-range electrostatic attraction toward substrates of concentrated negative charge governs substrate discrimination, which explains CTRC selectivity in regulating active digestive enzyme levels.

Substrate Specificity and Possible Heterologous Targets of Phytaspase, a Plant Cell Death Protease.

PubMed

Galiullina, Raisa A; Kasperkiewicz, Paulina; Chichkova, Nina V; Szalek, Aleksandra; Serebryakova, Marina V; Poreba, Marcin; Drag, Marcin; Vartapetian, Andrey B

2015-10-09

Plants lack aspartate-specific cell death proteases homologous to animal caspases. Instead, a subtilisin-like serine-dependent plant protease named phytaspase shown to be involved in the accomplishment of programmed death of plant cells is able to hydrolyze a number of peptide-based caspase substrates. Here, we determined the substrate specificity of rice (Oryza sativa) phytaspase by using the positional scanning substrate combinatorial library approach. Phytaspase was shown to display an absolute specificity of hydrolysis after an aspartic acid residue. The preceding amino acid residues, however, significantly influence the efficiency of hydrolysis. Efficient phytaspase substrates demonstrated a remarkable preference for an aromatic amino acid residue in the P3 position. The deduced optimum phytaspase recognition motif has the sequence IWLD and is strikingly hydrophobic. The established pattern was confirmed through synthesis and kinetic analysis of cleavage of a set of optimized peptide substrates. An amino acid motif similar to the phytaspase cleavage site is shared by the human gastrointestinal peptide hormones gastrin and cholecystokinin. In agreement with the established enzyme specificity, phytaspase was shown to hydrolyze gastrin-1 and cholecystokinin at the predicted sites in vitro, thus destroying the active moieties of the hormones. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Structural and Kinetic Basis for Substrate Selectivity in Populus tremuloides Sinapyl Alcohol Dehydrogenase

PubMed Central

Bomati, Erin K.; Noel, Joseph P.

2005-01-01

We describe the three-dimensional structure of sinapyl alcohol dehydrogenase (SAD) from Populus tremuloides (aspen), a member of the NADP(H)-dependent dehydrogenase family that catalyzes the last reductive step in the formation of monolignols. The active site topology revealed by the crystal structure substantiates kinetic results indicating that SAD maintains highest specificity for the substrate sinapaldehyde. We also report substantial substrate inhibition kinetics for the SAD-catalyzed reduction of hydroxycinnamaldehydes. Although SAD and classical cinnamyl alcohol dehydrogenases (CADs) catalyze the same reaction and share some sequence identity, the active site topology of SAD is strikingly different from that predicted for classical CADs. Kinetic analyses of wild-type SAD and several active site mutants demonstrate the complexity of defining determinants of substrate specificity in these enzymes. These results, along with a phylogenetic analysis, support the inclusion of SAD in a plant alcohol dehydrogenase subfamily that includes cinnamaldehyde and benzaldehyde dehydrogenases. We used the SAD three-dimensional structure to model several of these SAD-like enzymes, and although their active site topologies largely mirror that of SAD, we describe a correlation between substrate specificity and amino acid substitution patterns in their active sites. The SAD structure thus provides a framework for understanding substrate specificity in this family of enzymes and for engineering new enzyme specificities. PMID:15829607

Structural and kinetic basis for substrate selectivity in Populus tremuloides sinapyl alcohol dehydrogenase.

PubMed

Bomati, Erin K; Noel, Joseph P

2005-05-01

We describe the three-dimensional structure of sinapyl alcohol dehydrogenase (SAD) from Populus tremuloides (aspen), a member of the NADP(H)-dependent dehydrogenase family that catalyzes the last reductive step in the formation of monolignols. The active site topology revealed by the crystal structure substantiates kinetic results indicating that SAD maintains highest specificity for the substrate sinapaldehyde. We also report substantial substrate inhibition kinetics for the SAD-catalyzed reduction of hydroxycinnamaldehydes. Although SAD and classical cinnamyl alcohol dehydrogenases (CADs) catalyze the same reaction and share some sequence identity, the active site topology of SAD is strikingly different from that predicted for classical CADs. Kinetic analyses of wild-type SAD and several active site mutants demonstrate the complexity of defining determinants of substrate specificity in these enzymes. These results, along with a phylogenetic analysis, support the inclusion of SAD in a plant alcohol dehydrogenase subfamily that includes cinnamaldehyde and benzaldehyde dehydrogenases. We used the SAD three-dimensional structure to model several of these SAD-like enzymes, and although their active site topologies largely mirror that of SAD, we describe a correlation between substrate specificity and amino acid substitution patterns in their active sites. The SAD structure thus provides a framework for understanding substrate specificity in this family of enzymes and for engineering new enzyme specificities.

Dose-dependent fate of GFP-E. coli in the alimentary canal of adult house flies

USDA-ARS?s Scientific Manuscript database

Adult house flies (Diptera: Muscidae; Musca domestica L.) disseminate bacteria from microbe-rich substrates to areas where humans and domesticated animals reside. Because bacterial abundance fluctuates widely across substrates, flies encounter and ingest varying amounts of bacteria. We investigated ...

Wide-angle Spectrally Selective Perfect Absorber by Utilizing Dispersionless Tamm Plasmon Polaritons

PubMed Central

Xue, Chun-hua; Wu, Feng; Jiang, Hai-tao; Li, Yunhui; Zhang, Ye-wen; Chen, Hong

2016-01-01

We theoretically investigate wide-angle spectrally selective absorber by utilizing dispersionless Tamm plasmon polaritons (TPPs) under TM polarization. TPPs are resonant tunneling effects occurring on the interface between one-dimensional photonic crystals (1DPCs) and metal slab, and their dispersion properties are essentially determined by that of 1DPCs. Our investigations show that dispersionless TPPs can be excited in 1DPCs containing hyperbolic metamaterials (HMMs) on metal substrate. Based on dispersionless TPPs, electromagnetic waves penetrate into metal substrate and are absorbed entirely by lossy metal, exhibiting a narrow-band and wide-angle perfect absorption for TM polarization. Our results exhibit nearly perfect absorption with a value over 98% in the angle of incidence region of 0–80 degree. PMID:27991565

Surface plasmon-enhanced fluorescence on Au nanohole array for prostate-specific antigen detection

PubMed Central

Zhang, Qingwen; Wu, Lin; Wong, Ten It; Zhang, Jinling; Liu, Xiaohu; Zhou, Xiaodong; Bai, Ping; Liedberg, Bo; Wang, Yi

2017-01-01

Localized surface plasmon (LSP) has been widely applied for the enhancement of fluorescence emission for biosensing owing to its potential for strong field enhancement. However, due to its small penetration depth, LSP offers limited fluorescence enhancement over a whole sensor chip and, therefore, insufficient sensitivity for the detection of biomolecules, especially large molecules. We demonstrate the simultaneous excitation of LSP and propagating surface plasmon (PSP) on an Au nanohole array under Kretschmann configuration for the detection of prostate-specific antigen with a sandwich immunoassay. The proposed method combines the advantages of high field enhancement by LSP and large surface area probed by PSP field. The simulated results indicated that a maximum enhancement of electric field intensity up to 1,600 times can be achieved under the simultaneous excitation of LSP and PSP modes. The sandwich assay of PSA carried out on gold nanohole array substrate showed a limit of detection of 140 fM supporting coexcitation of LSP and PSP modes. The limit of detection was approximately sevenfold lower than that when only LSP was resonantly excited on the same substrate. The results of this study demonstrate high fluorescence enhancement through the coexcitation of LSP and PSP modes and pave a way for its implementation as a highly sensitive bioassay. PMID:28392689

Tree species diversity affects decomposition through modified micro-environmental conditions across European forests.

PubMed

Joly, François-Xavier; Milcu, Alexandru; Scherer-Lorenzen, Michael; Jean, Loreline-Katia; Bussotti, Filippo; Dawud, Seid Muhie; Müller, Sandra; Pollastrini, Martina; Raulund-Rasmussen, Karsten; Vesterdal, Lars; Hättenschwiler, Stephan

2017-05-01

Different tree species influence litter decomposition directly through species-specific litter traits, and indirectly through distinct modifications of the local decomposition environment. Whether these indirect effects on decomposition are influenced by tree species diversity is presently not clear. We addressed this question by studying the decomposition of two common substrates, cellulose paper and wood sticks, in a total of 209 forest stands of varying tree species diversity across six major forest types at the scale of Europe. Tree species richness showed a weak but positive correlation with the decomposition of cellulose but not with that of wood. Surprisingly, macroclimate had only a minor effect on cellulose decomposition and no effect on wood decomposition despite the wide range in climatic conditions among sites from Mediterranean to boreal forests. Instead, forest canopy density and stand-specific litter traits affected the decomposition of both substrates, with a particularly clear negative effect of the proportion of evergreen tree litter. Our study suggests that species richness and composition of tree canopies modify decomposition indirectly through changes in microenvironmental conditions. These canopy-induced differences in the local decomposition environment control decomposition to a greater extent than continental-scale differences in macroclimatic conditions. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

The role of nonbonded interactions in the conformational dynamics of organophosphorous hydrolase adsorbed onto functionalized mesoporous silica surfaces.

PubMed

Gomes, Diego E B; Lins, Roberto D; Pascutti, Pedro G; Lei, Chenghong; Soares, Thereza A

2010-01-14

The enzyme organophosphorous hydrolase (OPH) catalyzes the hydrolysis of a wide variety of organophosphorous compounds with high catalytic efficiency and broad substrate specificity. The immobilization of OPH in functionalized mesoporous silica (FMS) surfaces increases significantly its catalytic specific activity, as compared to the enzyme in solution, with important applications for the detection and decontamination of insecticides and chemical warfare agents. Experimental measurements of immobilization efficiency as a function of the charge and coverage percentage of different functional groups have been interpreted as electrostatic forces being the predominant interactions underlying the adsorption of OPH onto FMS surfaces. Explicit solvent molecular dynamics simulations have been performed for OPH in bulk solution and adsorbed onto two distinct interaction potential models of the FMS functional groups to investigate the relative contributions of nonbonded interactions to the conformational dynamics and adsorption of the protein. Our results support the conclusion that electrostatic interactions are responsible for the binding of OPH to the FMS surface. However, these results also show that van der Waals forces are detrimental for interfacial adhesion. In addition, it is found that OPH adsorption onto the FMS models favors a protein conformation whose active site is fully accessible to the substrate, in contrast to the unconfined protein.

Co-Immobilization of Proteins and DNA Origami Nanoplates to Produce High-Contrast Biomolecular Nanoarrays.

PubMed

Hager, Roland; Burns, Jonathan R; Grydlik, Martyna J; Halilovic, Alma; Haselgrübler, Thomas; Schäffler, Friedrich; Howorka, Stefan

2016-06-01

The biofunctionalization of nanopatterned surfaces with DNA origami nanostructures is an important topic in nanobiotechnology. An unexplored challenge is, however, to co-immobilize proteins with DNA origami at pre-determined substrate sites in high contrast relative to the nontarget areas. The immobilization should, in addition, preferably be achieved on a transparent substrate to allow ultrasensitive optical detection. If successful, specific co-binding would be a step towards stoichiometrically defined arrays with few to individual protein molecules per site. Here, we successfully immobilize with high specificity positively charged avidin proteins and negatively charged DNA origami nanoplates on 100 nm-wide carbon nanoislands while suppressing undesired adsorption to surrounding nontarget areas. The arrays on glass slides achieve unprecedented selectivity factors of up to 4000 and allow ultrasensitive fluorescence read-out. The co-immobilization onto the nanoislands leads to layered biomolecular architectures, which are functional because bound DNA origami influences the number of capturing sites on the nanopatches for other proteins. The novel hybrid DNA origami-protein nanoarrays allow the fabrication of versatile research platforms for applications in biosensing, biophysics, and cell biology, and, in addition, represent an important step towards single-molecule protein arrays. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Computer Simulations Reveal Substrate Specificity of Glycosidic Bond Cleavage in Native and Mutant Human Purine Nucleoside Phosphorylase.

PubMed

Isaksen, Geir Villy; Hopmann, Kathrin Helen; Åqvist, Johan; Brandsdal, Bjørn Olav

2016-04-12

Purine nucleoside phosphorylase (PNP) catalyzes the reversible phosphorolysis of purine ribonucleosides and 2'-deoxyribonucleosides, yielding the purine base and (2'-deoxy)ribose 1-phosphate as products. While this enzyme has been extensively studied, several questions with respect to the catalytic mechanism have remained largely unanswered. The role of the phosphate and key amino acid residues in the catalytic reaction as well as the purine ring protonation state is elucidated using density functional theory calculations and extensive empirical valence bond (EVB) simulations. Free energy surfaces for adenosine, inosine, and guanosine are fitted to ab initio data and yield quantitative agreement with experimental data when the surfaces are used to model the corresponding enzymatic reactions. The cognate substrates 6-aminopurines (inosine and guanosine) interact with PNP through extensive hydrogen bonding, but the substrate specificity is found to be a direct result of the electrostatic preorganization energy along the reaction coordinate. Asn243 has previously been identified as a key residue providing substrate specificity. Mutation of Asn243 to Asp has dramatic effects on the substrate specificity, making 6-amino- and 6-oxopurines equally good as substrates. The principal effect of this particular mutation is the change in the electrostatic preorganization energy between the native enzyme and the Asn243Asp mutant, clearly favoring adenosine over inosine and guanosine. Thus, the EVB simulations show that this particular mutation affects the electrostatic preorganization of the active site, which in turn can explain the substrate specificity.

Expression-based clustering of CAZyme-encoding genes of Aspergillus niger.

PubMed

Gruben, Birgit S; Mäkelä, Miia R; Kowalczyk, Joanna E; Zhou, Miaomiao; Benoit-Gelber, Isabelle; De Vries, Ronald P

2017-11-23

The Aspergillus niger genome contains a large repertoire of genes encoding carbohydrate active enzymes (CAZymes) that are targeted to plant polysaccharide degradation enabling A. niger to grow on a wide range of plant biomass substrates. Which genes need to be activated in certain environmental conditions depends on the composition of the available substrate. Previous studies have demonstrated the involvement of a number of transcriptional regulators in plant biomass degradation and have identified sets of target genes for each regulator. In this study, a broad transcriptional analysis was performed of the A. niger genes encoding (putative) plant polysaccharide degrading enzymes. Microarray data focusing on the initial response of A. niger to the presence of plant biomass related carbon sources were analyzed of a wild-type strain N402 that was grown on a large range of carbon sources and of the regulatory mutant strains ΔxlnR, ΔaraR, ΔamyR, ΔrhaR and ΔgalX that were grown on their specific inducing compounds. The cluster analysis of the expression data revealed several groups of co-regulated genes, which goes beyond the traditionally described co-regulated gene sets. Additional putative target genes of the selected regulators were identified, based on their expression profile. Notably, in several cases the expression profile puts questions on the function assignment of uncharacterized genes that was based on homology searches, highlighting the need for more extensive biochemical studies into the substrate specificity of enzymes encoded by these non-characterized genes. The data also revealed sets of genes that were upregulated in the regulatory mutants, suggesting interaction between the regulatory systems and a therefore even more complex overall regulatory network than has been reported so far. Expression profiling on a large number of substrates provides better insight in the complex regulatory systems that drive the conversion of plant biomass by fungi. In addition, the data provides additional evidence in favor of and against the similarity-based functions assigned to uncharacterized genes.

Is the molecular diversity of marine dissolved organic matter already imprinted in the exometabolome of single strains?

NASA Astrophysics Data System (ADS)

Noriega-Ortega, B. E.; Wienhausen, G.; Dittmar, T.; Simon, M.; Niggemann, J.

2016-02-01

Dissolved organic matter (DOM) in the ocean, the marine geometabolome, is an extremely complex mixture composed of a wide variety of compounds. The molecular chemodiversity affects the function and turnover rate of DOM in the ocean. We hypothesize that the active microbial community essentially contributes to the complexity of the DOM pool through uptake and excretion of compounds. We tested this hypothesis in culture experiments with fully-sequenced strains of the Roseobacter clade. Bacteria of the Roseobacter clade are among the most abundant microbial players in the ocean. We studied the exometabolome of two representatives of the Roseobacter clade, Phaeobacter inhibens DSM 17395 and Dinoroseobacter shibae. The organisms were grown separately in cultures on defined single model substrates (acetate, succinate, glutamate, glucose). We used a non-targeted analytical approach via Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) to characterize the exometabolome at the molecular level, complemented by compound-specific analyses of free and combined amino acids and carbohydrates. The exometabolome composition varied between the tested strains, which released a different suite of compounds depending on the growth phase as well as on growth conditions (substrate). Both organisms exhibited a core exometabolome with compounds released when growing on either substrate and at all growth phases, and a variable exometabolome specific for different substrates and growth phases. However, only a small fraction of the exometabolites detected by FT-ICR-MS could be directly linked to the genome or transcriptome. We interpret these findings as evidence for the excretion of molecularly highly-diverse metabolic waste, whose composition is dependent on the metabolic state and genetic repertoire of the organisms. The molecular diversity of compounds excreted by a single strain is extraordinary and is likely the reason for the molecular diversity of natural DOM in the ocean.

Proteomic Analysis of Arginine Methylation Sites in Human Cells Reveals Dynamic Regulation During Transcriptional Arrest*

PubMed Central

Sylvestersen, Kathrine B.; Horn, Heiko; Jungmichel, Stephanie; Jensen, Lars J.; Nielsen, Michael L.

2014-01-01

The covalent attachment of methyl groups to the side-chain of arginine residues is known to play essential roles in regulation of transcription, protein function, and RNA metabolism. The specific N-methylation of arginine residues is catalyzed by a small family of gene products known as protein arginine methyltransferases; however, very little is known about which arginine residues become methylated on target substrates. Here we describe a proteomics methodology that combines single-step immunoenrichment of methylated peptides with high-resolution mass spectrometry to identify endogenous arginine mono-methylation (MMA) sites. We thereby identify 1027 site-specific MMA sites on 494 human proteins, discovering numerous novel mono-methylation targets and confirming the majority of currently known MMA substrates. Nuclear RNA-binding proteins involved in RNA processing, RNA localization, transcription, and chromatin remodeling are predominantly found modified with MMA. Despite this, MMA sites prominently are located outside RNA-binding domains as compared with the proteome-wide distribution of arginine residues. Quantification of arginine methylation in cells treated with Actinomycin D uncovers strong site-specific regulation of MMA sites during transcriptional arrest. Interestingly, several MMA sites are down-regulated after a few hours of transcriptional arrest. In contrast, the corresponding di-methylation or protein expression levels are not altered, confirming that MMA sites contain regulated functions on their own. Collectively, we present a site-specific MMA data set in human cells and demonstrate for the first time that MMA is a dynamic post-translational modification regulated during transcriptional arrest by a hitherto uncharacterized arginine demethylase. PMID:24563534

Scale-up of 2G wire manufacturing at American Superconductor Corporation

NASA Astrophysics Data System (ADS)

Fleshler, S.; Buczek, D.; Carter, B.; Cedrone, P.; DeMoranville, K.; Gannon, J.; Inch, J.; Li, X.; Lynch, J.; Otto, A.; Podtburg, E.; Roy, D.; Rupich, M.; Sathyamurthy, S.; Schreiber, J.; Thieme, C.; Thompson, E.; Tucker, D.; Nagashima, K.; Ogata, M.

2009-10-01

American Superconductor Corporation (AMSC) has developed the base technology and a manufacturing line for initial volume production of low-cost second generation high temperature superconductor (2G HTS) wire for commercial and military applications. The manufacturing line is based on reel-to-reel processing of wide HTS strips using rolling assisted bi-axially textured substrate (RABiTS™) for the template and Metal Organic Deposition (MOD) for the HTS layer. AMSC’s wide strip process is a low cost manufacturing technology since multiple wires are produced in a single manufacturing pass by slitting the wide strip to narrower width in the last stage of the manufacturing process. Industry standard 4.4 mm wide wires are produced by laminating metallic foils, such as copper, stainless steel or any other material, to the HTS insert wire, and are chosen to tailor the electrical, thermal and mechanical properties of the wire for specific applications. The laminated, 4.4 mm wide wires are known as “344 superconductors.” In this paper, we summarize the status of AMSC’s manufacturing capability, the performance of the wire presently being produced, as well as the cost and technical advantages of AMSC’s manufacturing approach. In addition, future direction for research and development to improve electrical performance is presented.

Strategies in the design of small-molecule fluorescent probes for peptidases.

PubMed

Chen, Laizhong; Li, Jing; Du, Lupei; Li, Minyong

2014-11-01

Peptidases, which can cleave specific peptide bonds in innumerable categories of substrates, usually present pivotal positions in protein activation, cell signaling and regulation as well as in the origination of amino acids for protein generation or application in other metabolic pathways. They are also involved in many pathological conditions, such as cancer, atherosclerosis, arthritis, and neurodegenerative disorders. This review article aims to conduct a wide-ranging survey on the development of small-molecule fluorescent probes for peptidases, as well as to realize the state of the art in the tailor-made probes for diverse types of peptidases. © 2014 Wiley Periodicals, Inc.

Kinetic Studies that Evaluate the Solvolytic Mechanisms of Allyl and Vinyl Chloroformate Esters

PubMed Central

D’Souza, Malcolm J.; Givens, Aaron F.; Lorchak, Peter A.; Greenwood, Abigail E.; Gottschall, Stacey L.; Carter, Shannon E.; Kevill, Dennis N.

2013-01-01

At 25.0 °C the specific rates of solvolysis for allyl and vinyl chloroformates have been determined in a wide mix of pure and aqueous organic mixtures. In all the solvents studied, vinyl chloroformate was found to react significantly faster than allyl chloroformate. Multiple correlation analyses of these rates are completed using the extended (two-term) Grunwald-Winstein equation with incorporation of literature values for solvent nucleophilicity (NT) and solvent ionizing power (YCl). Both substrates were found to solvolyze by similar dual bimolecular carbonyl-addition and unimolecular ionization channels, each heavily dependent upon the solvents nucleophilicity and ionizing ability. PMID:23549265

Protein crystallography and site-direct mutagenesis analysis of the poly(ethylene terephthalate) hydrolase PETase from Ideonella sakaiensis.

PubMed

Liu, Bing; He, Lihui; Wang, Liping; Li, Tao; Li, Changcheng; Liu, Huayi; Luo, Yunzi; Bao, Rui

2018-03-30

Compared with traditional recycle strategies, biodegradation provides a sustainable solution for poly (ethylene terephthalate) (PET) wastes disposal. PETase, a newly identified enzyme from Ideonella sakaiensis, has high efficiency and specificity towards PET, which provides a prominent prospect on PET degradation. Based on the biochemical analysis, we propose that the wide substrate-binding pocket is critical for its excellent property on crystallized PET hydrolysis. Structure-guided site-directed mutagenesis exhibited improvement in PETase catalytic efficiency, providing valuable insight on how the molecular engineering of PETase can optimize its application in biocatalysis. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

L-Rhamnose isomerase and its use for biotechnological production of rare sugars.

PubMed

Xu, Wei; Zhang, Wenli; Zhang, Tao; Jiang, Bo; Mu, Wanmeng

2016-04-01

L-Rhamnose isomerase (L-RI, EC 5.3.1.14), catalyzing the isomerization between L-rhamnose and L-rhamnulose, plays an important role in microbial L-rhamnose metabolism and thus occurs in a wide range of microorganisms. It attracts more and more attention because of its broad substrate specificity and its great potential in enzymatic production of various rare sugars. In this article, the enzymatic properties of various reported L-RIs were compared in detail, and their applications in the production of L-rhamnulose and various rare sugars including D-allose, D-gulose, L-lyxose, L-mannose, L-talose, and L-galactose were also reviewed.

Resveratrol serves as a protein-substrate interaction stabilizer in human SIRT1 activation

NASA Astrophysics Data System (ADS)

Hou, Xuben; Rooklin, David; Fang, Hao; Zhang, Yingkai

2016-11-01

Resveratrol is a natural compound found in red wine that has been suggested to exert its potential health benefit through the activation of SIRT1, a crucial member of the mammalian NAD+-dependent deacetylases. SIRT1 has emerged as an attractive therapeutic target for many aging related diseases, however, how its activity can only be activated toward some specific substrates by resveratrol has been poorly understood. Herein, by employing extensive molecular dynamics simulations as well as fragment-centric topographical mapping of binding interfaces, we have clarified current controversies in the literature and elucidated that resveratrol plays an important activation role by stabilizing SIRT1/peptide interactions in a substrate-specific manner. This new mechanism highlights the importance of the N-terminal domain in substrate recognition, explains the activity restoration role of resveratrol toward some “loose-binding” substrates of SIRT1, and has significant implications for the rational design of new substrate-specific SIRT1 modulators.

Measuring the Global Substrate Specificity of Mycobacterial Serine Hydrolases Using a Library of Fluorogenic Ester Substrates.

PubMed

Bassett, Braden; Waibel, Brent; White, Alex; Hansen, Heather; Stephens, Dominique; Koelper, Andrew; Larsen, Erik M; Kim, Charles; Glanzer, Adam; Lavis, Luke D; Hoops, Geoffrey C; Johnson, R Jeremy

2018-04-16

Among the proteins required for lipid metabolism in Mycobacterium tuberculosis are a significant number of uncharacterized serine hydrolases, especially lipases and esterases. Using a streamlined synthetic method, a library of immolative fluorogenic ester substrates was expanded to better represent the natural lipidomic diversity of Mycobacterium. This expanded fluorogenic library was then used to rapidly characterize the global structure activity relationship (SAR) of mycobacterial serine hydrolases in M. smegmatis under different growth conditions. Confirmation of fluorogenic substrate activation by mycobacterial serine hydrolases was performed using nonspecific serine hydrolase inhibitors and reinforced the biological significance of the SAR. The hydrolases responsible for the global SAR were then assigned using gel-resolved activity measurements, and these assignments were used to rapidly identify the relative substrate specificity of previously uncharacterized mycobacterial hydrolases. These measurements provide a global SAR of mycobacterial hydrolase activity, a picture of cycling hydrolase activity, and a detailed substrate specificity profile for previously uncharacterized hydrolases.

Resveratrol serves as a protein-substrate interaction stabilizer in human SIRT1 activation.

PubMed

Hou, Xuben; Rooklin, David; Fang, Hao; Zhang, Yingkai

2016-11-30

Resveratrol is a natural compound found in red wine that has been suggested to exert its potential health benefit through the activation of SIRT1, a crucial member of the mammalian NAD + -dependent deacetylases. SIRT1 has emerged as an attractive therapeutic target for many aging related diseases, however, how its activity can only be activated toward some specific substrates by resveratrol has been poorly understood. Herein, by employing extensive molecular dynamics simulations as well as fragment-centric topographical mapping of binding interfaces, we have clarified current controversies in the literature and elucidated that resveratrol plays an important activation role by stabilizing SIRT1/peptide interactions in a substrate-specific manner. This new mechanism highlights the importance of the N-terminal domain in substrate recognition, explains the activity restoration role of resveratrol toward some "loose-binding" substrates of SIRT1, and has significant implications for the rational design of new substrate-specific SIRT1 modulators.

Engineering the substrate specificity of the DhbE adenylation domain by yeast cell surface display.

PubMed

Zhang, Keya; Nelson, Kathryn M; Bhuripanyo, Karan; Grimes, Kimberly D; Zhao, Bo; Aldrich, Courtney C; Yin, Jun

2013-01-24

The adenylation (A) domains of nonribosomal peptide synthetases (NRPSs) activate aryl acids or amino acids to launch their transfer through the NRPS assembly line for the biosynthesis of many medicinally important natural products. In order to expand the substrate pool of NRPSs, we developed a method based on yeast cell surface display to engineer the substrate specificities of the A-domains. We acquired A-domain mutants of DhbE that have 11- and 6-fold increases in k(cat)/K(m) with nonnative substrates 3-hydroxybenzoic acid and 2-aminobenzoic acid, respectively and corresponding 3- and 33-fold decreases in k(cat)/K(m) values with the native substrate 2,3-dihydroxybenzoic acid, resulting in a dramatic switch in substrate specificity of up to 200-fold. Our study demonstrates that yeast display can be used as a high throughput selection platform to reprogram the "nonribosomal code" of A-domains. Copyright © 2013 Elsevier Ltd. All rights reserved.

Optical and Interface-Based Methods of Defect Engineering in Silicon

ERIC Educational Resources Information Center

Kondratenko, Yevgeniy Vladimirovich

2009-01-01

Ion implantation is widely used in the microelectronics industry for fabrication of source and drain transistor regions. Unfortunately, implantation causes considerable damage to the substrate lattice rendering most of the implanted dopant electrically inactive. Rapid thermal annealing (RTA) heals the damage by rapidly heating the substrate with a…

Substrate-Driven Mapping of the Degradome by Comparison of Sequence Logos

PubMed Central

Fuchs, Julian E.; von Grafenstein, Susanne; Huber, Roland G.; Kramer, Christian; Liedl, Klaus R.

2013-01-01

Sequence logos are frequently used to illustrate substrate preferences and specificity of proteases. Here, we employed the compiled substrates of the MEROPS database to introduce a novel metric for comparison of protease substrate preferences. The constructed similarity matrix of 62 proteases can be used to intuitively visualize similarities in protease substrate readout via principal component analysis and construction of protease specificity trees. Since our new metric is solely based on substrate data, we can engraft the protease tree including proteolytic enzymes of different evolutionary origin. Thereby, our analyses confirm pronounced overlaps in substrate recognition not only between proteases closely related on sequence basis but also between proteolytic enzymes of different evolutionary origin and catalytic type. To illustrate the applicability of our approach we analyze the distribution of targets of small molecules from the ChEMBL database in our substrate-based protease specificity trees. We observe a striking clustering of annotated targets in tree branches even though these grouped targets do not necessarily share similarity on protein sequence level. This highlights the value and applicability of knowledge acquired from peptide substrates in drug design of small molecules, e.g., for the prediction of off-target effects or drug repurposing. Consequently, our similarity metric allows to map the degradome and its associated drug target network via comparison of known substrate peptides. The substrate-driven view of protein-protein interfaces is not limited to the field of proteases but can be applied to any target class where a sufficient amount of known substrate data is available. PMID:24244149

Lightweight Electrode For Nickel/Hydrogen Cell

NASA Technical Reports Server (NTRS)

Britton, Doris L.

1994-01-01

Improved substrate for nickel electrode increases specific energy of nickel/hydrogen cell. Consists of 50 percent by weight nickel fiber, 35 percent nickel powder, and 15 percent cobalt powder. Porosity and thickness of nickel electrodes affect specific energy, initial performance, and cycle life of cell. Substrate easily manufactured with much larger porosities than those of heavy-sintered state-of-art nickel substrate.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Crosby, Heidi A; Pelletier, Dale A; Hurst, Gregory

Background: Protein acetylation is widespread in prokaryotes. Results: Six new acyl-CoA synthetases whose activities are controlled by acetylation were identified, and their substrate preference established. A new protein acetyltransferase was also identified and its substrate specificity determined. Conclusion: Protein acetyltransferases acetylate a conserved lysine residue in protein substrates. Significance: The R. palustris Pat enzyme specifically acetylates AMP-forming acyl-CoA synthetases and regulates fatty acid metabolism.

High-gain dipole antenna using polydimethylsiloxane-glass microsphere (PDMS-GM) substrate for 5G applications

NASA Astrophysics Data System (ADS)

Muhamad, Wan Asilah Wan; Ngah, Razali; Jamlos, Mohd Faizal; Soh, Ping Jack; Ali, Mohd Tarmizi

2017-01-01

A new dipole antenna designed using polydimethylsiloxane-glass microsphere (PDMS-GM) substrate is presented. The PDMS-GM substrate offered a lower permittivity of 1.85 compared to pure PDMS of 2.7. This resulted in a wide operating frequency range from 19 GHz up to more than 45 GHz, indicating a bandwidth of more than 28 GHz. The proposed PDMS-GM antenna featured a gain of up to 13.3 dB compared to pure PDMS which only produced 13 GHz of bandwidth and 5.5 dB gain. Instead of wide bandwidth and high gain, the proposed antenna is capable of becoming water resistant by covering its radiator and SMA connector. Such capabilities of the new PDMS-GM antenna indicated suitability for the fifth-generation (5G) wireless communication systems.

Identification of Explosives from Porous Materials: Applications Using Reverse Phase High Performance Liquid Chromatography and Gas Chromatography

DOE Office of Scientific and Technical Information (OSTI.GOV)

C.J. Miller; G. Elias; N.C. Schmitt

2010-06-01

High performance liquid chromatography and gas chromatography techniques are well documented and widely used for the detection of trace explosives from organic solvents. These techniques were modified to specifically identify and quantify explosives extracted from various materials taken from people who had recently handled explosives. Documented techniques were modified to specifically detect and quantify RDX, TNT, and PETN from denim, colored flannel, vinyl, and canvas extracted in methanol using no sample cleanup prior to analysis. The methanol extracts were injected directly into several different column types and analyzed by HPLC-UV and/or GC-ECD. This paper describes general screening methods that weremore » used to determine the presence of explosives in unknown samples and techniques that have been optimized for quantification of each explosive from the substrate extracts.« less

Fluorescent Dendritic Micro-Hydrogels: Synthesis, Analysis and Use in Single-Cell Detection.

PubMed

Christadore, Lisa; Grinstaff, Mark W; Schaus, Scott E

2018-04-18

Hydrogels are of keen interest for a wide range of medical and biotechnological applications including as 3D substrate structures for the detection of proteins, nucleic acids, and cells. Hydrogel parameters such as polymer wt % and crosslink density are typically altered for a specific application; now, fluorescence can be incorporated into such criteria by specific macromonomer selection. Intrinsic fluorescence was observed at λ max 445 nm from hydrogels polymerized from lysine and aldehyde- terminated poly(ethylene glycol) macromonomers upon excitation with visible light. The hydrogel’s photochemical properties are consistent with formation of a nitrone functionality. Printed hydrogels of 150 μm were used to detect individual cell adherence via a decreased in fluorescence. The use of such intrinsically fluorescent hydrogels as a platform for cell sorting and detection expands the current repertoire of tools available.

Keratinolytic protease: a green biocatalyst for leather industry.

PubMed

Fang, Zhen; Yong, Yang-Chun; Zhang, Juan; Du, Guocheng; Chen, Jian

2017-11-01

Depilation/unhairing is the crucial but heavy pollution process in leather industry. Traditional inorganic sulfide treatment was the most widely used depilation technique in the past decades, which was usually detrimental to leather quality and resulted in serious environmental pollution. Using biocatalysts to substitute inorganic sulfide showed great advantages in environment protection and unhairing efficiency. Keratinolytic protease is one of the excellent biocatalysts to hydrolyze disulfide bond-rich proteins of hair and has little damage to leather. Biological treatment with keratinolytic proteases could largely reduce the quantity and toxicity of wastewater effluent from the leather industry. But low thermostability and substrate specificity or specific activity of these enzymes limited their practical application. Therefore, recent progresses on protein engineering strategies (site-directed mutagenesis, protein fusion, N/C-terminus truncation, and domain swapping) used to enhance the keratinolytic enzyme performance were presented.

Challenges and the state of the technology for printed sensor arrays for structural monitoring

NASA Astrophysics Data System (ADS)

Joshi, Shiv; Bland, Scott; DeMott, Robert; Anderson, Nickolas; Jursich, Gregory

2017-04-01

Printed sensor arrays are attractive for reliable, low-cost, and large-area mapping of structural systems. These sensor arrays can be printed on flexible substrates or directly on monitored structural parts. This technology is sought for continuous or on-demand real-time diagnosis and prognosis of complex structural components. In the past decade, many innovative technologies and functional materials have been explored to develop printed electronics and sensors. For example, an all-printed strain sensor array is a recent example of a low-cost, flexible and light-weight system that provides a reliable method for monitoring the state of aircraft structural parts. Among all-printing techniques, screen and inkjet printing methods are well suited for smaller-scale prototyping and have drawn much interest due to maturity of printing procedures and availability of compatible inks and substrates. Screen printing relies on a mask (screen) to transfer a pattern onto a substrate. Screen printing is widely used because of the high printing speed, large selection of ink/substrate materials, and capability of making complex multilayer devices. The complexity of collecting signals from a large number of sensors over a large area necessitates signal multiplexing electronics that need to be printed on flexible substrate or structure. As a result, these components are subjected to same deformation, temperature and other parameters for which sensor arrays are designed. The characteristics of these electronic components, such as transistors, are affected by deformation and other environmental parameters which can lead to erroneous sensed parameters. The manufacturing and functional challenges of the technology of printed sensor array systems for structural state monitoring are the focus of this presentation. Specific examples of strain sensor arrays will be presented to highlight the technical challenges.

Synthesis of 1-[bis(trifluoromethyl)phosphine]-1'-oxazolinylferrocene ligands and their application in regio- and enantioselective Pd-catalyzed allylic alkylation of monosubstituted allyl substrates.

PubMed

Lai, Zeng-Wei; Yang, Rong-Fei; Ye, Ke-Yin; Sun, Hongbin; You, Shu-Li

2014-01-01

A class of novel, easily accessible and air-stable 1-[bis(trifluoromethyl)phosphine]-1'-oxazolinylferrocene ligands has been synthesized from ferrocene. It became apparent that these ligands can be used in the regio- and enantioselective Pd-catalyzed allylic alkylation of monosubstituted allyl substrates in a highly efficient manner. Excellent regio- and enantioselectivity could be obtained for a wide range of substrates.

Crystal structure of low-molecular-weight protein tyrosine phosphatase from Mycobacterium tuberculosis at 1.9-A resolution.

PubMed

Madhurantakam, Chaithanya; Rajakumara, Eerappa; Mazumdar, Pooja Anjali; Saha, Baisakhee; Mitra, Devrani; Wiker, Harald G; Sankaranarayanan, Rajan; Das, Amit Kumar

2005-03-01

The low-molecular-weight protein tyrosine phosphatase (LMWPTPase) belongs to a distinctive class of phosphotyrosine phosphatases widely distributed among prokaryotes and eukaryotes. We report here the crystal structure of LMWPTPase of microbial origin, the first of its kind from Mycobacterium tuberculosis. The structure was determined to be two crystal forms at 1.9- and 2.5-A resolutions. These structural forms are compared with those of the LMWPTPases of eukaryotes. Though the overall structure resembles that of the eukaryotic LMWPTPases, there are significant changes around the active site and the protein tyrosine phosphatase (PTP) loop. The variable loop forming the wall of the crevice leading to the active site is conformationally unchanged from that of mammalian LMWPTPase; however, differences are observed in the residues involved, suggesting that they have a role in influencing different substrate specificities. The single amino acid substitution (Leu12Thr [underlined below]) in the consensus sequence of the PTP loop, CTGNICRS, has a major role in the stabilization of the PTP loop, unlike what occurs in mammalian LMWPTPases. A chloride ion and a glycerol molecule were modeled in the active site where the chloride ion interacts in a manner similar to that of phosphate with the main chain nitrogens of the PTP loop. This structural study, in addition to identifying specific mycobacterial features, may also form the basis for exploring the mechanism of the substrate specificities of bacterial LMWPTPases.

Identification and Characterization of a Novel Galactofuranose-Specific β-D-Galactofuranosidase from Streptomyces Species

PubMed Central

Matsunaga, Emiko; Higuchi, Yujiro; Mori, Kazuki; Yairo, Nao; Oka, Takuji; Shinozuka, Saki; Tashiro, Kosuke; Izumi, Minoru; Kuhara, Satoru; Takegawa, Kaoru

2015-01-01

β-D-galactofuranose (Galf) is a component of polysaccharides and glycoconjugates and its transferase has been well analyzed. However, no β-D-galactofuranosidase (Galf-ase) gene has been identified in any organism. To search for a Galf-ase gene we screened soil samples and discovered a strain, identified as a Streptomyces species by the 16S ribosomal RNA gene analysis, that exhibits Galf-ase activity for 4-nitrophenyl β-D-galactofuranoside (pNP-β-D-Galf) in culture supernatants. By draft genome sequencing of the strain, named JHA19, we found four candidate genes encoding Galf-ases. Using recombinant proteins expressed in Escherichia coli, we found that three out of four candidates displayed the activity of not only Galf-ase but also α-L-arabinofuranosidase (Araf-ase), whereas the other one showed only the Galf-ase activity. This novel Galf-specific hydrolase is encoded by ORF1110 and has an optimum pH of 5.5 and a Km of 4.4 mM for the substrate pNP-β-D-Galf. In addition, this enzyme was able to release galactose residue from galactomannan prepared from the filamentous fungus Aspergillus fumigatus, suggesting that natural polysaccharides could be also substrates. By the BLAST search using the amino acid sequence of ORF1110 Galf-ase, we found that there are homolog genes in both prokaryotes and eukaryotes, indicating that Galf-specific Galf-ases widely exist in microorganisms. PMID:26340350

Specificity of hammerhead ribozyme cleavage.

PubMed Central

Hertel, K J; Herschlag, D; Uhlenbeck, O C

1996-01-01

To be effective in gene inactivation, the hammerhead ribozyme must cleave a complementary RNA target without deleterious effects from cleaving non-target RNAs that contain mismatches and shorter stretches of complementarity. The specificity of hammerhead cleavage was evaluated using HH16, a well-characterized ribozyme designed to cleave a target of 17 residues. Under standard reaction conditions, HH16 is unable to discriminate between its full-length substrate and 3'-truncated substrates, even when six fewer base pairs are formed between HH16 and the substrate. This striking lack of specificity arises because all the substrates bind to the ribozyme with sufficient affinity so that cleavage occurs before their affinity differences are manifested. In contrast, HH16 does exhibit high specificity towards certain 3'-truncated versions of altered substrates that either also contain a single base mismatch or are shortened at the 5' end. In addition, the specificity of HH16 is improved in the presence of p7 nucleocapsid protein from human immunodeficiency virus (HIV)-1, which accelerates the association and dissociation of RNA helices. These results support the view that the hammerhead has an intrinsic ability to discriminate against incorrect bases, but emphasizes that the high specificity is only observed in a certain range of helix lengths. Images PMID:8670879

Long-range Electrostatic Complementarity Governs Substrate Recognition by Human Chymotrypsin C, a Key Regulator of Digestive Enzyme Activation*

PubMed Central

Batra, Jyotica; Szabó, András; Caulfield, Thomas R.; Soares, Alexei S.; Sahin-Tóth, Miklós; Radisky, Evette S.

2013-01-01

Human chymotrypsin C (CTRC) is a pancreatic serine protease that regulates activation and degradation of trypsinogens and procarboxypeptidases by targeting specific cleavage sites within their zymogen precursors. In cleaving these regulatory sites, which are characterized by multiple flanking acidic residues, CTRC shows substrate specificity that is distinct from that of other isoforms of chymotrypsin and elastase. Here, we report the first crystal structure of active CTRC, determined at 1.9-Å resolution, revealing the structural basis for binding specificity. The structure shows human CTRC bound to the small protein protease inhibitor eglin c, which binds in a substrate-like manner filling the S6-S5′ subsites of the substrate binding cleft. Significant binding affinity derives from burial of preferred hydrophobic residues at the P1, P4, and P2′ positions of CTRC, although acidic P2′ residues can also be accommodated by formation of an interfacial salt bridge. Acidic residues may also be specifically accommodated in the P6 position. The most unique structural feature of CTRC is a ring of intense positive electrostatic surface potential surrounding the primarily hydrophobic substrate binding site. Our results indicate that long-range electrostatic attraction toward substrates of concentrated negative charge governs substrate discrimination, which explains CTRC selectivity in regulating active digestive enzyme levels. PMID:23430245

Substrate specificity of xenobiotic metabolizing esterases in the liver of two catfish species

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jaiswal, R.G.; Huang, T.L.; Obih, P.O.

1994-12-31

The preliminary studies were conducted on the characterization of substrate specificity in the liver microsomes and cytosol of two catfish species, Ictalurus punctatus and Ictalurus natalie. A series of five esters of p-nitrophenol were used as calorimetric substrates to assay the carboxylesterases. The substrate specificity of liver microsomal and cytosolic carboxylesterases were remarkably different from each other. The valerate ester of p-nitrophenol was most rapidly hydrolyzed by the microsomal carboxylesterases, whereas the prioponate ester was the best substrate for cytosolic carboxylesterases. The Ictalurus natalie catfish species were obtained from the Devil Swamp site of the Mississippi River Basin which ismore » known to be heavily contaminated with toxic and hazardous industrial wastes. These results will be discussed in relation to the responses of xenobiotic metabolizing esterases to environmental pollutants and their possible use as biomarkers.« less

Analysis of substrate specificity of human DHHC protein acyltransferases using a yeast expression system

PubMed Central

Ohno, Yusuke; Kashio, Atsushi; Ogata, Ren; Ishitomi, Akihiro; Yamazaki, Yuki; Kihara, Akio

2012-01-01

Palmitoylation plays important roles in the regulation of protein localization, stability, and activity. The protein acyltransferases (PATs) have a common DHHC Cys-rich domain. Twenty-three DHHC proteins have been identified in humans. However, it is unclear whether all of these DHHC proteins function as PATs. In addition, their substrate specificities remain largely unknown. Here we develop a useful method to examine substrate specificities of PATs using a yeast expression system with six distinct model substrates. We identify 17 human DHHC proteins as PATs. Moreover, we classify 11 human and 5 yeast DHHC proteins into three classes (I, II, and III), based on the cellular localization of their respective substrates (class I, soluble proteins; class II, integral membrane proteins; class III, lipidated proteins). Our results may provide an important clue for understanding the function of individual DHHC proteins. PMID:23034182

Dissecting substrate specificities of the mitochondrial AFG3L2 protease.

PubMed

Ding, Bojian; Martin, Dwight W; Rampello, Anthony J; Glynn, Steven E

2018-06-22

Human AFG3L2 is a compartmental AAA+ protease that performs ATP-fueled degradation at the matrix face of the inner mitochondrial membrane. Identifying how AFG3L2 selects substrates from the diverse complement of matrix-localized proteins is essential for understanding mitochondrial protein biogenesis and quality control. Here, we create solubilized forms of AFG3L2 to examine the enzyme's substrate specificity mechanisms. We show that conserved residues within the pre-sequence of the mitochondrial ribosomal protein, MrpL32, target the subunit to the protease for processing into a mature form. Moreover, these residues can act as a degron, delivering diverse model proteins to AFG3L2 for degradation. By determining the sequence of degra-dation products from multiple substrates using mass spectrometry, we construct a peptidase specificity pro-file that displays constrained product lengths and is dominated by the identity of the residue at the P1' posi-tion, with a strong preference for hydrophobic and small polar residues. This specificity profile is validated by examining the cleavage of both fluorogenic reporter peptides and full polypeptide substrates bearing different P1' residues. Together, these results demonstrate that AFG3L2 contains multiple modes of specificity, dis-criminating between potential substrates by recognizing accessible degron sequences, and performing peptide bond cleavage at preferred patterns of residues within the compartmental chamber.

Critical Amino Acids in the Active Site of Meprin Metalloproteinases for Substrate and Peptide Bond Specificity*

PubMed Central

Villa, James P.; Bertenshaw, Greg P.; Bond, Judith S.

2008-01-01

SUMMARY The protease domains of the evolutionarily-related α and ß subunits of meprin metalloproteases are approximately 55% identical at the amino acid level, however, their substrate and peptide bond specificities differ markedly. The meprin ß subunit favors acidic residues proximal to the scissile bond, while the α subunit prefers small or aromatic amino acids flanking the scissile bond. Thus gastrin, a peptide that contains a string of five Glu residues, is an excellent substrate for meprin ß while it is not hydrolyzed by meprin α. Work herein aimed to identify critical amino acids in the meprin active sites that determine the substrate specificity differences. Sequence alignments and homology models, based on the crystal structure of the crayfish astacin, showed electrostatic differences within the meprin active sites. Site-directed mutagenesis of active site residues demonstrated that replacement of a hydrophobic residue by a basic amino acid enabled the meprin α protease to cleave gastrin. The meprin αY199K mutant was most effective; the corresponding mutation of meprin ßK185Y resulted in decreased activity toward gastrin. Peptide cleavage site determinations and kinetic analyses using a variety of peptides extended evidence that meprin αTyr199/ßLys185 are substrate specificity determinants in meprin active sites. These studies shed light on the molecular basis for the substrate specificity differences of astacin metalloproteinases. PMID:12888571

Computational Study on Substrate Specificity of a Novel Cysteine Protease 1 Precursor from Zea mays

PubMed Central

Liu, Huimin; Chen, Liangcheng; Li, Quan; Zheng, Mingzhu; Liu, Jingsheng

2014-01-01

Cysteine protease 1 precursor from Zea mays (zmCP1) is classified as a member of the C1A family of peptidases (papain-like cysteine protease) in MEROPS (the Peptidase Database). The 3D structure and substrate specificity of the zmCP1 is still unknown. This study is the first one to build the 3D structure of zmCP1 by computer-assisted homology modeling. In order to determine the substrate specificity of zmCP1, docking study is used for rapid and convenient analysis of large populations of ligand–enzyme complexes. Docking results show that zmCP1 has preference for P1 position and P2 position for Arg and a large hydrophobic residue (such as Phe). Gly147, Gly191, Cys189, and Asp190 are predicted to function as active residues at the S1 subsite, and the S2 subsite contains Leu283, Leu193, Ala259, Met194, and Ala286. SIFt results indicate that Gly144, Arg268, Trp308, and Ser311 play important roles in substrate binding. Then Molecular Mechanics-Poisson-Boltzmann Surface Area (MM-PBSA) method was used to explain the substrate specificity for P1 position of zmCp1. This study provides insights into the molecular basis of zmCP1 activity and substrate specificity. PMID:24921705

A multiwell format assay for heparanase.

PubMed

Behzad, Farhad; Brenchley, Paul E C

2003-09-15

This assay employs a biotinylated heparan sulfate glycosaminoglycan (HSGAG) substrate that is covalently linked to the surface of 96-well immunoassay plates. The ratio of biotin:HSGAG and the coating concentration of substrate bound to the wells have been optimized and allow removal of biotin HSGAG within 60 min of incubation at 37 degrees C in assay buffer with a standard dilution of bacterial heparitinase or platelet heparanase. Loss of biotin signal from the well surface is detected on incubation with peroxidase-streptavidin followed by color development using 3,3',5,5'-tetramethylbenzidine as the peroxidase substrate. The new assay allows specific detection of heparanase activity in multiple samples in a total time of 3 h including a 1-h substrate digestion step and is a significant improvement with regard to sensitivity, specificity, and ease of handling of multiple samples compared to other described assays. Heparanase specifically degrades the biotinylated HSGAG substrate, when used with an optimized assay buffer. A range of enzymes including collagenase, trypsin, plasmin, pepsin, chondroitinases, hyaluronidase, and neuraminidase show no effect on the substrate under optimized assay conditions. The covalent linkage of the substrate to the well prevents leaching of substrate and allows preparation and long-term storage of substrate-coated plates. The assay can be used to detect heparanase levels in clinical samples and cell culture supernatants and is ideal as a screening method for antagonists of enzyme activity.

Nanoengineered Polystyrene Surfaces with Nanopore Array Pattern Alters Cytoskeleton Organization and Enhances Induction of Neural Differentiation of Human Adipose-Derived Stem Cells.

PubMed

Jung, Ae Ryang; Kim, Richard Y; Kim, Hyung Woo; Shrestha, Kshitiz Raj; Jeon, Seung Hwan; Cha, Kyoung Je; Park, Yong Hyun; Kim, Dong Sung; Lee, Ji Youl

2015-07-01

Human adipose-derived stem cells (hADSCs) can differentiate into various cell types depending on chemical and topographical cues. One topographical cue recently noted to be successful in inducing differentiation is the nanoengineered polystyrene surface containing nanopore array-patterned substrate (NP substrate), which is designed to mimic the nanoscale topographical features of the extracellular matrix. In this study, efficacies of NP and flat substrates in inducing neural differentiation of hADSCs were examined by comparing their substrate-cell adhesion rates, filopodia growth, nuclei elongation, and expression of neural-specific markers. The polystyrene nano Petri dishes containing NP substrates were fabricated by a nano injection molding process using a nickel electroformed nano-mold insert (Diameter: 200 nm. Depth of pore: 500 nm. Center-to-center distance: 500 nm). Cytoskeleton and filopodia structures were observed by scanning electron microscopy and F-actin staining, while cell adhesion was tested by vinculin staining after 24 and 48 h of seeding. Expression of neural specific markers was examined by real-time quantitative polymerase chain reaction and immunocytochemistry. Results showed that NP substrates lead to greater substrate-cell adhesion, filopodia growth, nuclei elongation, and expression of neural specific markers compared to flat substrates. These results not only show the advantages of NP substrates, but they also suggest that further study into cell-substrate interactions may yield great benefits for biomaterial engineering.

Visual cues for woodpeckers: light reflectance of decayed wood varies by decay fungus

USGS Publications Warehouse

O'Daniels, Sean T.; Kesler, Dylan C.; Mihail, Jeanne D.; Webb, Elisabeth B.; Werner, Scott J.

2018-01-01

The appearance of wood substrates is likely relevant to bird species with life histories that require regular interactions with wood for food and shelter. Woodpeckers detect decayed wood for cavity placement or foraging, and some species may be capable of detecting trees decayed by specific fungi; however, a mechanism allowing for such specificity remains unidentified. We hypothesized that decay fungi associated with woodpecker cavity sites alter the substrate reflectance in a species-specific manner that is visually discriminable by woodpeckers. We grew 10 species of wood decay fungi from pure cultures on sterile wood substrates of 3 tree species. We then measured the relative reflectance spectra of decayed and control wood wafers and compared them using the receptor noise-limited (RNL) color discrimination model. The RNL model has been used in studies of feather coloration, egg shells, flowers, and fruit to model how the colors of objects appear to birds. Our analyses indicated 6 of 10 decayed substrate/control comparisons were above the threshold of discrimination (i.e., indicating differences discriminable by avian viewers), and 12 of 13 decayed substrate comparisons were also above threshold for a hypothetical woodpecker. We conclude that woodpeckers should be capable of visually detecting decayed wood on trees where bark is absent, and they should also be able to detect visually species-specific differences in wood substrates decayed by fungi used in this study. Our results provide evidence for a visual mechanism by which woodpeckers could identify and select substrates decayed by specific fungi, which has implications for understanding ecologically important woodpecker–fungus interactions.

Substrate specificity effects of lipoxygenase products and inhibitors on soybean lipoxygenase-1.

PubMed

Wecksler, Aaron T; Garcia, Natalie K; Holman, Theodore R

2009-09-15

Recently, it has been shown that lipoxygenase (LO) products affect the substrate specificity of human 15-LO. In the current paper, we demonstrate that soybean LO-1 (sLO-1) is not affected by its own products, however, inhibitors which bind the allosteric site, oleyl sulfate (OS) and palmitoleyl sulfate (PS), not only lower catalytic activity, but also change the substrate specificity, by increasing the arachidonic acid (AA)/linoleic acid (LA) ratio to 4.8 and 4.0, respectively. The fact that LO inhibitors can lower activity and also change the LO product ratio is a new concept in lipoxygenase inhibition, where the goal is to not only reduce the catalytic activity but also alter substrate selectivity towards a physiologically beneficial product.

Identification of ribozymes within a ribozyme library that efficiently cleave a long substrate RNA.

PubMed Central

Campbell, T B; Cech, T R

1995-01-01

Positions 2-6 of the substrate-binding internal guide sequence (IGS) of the L-21 Sca I form of the Tetrahymena thermophila intron were mutagenized to produce a GN5 IGS library. Ribozymes within the GN5 library capable of efficient cleavage of an 818-nt human immunodeficiency virus type 1 vif-vpr RNA, at 37 degrees C, were identified by ribozyme-catalyzed guanosine addition to the 3' cleavage product. Three ribozymes (IGS = GGGGCU, GGCUCC, and GUGGCU) within the GN5 library that actively cleaved the long substrate were characterized kinetically and compared to the wild-type ribozyme (GGAGGG) and two control ribozymes (GGAGUC and GGAGAU). The two control ribozymes have specific sites within the long substrate, but were not identified during screening of the library. Under single-turnover conditions, ribozymes GGGGCU, GGCUCC, and GUGGCU cleaved the 818-nt substrate 4- to 200-fold faster than control ribozymes. Short cognate substrates, which should be structureless and therefore accessible to ribozyme binding, were cleaved at similar rates by all ribozymes except GGGGCU, which showed a fourfold rate enhancement. The rate of cleavage of long relative to short substrate under single-turnover conditions suggests that GGCUCC and GUGGCU were identified because of accessibility to their specific cleavage sites within the long substrate (substrate-specific effects), whereas GGGGCU was identified because of an enhanced rate of substrate binding despite a less accessible site in the long substrate. Even though screening was performed with 100-fold excess substrate (relative to total ribozyme), the rate of multiple-turnover catalysis did not contribute to identification of trans-cleaving ribozymes in the GN5 library. PMID:7489519

Epitaxial growth of 100-μm thick M-type hexaferrite crystals on wide bandgap semiconductor GaN/Al{sub 2}O{sub 3} substrates

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hu, Bolin; Su, Zhijuan; Bennett, Steve

2014-05-07

Thick barium hexaferrite BaFe{sub 12}O{sub 19} (BaM) films having thicknesses of ∼100 μm were epitaxially grown on GaN/Al{sub 2}O{sub 3} substrates from a molten-salt solution by vaporizing the solvent. X-ray diffraction measurement verified the growth of BaM (001) textured growth of thick films. Saturation magnetization, 4πM{sub s}, was measured for as-grown films to be 4.6 ± 0.2 kG and ferromagnetic resonance measurements revealed a microwave linewidth of ∼100 Oe at X-band. Scanning electron microscopy indicated clear hexagonal crystals distributed on the semiconductor substrate. These results demonstrate feasibility of growing M-type hexaferrite crystal films on wide bandgap semiconductor substrates by using a simplemore » powder melting method. It also presents a potential pathway for the integration of ferrite microwave passive devices with active semiconductor circuit elements creating system-on-a-wafer architectures.« less

Monitoring of protease catalyzed reactions by quantitative MALDI MS using metal labeling.

PubMed

Gregorius, Barbara; Jakoby, Thomas; Schaumlöffel, Dirk; Tholey, Andreas

2013-05-21

Quantitative mass spectrometry is a powerful tool for the determination of enzyme activities as it does not require labeled substrates and simultaneously allows for the identification of reaction products. However, major restrictions are the limited number of samples which can be measured in parallel due to the need for isotope labeled internal standards. Here we describe the use of metal labeling of peptides for the setup of multiplexed enzyme activity assays. After proteolytic reaction, using the protease trypsin, remaining substrates and peptide products formed in the reaction were labeled with metal chelators complexing rare earth metal ions. Labeled peptides were quantified with high accuracy and over a wide dynamic range (at least 2 orders of magnitude) using MALDI MS in case of simple peptide mixtures or by LC-MALDI MS for complex substrate mixtures and used for the monitoring of time-dependent product formation and substrate consumption. Due to multiplexing capabilities and accuracy, the presented approach will be useful for the determination of enzyme activities with a wide range of biochemical and biotechnological applications.

Effect of oxidation of the non-catalytic β-propeller domain on the substrate specificity of prolyl oligopeptidase from Pleurotus eryngii.

PubMed

Tokai, Shota; Bito, Tomohiro; Shimizu, Katsuhiko; Arima, Jiro

2017-05-27

Enzymes belonging to the S9 family of prolyl oligopeptidases are of interest because of their pharmacological importance and have a non-catalytic β-propeller domain. In this study, we found that the oxidation of Met203, which lies on surface of the β-propeller domain, leads to change in the substrate specificity of eryngase, an enzyme from Pleurotus eryngii and a member of the S9 family of prolyl oligopeptidases. The activity of eryngase for L-Phe-p-nitroanilide was maintained following hydrogen peroxide treatment but was dramatically reduced for other p-nitroanilide substrates. MALDI-TOF MS analysis using tryptic peptides of eryngase indicated that the change in substrate specificity was triggered by oxidizing Met203 to methionine sulfoxide. In addition, mutations of Met203 to smaller residues provided specificities similar to those observed following oxidation of the wild-type enzyme. Substitution of Met203 with Phe significantly decreased activity, indicating that Met203 may be involved in substrate gating. Copyright © 2017 Elsevier Inc. All rights reserved.

Interference effects in the sum frequency generation spectra of thin organic films. II: Applications to different thin-film systems.

PubMed

Tong, Yujin; Zhao, Yanbao; Li, Na; Ma, Yunsheng; Osawa, Masatoshi; Davies, Paul B; Ye, Shen

2010-07-21

In this paper, the results of the modeling calculations carried out for predicting the interference effects expected in the sum frequency generation (SFG) spectra of a specific thin-layer system, described in the accompanying paper, are tested by comparing them with the experimental spectra obtained for a real thin-layer film comprising an organic monolayer/variable thickness dielectric layer/gold substrate. In this system, two contributions to the SFG spectra arise, a resonant contribution from the organic film and a nonresonant contribution from the gold substrate. The modeling calculations are in excellent agreement with the experimental spectra over a wide range of thicknesses and for different polarization combinations. The introduction of another resonant monolayer adjacent to the gold substrate and with the molecules having a reverse orientation has a significant affect on the spectral shapes which is predicted. If a dielectric substrate such as CaF(2) is used instead of a gold substrate, only the spectral intensities vary with the film thickness but not the spectral shapes. The counterpropagating beam geometry will change both the thickness dependent spectral shapes and the intensity of different vibrational modes in comparison with a copropagating geometry. The influences of these experimental factors, i.e., the molecular orientational structure in the thin film, the nature of the substrate, and the selected incident beam geometry, on the experimental SFG spectra are quantitatively predicted by the calculations. The thickness effects on the signals from a SFG active monolayer contained in a thin liquid-layer cell of the type frequently used for in situ electrochemical measurements is also discussed. The modeling calculation is also valid for application to other thin-film systems comprising more than two resonant SFG active interfaces by appropriate choice of optical geometries and relevant optical properties.

Confinement induced ordering in dewetting of ultra-thin polymer bilayers on nanopatterned substrates.

PubMed

Bhandaru, Nandini; Das, Anuja; Mukherjee, Rabibrata

2016-01-14

We report the dewetting of a thin bilayer of polystyrene (PS) and poly(methylmethacrylate) (PMMA) on a topographically patterned nonwettable substrate comprising an array of pillars, arranged in a square lattice. With a gradual increase in the concentration of the PMMA solution (Cn-PMMA), the morphology of the bottom layer changes to: (1) an aligned array of spin dewetted droplets arranged along substrate grooves at very low Cn-PMMA; (2) an interconnected network of threads surrounding each pillar at intermediate Cn-PMMA; and (3) a continuous bottom layer at higher Cn-PMMA. On the other hand the morphology of the PS top layer depends largely on the nature of the pre-existing bottom layer, in addition to Cn-PS. An ordered array of PMMA core-PS shell droplets forms right after spin coating when both Cn-PMMA and Cn-PS are very low. Bilayers with all other initial configurations evolve during thermal annealing, resulting in a variety of ordered structures. Unique morphologies realized include laterally coexisting structures of the two polymers confined within the substrate grooves due to initial rupture of the bottom layer on the substrate followed by a squeezing flow of the top layer; an array of core-shell and single polymer droplets arranged in an alternating order etc., to highlight a few. Such structures cannot be fabricated by any stand-alone lithography technique. On the other hand, in some cases the partially dewetted bottom layer imparts stability to an intact top PS layer against dewetting. Apart from ordering, under certain specific conditions significant miniaturization and downsizing of dewetted feature periodicity and dimension as compared to dewetting of a single layer on a flat substrate is observed. With the help of a morphology phase diagram we show that ordering is achieved over a wide combination of Cn-PMMA and Cn-PS, though the morphology and dewetting pathway differs significantly with variation in the thickness of the individual layers.

Design of ultrasensitive probes for human neutrophil elastase through hybrid combinatorial substrate library profiling

PubMed Central

Kasperkiewicz, Paulina; Poreba, Marcin; Snipas, Scott J.; Parker, Heather; Winterbourn, Christine C.; Salvesen, Guy S.; Drag, Marcin

2014-01-01

The exploration of protease substrate specificity is generally restricted to naturally occurring amino acids, limiting the degree of conformational space that can be surveyed. We substantially enhanced this by incorporating 102 unnatural amino acids to explore the S1–S4 pockets of human neutrophil elastase. This approach provides hybrid natural and unnatural amino acid sequences, and thus we termed it the Hybrid Combinatorial Substrate Library. Library results were validated by the synthesis of individual tetrapeptide substrates, with the optimal substrate demonstrating more than three orders of magnitude higher catalytic efficiency than commonly used substrates of elastase. This optimal substrate was converted to an activity-based probe that demonstrated high selectivity and revealed the specific presence of active elastase during the process of neutrophil extracellular trap formation. We propose that this approach can be successfully used for any type of endopeptidase to deliver high activity and selectivity in substrates and probes. PMID:24550277

A Novel Variable Wide Bandgap Material for High Power, High Frequency Devices

DTIC Science & Technology

2011-01-13

temperature above 1300 °C caused the back side of the Si substrates to soften and form molybdenum silicides with the holder or to simply sublime...copper while Figures 7b, 7d, and 7f show the measured impurity levels of aluminum and sodium in the 4H-SiC substrate, RF sputtered film, and DC... sodium which are completely absent in the 4H- SiC substrate. These impurities are also attributed to the aluminum silicate shell that is evidently

Modified methods for growing 3-D skin equivalents: an update.

PubMed

Lamb, Rebecca; Ambler, Carrie A

2014-01-01

Artificial epidermis can be reconstituted in vitro by seeding primary epidermal cells (keratinocytes) onto a supportive substrate and then growing the developing skin equivalent at the air-liquid interface. In vitro skin models are widely used to study skin biology and for industrial drug and cosmetic testing. Here, we describe updated methods for growing 3-dimensional skin equivalents using de-vitalized, de-epidermalized dermis (DED) substrates including methods for DED substrate preparation, cell seeding, growth conditions, and fixation procedures.

Synthesis of 1-[bis(trifluoromethyl)phosphine]-1’-oxazolinylferrocene ligands and their application in regio- and enantioselective Pd-catalyzed allylic alkylation of monosubstituted allyl substrates

PubMed Central

Lai, Zeng-Wei; Yang, Rong-Fei; Ye, Ke-Yin

2014-01-01

Summary A class of novel, easily accessible and air-stable 1-[bis(trifluoromethyl)phosphine]-1’-oxazolinylferrocene ligands has been synthesized from ferrocene. It became apparent that these ligands can be used in the regio- and enantioselective Pd-catalyzed allylic alkylation of monosubstituted allyl substrates in a highly efficient manner. Excellent regio- and enantioselectivity could be obtained for a wide range of substrates. PMID:24991277

Overlapping protective roles for glutathione transferase gene family members in chemical and oxidative stress response in Agrobacterium tumefaciens.

PubMed

Skopelitou, Katholiki; Muleta, Abdi W; Pavli, Ourania; Skaracis, Georgios N; Flemetakis, Emmanouil; Papageorgiou, Anastassios C; Labrou, Nikolaos E

2012-03-01

In the present work, we describe the characterisation of the glutathione transferase (GST) gene family from Agrobacterium tumefaciens C58. A genome survey revealed the presence of eight GST-like proteins in A. tumefaciens (AtuGSTs). Comparison by multiple sequence alignment generated a dendrogram revealing the phylogenetic relationships of AtuGSTs-like proteins. The beta and theta classes identified in other bacterial species are represented by five members in A. tumefaciens C58. In addition, there are three "orphan" sequences that do not fit into any previously recognised GST classes. The eight GST-like genes were cloned, expressed in Escherichia coli and their substrate specificity was determined towards 17 different substrates. The results showed that AtuGSTs catalyse a broad range of reactions, with different members of the family exhibiting quite varied substrate specificity. The 3D structures of AtuGSTs were predicted using molecular modelling. The use of comparative sequence and structural analysis of the AtuGST isoenzymes allowed us to identify local sequence and structural characteristics between different GST isoenzymes and classes. Gene expression profiling was conducted under normal culture conditions as well as under abiotic stress conditions (addition of xenobiotics, osmotic stress and cold and heat shock) to induce and monitor early stress-response mechanisms. The results reveal the constitutive expression of GSTs in A. tumefaciens and a modulation of GST activity after treatments, indicating that AtuGSTs presumably participate in a wide range of functions, many of which are important in counteracting stress conditions. These functions may be relevant to maintaining cellular homeostasis as well as in the direct detoxification of toxic compounds.

Hydrophobic fluorescent probes introduce artifacts into single molecule tracking experiments due to non-specific binding.

PubMed

Zanetti-Domingues, Laura C; Tynan, Christopher J; Rolfe, Daniel J; Clarke, David T; Martin-Fernandez, Marisa

2013-01-01

Single-molecule techniques are powerful tools to investigate the structure and dynamics of macromolecular complexes; however, data quality can suffer because of weak specific signal, background noise and dye bleaching and blinking. It is less well-known, but equally important, that non-specific binding of probe to substrates results in a large number of immobile fluorescent molecules, introducing significant artifacts in live cell experiments. Following from our previous work in which we investigated glass coating substrates and demonstrated that the main contribution to this non-specific probe adhesion comes from the dye, we carried out a systematic investigation of how different dye chemistries influence the behaviour of spectrally similar fluorescent probes. Single-molecule brightness, bleaching and probe mobility on the surface of live breast cancer cells cultured on a non-adhesive substrate were assessed for anti-EGFR affibody conjugates with 14 different dyes from 5 different manufacturers, belonging to 3 spectrally homogeneous bands (491 nm, 561 nm and 638 nm laser lines excitation). Our results indicate that, as well as influencing their photophysical properties, dye chemistry has a strong influence on the propensity of dye-protein conjugates to adhere non-specifically to the substrate. In particular, hydrophobicity has a strong influence on interactions with the substrate, with hydrophobic dyes showing much greater levels of binding. Crucially, high levels of non-specific substrate binding result in calculated diffusion coefficients significantly lower than the true values. We conclude that the physic-chemical properties of the dyes should be considered carefully when planning single-molecule experiments. Favourable dye characteristics such as photostability and brightness can be offset by the propensity of a conjugate for non-specific adhesion.

Hydrophobic Fluorescent Probes Introduce Artifacts into Single Molecule Tracking Experiments Due to Non-Specific Binding

PubMed Central

Rolfe, Daniel J.; Clarke, David T.; Martin-Fernandez, Marisa

2013-01-01

Single-molecule techniques are powerful tools to investigate the structure and dynamics of macromolecular complexes; however, data quality can suffer because of weak specific signal, background noise and dye bleaching and blinking. It is less well-known, but equally important, that non-specific binding of probe to substrates results in a large number of immobile fluorescent molecules, introducing significant artifacts in live cell experiments. Following from our previous work in which we investigated glass coating substrates and demonstrated that the main contribution to this non-specific probe adhesion comes from the dye, we carried out a systematic investigation of how different dye chemistries influence the behaviour of spectrally similar fluorescent probes. Single-molecule brightness, bleaching and probe mobility on the surface of live breast cancer cells cultured on a non-adhesive substrate were assessed for anti-EGFR affibody conjugates with 14 different dyes from 5 different manufacturers, belonging to 3 spectrally homogeneous bands (491 nm, 561 nm and 638 nm laser lines excitation). Our results indicate that, as well as influencing their photophysical properties, dye chemistry has a strong influence on the propensity of dye-protein conjugates to adhere non-specifically to the substrate. In particular, hydrophobicity has a strong influence on interactions with the substrate, with hydrophobic dyes showing much greater levels of binding. Crucially, high levels of non-specific substrate binding result in calculated diffusion coefficients significantly lower than the true values. We conclude that the physic-chemical properties of the dyes should be considered carefully when planning single-molecule experiments. Favourable dye characteristics such as photostability and brightness can be offset by the propensity of a conjugate for non-specific adhesion. PMID:24066121

Gap-mode enhancement on MoS2 probed by functionalized tip-enhanced Raman spectroscopy

NASA Astrophysics Data System (ADS)

Alajlan, Abdulrahman M.; Voronine, Dmitri V.; Sinyukov, Alexander M.; Zhang, Zhenrong; Sokolov, Alexei V.; Scully, Marlan O.

2016-09-01

Surface enhancement of molecular spectroscopic signals has been widely used for sensing and nanoscale imaging. Because of the weak electromagnetic enhancement of Raman signals on semiconductors, it is motivating but challenging to study the electromagnetic effect separately from the chemical effects. We report tip-enhanced Raman scattering measurements on Au and bulk MoS2 substrates using a metallic tip functionalized with copper phthalocyanine molecules and demonstrate similar gap-mode enhancement on both substrates. We compare the experimental results with theoretical calculations to confirm the gap-mode enhancement on MoS2 using a well-established electrostatic model. The functionalized tip approach allows for suppressing the background and is ideal for separating electromagnetic and chemical enhancement mechanisms on various substrates. Our results may find a wide range of applications in MoS2-based devices, sensors, and metal-free nanoscale bio-imaging.

A Bottom-Up Proteomic Approach to Identify Substrate Specificity of Outer-Membrane Protease OmpT.

PubMed

Wood, Sarah E; Sinsinbar, Gaurav; Gudlur, Sushanth; Nallani, Madhavan; Huang, Che-Fan; Liedberg, Bo; Mrksich, Milan

2017-12-22

Identifying peptide substrates that are efficiently cleaved by proteases gives insights into substrate recognition and specificity, guides development of inhibitors, and improves assay sensitivity. Peptide arrays and SAMDI mass spectrometry were used to identify a tetrapeptide substrate exhibiting high activity for the bacterial outer-membrane protease (OmpT). Analysis of protease activity for the preferred residues at the cleavage site (P1, P1') and nearest-neighbor positions (P2, P2') and their positional interdependence revealed FRRV as the optimal peptide with the highest OmpT activity. Substituting FRRV into a fragment of LL37, a natural substrate of OmpT, led to a greater than 400-fold improvement in OmpT catalytic efficiency, with a k cat /K m value of 6.1×10 6  L mol -1  s -1 . Wild-type and mutant OmpT displayed significant differences in their substrate specificities, demonstrating that even modest mutants may not be suitable substitutes for the native enzyme. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Two distinct domains contribute to the substrate acyl chain length selectivity of plant acyl-ACP thioesterase.

PubMed

Jing, Fuyuan; Zhao, Le; Yandeau-Nelson, Marna D; Nikolau, Basil J

2018-02-28

The substrate specificity of acyl-ACP thioesterase (TE) plays an essential role in controlling the fatty acid profile produced by type II fatty acid synthases. Here we identify two groups of residues that synergistically determine different substrate specificities of two acyl-ACP TEs from Cuphea viscosissima (CvFatB1 and CvFatB2). One group (V194, V217, N223, R226, R227, and I268 in CvFatB2) is critical in determining the structure and depth of a hydrophobic cavity in the N-terminal hotdog domain that binds the substrate's acyl moiety. The other group (255-RKLSKI-260 and 285-RKLPKL-289 in CvFatB2) defines positively charged surface patches that may facilitate binding of the ACP moiety. Mutagenesis of residues within these two groups results in distinct synthetic acyl-ACP TEs that efficiently hydrolyze substrates with even shorter chains (C4- to C8-ACPs). These insights into structural determinants of acyl-ACP TE substrate specificity are useful in modifying this enzyme for tailored fatty acid production in engineered organisms.

[Substrate specificities of bile salt hydrolase 1 and its mutants from Lactobacillus salivarius].

PubMed

Bi, Jie; Fang, Fang; Qiu, Yuying; Yang, Qingli; Chen, Jian

2014-03-01

In order to analyze the correlation between critical residues in the catalytic centre of BSH and the enzyme substrate specificity, seven mutants of Lactobacillus salivarius bile salt hydrolase (BSH1) were constructed by using the Escherichia coli pET-20b(+) gene expression system, rational design and site-directed mutagenesis. These BSH1 mutants exhibited different hydrolytic activities against various conjugated bile salts through substrate specificities comparison. Among the residues being tested, Cys2 and Thr264 were deduced as key sites for BSH1 to catalyze taurocholic acid and glycocholic acid, respectively. Moreover, Cys2 and Thr264 were important for keeping the catalytic activity of BSH1. The high conservative Cys2 was not the only active site, other mutant amino acid sites were possibly involved in substrate binding. These mutant residues might influence the space and shape of the substrate-binding pockets or the channel size for substrate passing through and entering active site of BSH1, thus, the hydrolytic activity of BSH1 was changed to different conjugated bile salt.

Unnatural amino acids increase activity and specificity of synthetic substrates for human and malarial cathepsin C.

PubMed

Poreba, Marcin; Mihelic, Marko; Krai, Priscilla; Rajkovic, Jelena; Krezel, Artur; Pawelczak, Malgorzata; Klemba, Michael; Turk, Dusan; Turk, Boris; Latajka, Rafal; Drag, Marcin

2014-04-01

Mammalian cathepsin C is primarily responsible for the removal of N-terminal dipeptides and activation of several serine proteases in inflammatory or immune cells, while its malarial parasite ortholog dipeptidyl aminopeptidase 1 plays a crucial role in catabolizing the hemoglobin of its host erythrocyte. In this report, we describe the systematic substrate specificity analysis of three cathepsin C orthologs from Homo sapiens (human), Bos taurus (bovine) and Plasmodium falciparum (malaria parasite). Here, we present a new approach with a tailored fluorogenic substrate library designed and synthesized to probe the S1 and S2 pocket preferences of these enzymes with both natural and a broad range of unnatural amino acids. Our approach identified very efficiently hydrolyzed substrates containing unnatural amino acids, which resulted in the design of significantly better substrates than those previously known. Additionally, in this study significant differences in terms of the structures of optimal substrates for human and malarial orthologs are important from the therapeutic point of view. These data can be also used for the design of specific inhibitors or activity-based probes.

Charge-specific size-dependent separation of water-soluble organic molecules by fluorinated nanoporous networks

NASA Astrophysics Data System (ADS)

Byun, Jeehye; Patel, Hasmukh A.; Thirion, Damien; Yavuz, Cafer T.

2016-11-01

Molecular architecture in nanoscale spaces can lead to selective chemical interactions and separation of species with similar sizes and functionality. Substrate specific sorbent chemistry is well known through highly crystalline ordered structures such as zeolites, metal organic frameworks and widely available nanoporous carbons. Size and charge-dependent separation of aqueous molecular contaminants, on the contrary, have not been adequately developed. Here we report a charge-specific size-dependent separation of water-soluble molecules through an ultra-microporous polymeric network that features fluorines as the predominant surface functional groups. Treatment of similarly sized organic molecules with and without charges shows that fluorine interacts with charges favourably. Control experiments using similarly constructed frameworks with or without fluorines verify the fluorine-cation interactions. Lack of a σ-hole for fluorine atoms is suggested to be responsible for this distinct property, and future applications of this discovery, such as desalination and mixed matrix membranes, may be expected to follow.

Charge-specific size-dependent separation of water-soluble organic molecules by fluorinated nanoporous networks

PubMed Central

Byun, Jeehye; Patel, Hasmukh A.; Thirion, Damien; Yavuz, Cafer T.

2016-01-01

Molecular architecture in nanoscale spaces can lead to selective chemical interactions and separation of species with similar sizes and functionality. Substrate specific sorbent chemistry is well known through highly crystalline ordered structures such as zeolites, metal organic frameworks and widely available nanoporous carbons. Size and charge-dependent separation of aqueous molecular contaminants, on the contrary, have not been adequately developed. Here we report a charge-specific size-dependent separation of water-soluble molecules through an ultra-microporous polymeric network that features fluorines as the predominant surface functional groups. Treatment of similarly sized organic molecules with and without charges shows that fluorine interacts with charges favourably. Control experiments using similarly constructed frameworks with or without fluorines verify the fluorine-cation interactions. Lack of a σ-hole for fluorine atoms is suggested to be responsible for this distinct property, and future applications of this discovery, such as desalination and mixed matrix membranes, may be expected to follow. PMID:27830697

Fine-mapping inflammatory bowel disease loci to single variant resolution

PubMed Central

Huang, Hailiang; Fang, Ming; Jostins, Luke; Mirkov, Maša Umićević; Boucher, Gabrielle; Anderson, Carl A; Andersen, Vibeke; Cleynen, Isabelle; Cortes, Adrian; Crins, François; D'Amato, Mauro; Deffontaine, Valérie; Dimitrieva, Julia; Docampo, Elisa; Elansary, Mahmoud; Farh, Kyle Kai-How; Franke, Andre; Gori, Ann-Stephan; Goyette, Philippe; Halfvarson, Jonas; Haritunians, Talin; Knight, Jo; Lawrance, Ian C; Lees, Charlie W; Louis, Edouard; Mariman, Rob; Meuwissen, Theo; Mni, Myriam; Momozawa, Yukihide; Parkes, Miles; Spain, Sarah L; Théâtre, Emilie; Trynka, Gosia; Satsangi, Jack; van Sommeren, Suzanne; Vermeire, Severine; Xavier, Ramnik J; Weersma, Rinse K; Duerr, Richard H; Mathew, Christopher G; Rioux, John D; McGovern, Dermot PB; Cho, Judy H; Georges, Michel; Daly, Mark J; Barrett, Jeffrey C

2017-01-01

Summary The inflammatory bowel diseases (IBD) are chronic gastrointestinal inflammatory disorders that affect millions worldwide. Genome-wide association studies have identified 200 IBD-associated loci, but few have been conclusively resolved to specific functional variants. Here we report fine-mapping of 94 IBD loci using high-density genotyping in 67,852 individuals. We pinpointed 18 associations to a single causal variant with >95% certainty, and an additional 27 associations to a single variant with >50% certainty. These 45 variants are significantly enriched for protein-coding changes (n=13), direct disruption of transcription factor binding sites (n=3) and tissue specific epigenetic marks (n=10), with the latter category showing enrichment in specific immune cells among associations stronger in CD and in gut mucosa among associations stronger in UC. The results of this study suggest that high-resolution fine-mapping in large samples can convert many GWAS discoveries into statistically convincing causal variants, providing a powerful substrate for experimental elucidation of disease mechanisms. PMID:28658209

Interface thermal resistance of nanostructured FeCoCu film and Si substrate

NASA Astrophysics Data System (ADS)

Nikolaenko, Yuri M.; Medvedev, Yuri V.; Genenko, Yuri A.; Ghafari, Mohammad; Hahn, Horst

2006-05-01

Results of measurement of thermal resistance (RFS ) of film substrate interface of 10 nm (Fe1-x Cox )1-y Cuy film on Si substrate with 50 nm SiO2 sublayer are presented. The estimated magnitude is two orders greater then RFS of epitaxial manganite films on StTiO3 substrate with and without sublayer. The significant increase of RFS is explained by granular structure of film with average size of grain about 10 nm. In this case the additional thermal barier in the film-substrate interface is appeared. It provides the change of regime of phonons propagation from ballistic to diffusion one. The principle possibility of variation of RFS in wide range as a task of nanotechnology is discussed.

A study to improve the mechanical properties of silicon carbide ribbon fibers

NASA Technical Reports Server (NTRS)

Debolt, H. E.; Robey, R. J.

1976-01-01

Preliminary deposition studies of SiC ribbon on a carbon ribbon substrate showed that the dominant strength limiting flaws were at the substrate surface. Procedures for making the carbon ribbon substrate from polyimide film were improved, providing lengths up to 450 meters (1,500 ft.) of flat carbon ribbon substrate 1,900 microns (75 mils) wide by 25 microns (1 mil) thick. The flaws on the carbon ribbon were smaller and less frequent than on carbon ribbon used earlier. SiC ribbon made using the improved substrate, including a layer of pyrolytic graphite to reduce further the severity of substrate surface flaws, showed strength levels up to the 2,068 MPa (300 Ksi) target of the program, with average strength levels over 1,700 MPa (250 Ksi) with coefficient of variation as low as 10% for some runs.

The diversity and specificity of the extracellular proteome in the cellulolytic bacterium Caldicellulosiruptor bescii is driven by the nature of the cellulosic growth substrate

DOE PAGES

Poudel, Suresh; Giannone, Richard J.; Basen, Mirko; ...

2018-03-23

Background: Caldicellulosiruptor bescii is a thermophilic cellulolytic bacterium that efficiently deconstructs lignocellulosic biomass into sugars, which subsequently can be fermented into alcohols, such as ethanol, and other products. Deconstruction of complex substrates by C. bescii involves a myriad of highly abundant, substrate-specific extracellular solute binding proteins (ESBPs) and carbohydrate-active enzymes (CAZymes) containing carbohydrate-binding modules (CBMs). Mass spectrometry-based proteomics was employed to investigate how these substrate recognition proteins and enzymes vary as a function of lignocellulosic substrates.Results:Proteomic analysis revealed several key extracellular proteins that respond specifically to either C5 or C6 mono- and polysaccharides. These include proteins of unknown functions (PUFs),more » ESBPs, and CAZymes. ESBPs that were previously shown to interact more efficiently with hemicellulose and pectin were detected in high abundance during growth on complex C5 substrates, such as switchgrass and xylan. Some proteins, such as Athe_0614 and Athe_2368, whose functions are not well defined were predicted to be involved in xylan utilization and ABC transport and were significantly more abundant in complex and C5 substrates, respectively. The proteins encoded by the entire glucan degradation locus (GDL; Athe_1857, 1859, 1860, 1865, 1867, and 1866) were highly abundant under all growth conditions, particularly when C. bescii was grown on cellobiose, switchgrass, or xylan. In contrast, the glycoside hydrolases Athe_0609 (Pullulanase) and 0610, which both possess CBM20 and a starch binding domain, appear preferential to C5/complex substrate deconstruction. Some PUFs, such as Athe_2463 and 2464, were detected as highly abundant when grown on C5 substrates (xylan and xylose), also suggesting C5-substrate specificity. In conclusion, this study reveals the protein membership of the C. bescii secretome and demonstrates its plasticity based on the complexity (mono-/disaccharides vs. polysaccharides) and type of carbon (C5 vs. C6) available to the microorganism. The presence or increased abundance of extracellular proteins as a response to specific substrates helps to further elucidate C. bescii’s utilization and conversion of lignocellulosic biomass to biofuel and other valuable products. This includes improved characterization of extracellular proteins that lack discrete functional roles and are poorly/not annotated.« less

The diversity and specificity of the extracellular proteome in the cellulolytic bacterium Caldicellulosiruptor bescii is driven by the nature of the cellulosic growth substrate

DOE Office of Scientific and Technical Information (OSTI.GOV)

Poudel, Suresh; Giannone, Richard J.; Basen, Mirko

Background: Caldicellulosiruptor bescii is a thermophilic cellulolytic bacterium that efficiently deconstructs lignocellulosic biomass into sugars, which subsequently can be fermented into alcohols, such as ethanol, and other products. Deconstruction of complex substrates by C. bescii involves a myriad of highly abundant, substrate-specific extracellular solute binding proteins (ESBPs) and carbohydrate-active enzymes (CAZymes) containing carbohydrate-binding modules (CBMs). Mass spectrometry-based proteomics was employed to investigate how these substrate recognition proteins and enzymes vary as a function of lignocellulosic substrates.Results:Proteomic analysis revealed several key extracellular proteins that respond specifically to either C5 or C6 mono- and polysaccharides. These include proteins of unknown functions (PUFs),more » ESBPs, and CAZymes. ESBPs that were previously shown to interact more efficiently with hemicellulose and pectin were detected in high abundance during growth on complex C5 substrates, such as switchgrass and xylan. Some proteins, such as Athe_0614 and Athe_2368, whose functions are not well defined were predicted to be involved in xylan utilization and ABC transport and were significantly more abundant in complex and C5 substrates, respectively. The proteins encoded by the entire glucan degradation locus (GDL; Athe_1857, 1859, 1860, 1865, 1867, and 1866) were highly abundant under all growth conditions, particularly when C. bescii was grown on cellobiose, switchgrass, or xylan. In contrast, the glycoside hydrolases Athe_0609 (Pullulanase) and 0610, which both possess CBM20 and a starch binding domain, appear preferential to C5/complex substrate deconstruction. Some PUFs, such as Athe_2463 and 2464, were detected as highly abundant when grown on C5 substrates (xylan and xylose), also suggesting C5-substrate specificity. In conclusion, this study reveals the protein membership of the C. bescii secretome and demonstrates its plasticity based on the complexity (mono-/disaccharides vs. polysaccharides) and type of carbon (C5 vs. C6) available to the microorganism. The presence or increased abundance of extracellular proteins as a response to specific substrates helps to further elucidate C. bescii’s utilization and conversion of lignocellulosic biomass to biofuel and other valuable products. This includes improved characterization of extracellular proteins that lack discrete functional roles and are poorly/not annotated.« less

The diversity and specificity of the extracellular proteome in the cellulolytic bacterium Caldicellulosiruptor bescii is driven by the nature of the cellulosic growth substrate.

PubMed

Poudel, Suresh; Giannone, Richard J; Basen, Mirko; Nookaew, Intawat; Poole, Farris L; Kelly, Robert M; Adams, Michael W W; Hettich, Robert L

2018-01-01

Caldicellulosiruptor bescii is a thermophilic cellulolytic bacterium that efficiently deconstructs lignocellulosic biomass into sugars, which subsequently can be fermented into alcohols, such as ethanol, and other products. Deconstruction of complex substrates by C. bescii involves a myriad of highly abundant, substrate-specific extracellular solute binding proteins (ESBPs) and carbohydrate-active enzymes (CAZymes) containing carbohydrate-binding modules (CBMs). Mass spectrometry-based proteomics was employed to investigate how these substrate recognition proteins and enzymes vary as a function of lignocellulosic substrates. Proteomic analysis revealed several key extracellular proteins that respond specifically to either C5 or C6 mono- and polysaccharides. These include proteins of unknown functions (PUFs), ESBPs, and CAZymes. ESBPs that were previously shown to interact more efficiently with hemicellulose and pectin were detected in high abundance during growth on complex C5 substrates, such as switchgrass and xylan. Some proteins, such as Athe_0614 and Athe_2368, whose functions are not well defined were predicted to be involved in xylan utilization and ABC transport and were significantly more abundant in complex and C5 substrates, respectively. The proteins encoded by the entire glucan degradation locus (GDL; Athe_1857, 1859, 1860, 1865, 1867, and 1866) were highly abundant under all growth conditions, particularly when C. bescii was grown on cellobiose, switchgrass, or xylan. In contrast, the glycoside hydrolases Athe_0609 (Pullulanase) and 0610, which both possess CBM20 and a starch binding domain, appear preferential to C5/complex substrate deconstruction. Some PUFs, such as Athe_2463 and 2464, were detected as highly abundant when grown on C5 substrates (xylan and xylose), also suggesting C5-substrate specificity. This study reveals the protein membership of the C. bescii secretome and demonstrates its plasticity based on the complexity (mono-/disaccharides vs. polysaccharides) and type of carbon (C5 vs. C6) available to the microorganism. The presence or increased abundance of extracellular proteins as a response to specific substrates helps to further elucidate C. bescii 's utilization and conversion of lignocellulosic biomass to biofuel and other valuable products. This includes improved characterization of extracellular proteins that lack discrete functional roles and are poorly/not annotated.

High-performance carbon nanotube thin-film transistors on flexible paper substrates

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Na; Yun, Ki Nam; Yu, Hyun-Yong

Single-walled carbon nanotubes (SWCNTs) are promising materials as active channels for flexible transistors owing to their excellent electrical and mechanical properties. However, flexible SWCNT transistors have never been realized on paper substrates, which are widely used, inexpensive, and recyclable. In this study, we fabricated SWCNT thin-film transistors on photo paper substrates. The devices exhibited a high on/off current ratio of more than 10{sup 6} and a field-effect mobility of approximately 3 cm{sup 2}/V·s. The proof-of-concept demonstration indicates that SWCNT transistors on flexible paper substrates could be applied as low-cost and recyclable flexible electronics.

SPEER-SERVER: a web server for prediction of protein specificity determining sites

PubMed Central

Chakraborty, Abhijit; Mandloi, Sapan; Lanczycki, Christopher J.; Panchenko, Anna R.; Chakrabarti, Saikat

2012-01-01

Sites that show specific conservation patterns within subsets of proteins in a protein family are likely to be involved in the development of functional specificity. These sites, generally termed specificity determining sites (SDS), might play a crucial role in binding to a specific substrate or proteins. Identification of SDS through experimental techniques is a slow, difficult and tedious job. Hence, it is very important to develop efficient computational methods that can more expediently identify SDS. Herein, we present Specificity prediction using amino acids’ Properties, Entropy and Evolution Rate (SPEER)-SERVER, a web server that predicts SDS by analyzing quantitative measures of the conservation patterns of protein sites based on their physico-chemical properties and the heterogeneity of evolutionary changes between and within the protein subfamilies. This web server provides an improved representation of results, adds useful input and output options and integrates a wide range of analysis and data visualization tools when compared with the original standalone version of the SPEER algorithm. Extensive benchmarking finds that SPEER-SERVER exhibits sensitivity and precision performance that, on average, meets or exceeds that of other currently available methods. SPEER-SERVER is available at http://www.hpppi.iicb.res.in/ss/. PMID:22689646

SPEER-SERVER: a web server for prediction of protein specificity determining sites.

PubMed

Chakraborty, Abhijit; Mandloi, Sapan; Lanczycki, Christopher J; Panchenko, Anna R; Chakrabarti, Saikat

2012-07-01

Sites that show specific conservation patterns within subsets of proteins in a protein family are likely to be involved in the development of functional specificity. These sites, generally termed specificity determining sites (SDS), might play a crucial role in binding to a specific substrate or proteins. Identification of SDS through experimental techniques is a slow, difficult and tedious job. Hence, it is very important to develop efficient computational methods that can more expediently identify SDS. Herein, we present Specificity prediction using amino acids' Properties, Entropy and Evolution Rate (SPEER)-SERVER, a web server that predicts SDS by analyzing quantitative measures of the conservation patterns of protein sites based on their physico-chemical properties and the heterogeneity of evolutionary changes between and within the protein subfamilies. This web server provides an improved representation of results, adds useful input and output options and integrates a wide range of analysis and data visualization tools when compared with the original standalone version of the SPEER algorithm. Extensive benchmarking finds that SPEER-SERVER exhibits sensitivity and precision performance that, on average, meets or exceeds that of other currently available methods. SPEER-SERVER is available at http://www.hpppi.iicb.res.in/ss/.

Combining affinity proteomics and network context to identify new phosphatase substrates and adapters in growth pathways

PubMed Central

Sacco, Francesca; Boldt, Karsten; Calderone, Alberto; Panni, Simona; Paoluzi, Serena; Castagnoli, Luisa; Ueffing, Marius; Cesareni, Gianni

2014-01-01

Protein phosphorylation homoeostasis is tightly controlled and pathological conditions are caused by subtle alterations of the cell phosphorylation profile. Altered levels of kinase activities have already been associated to specific diseases. Less is known about the impact of phosphatases, the enzymes that down-regulate phosphorylation by removing the phosphate groups. This is partly due to our poor understanding of the phosphatase-substrate network. Much of phosphatase substrate specificity is not based on intrinsic enzyme specificity with the catalytic pocket recognizing the sequence/structure context of the phosphorylated residue. In addition many phosphatase catalytic subunits do not form a stable complex with their substrates. This makes the inference and validation of phosphatase substrates a non-trivial task. Here, we present a novel approach that builds on the observation that much of phosphatase substrate selection is based on the network of physical interactions linking the phosphatase to the substrate. We first used affinity proteomics coupled to quantitative mass spectrometry to saturate the interactome of eight phosphatases whose down regulations was shown to affect the activation of the RAS-PI3K pathway. By integrating information from functional siRNA with protein interaction information, we develop a strategy that aims at inferring phosphatase physiological substrates. Graph analysis is used to identify protein scaffolds that may link the catalytic subunits to their substrates. By this approach we rediscover several previously described phosphatase substrate interactions and characterize two new protein scaffolds that promote the dephosphorylation of PTPN11 and ERK by DUSP18 and DUSP26, respectively. PMID:24847354

Biochemistry Students' Ideas about How an Enzyme Interacts with a Substrate

ERIC Educational Resources Information Center

Linenberger, Kimberly J.; Bretz, Stacey Lowery

2015-01-01

Enzyme-substrate interactions are a fundamental concept of biochemistry that is built upon throughout multiple biochemistry courses. Central to understanding enzyme-substrate interactions is specific knowledge of exactly how an enzyme and substrate interact. Within this narrower topic, students must understand the various binding sites on an…

Heparin/heparan sulfate 6-O-sulfatase from Flavobacterium heparinum: integrated structural and biochemical investigation of enzyme active site and substrate specificity.

PubMed

Myette, James R; Soundararajan, Venkataramanan; Shriver, Zachary; Raman, Rahul; Sasisekharan, Ram

2009-12-11

Heparin and heparan sulfate glycosaminoglycans (HSGAGs) comprise a chemically heterogeneous class of sulfated polysaccharides. The development of structure-activity relationships for this class of polysaccharides requires the identification and characterization of degrading enzymes with defined substrate specificity and enzymatic activity. Toward this end, we report here the molecular cloning and extensive structure-function analysis of a 6-O-sulfatase from the Gram-negative bacterium Flavobacterium heparinum. In addition, we report the recombinant expression of this enzyme in Escherichia coli in a soluble, active form and identify it as a specific HSGAG sulfatase. We further define the mechanism of action of the enzyme through biochemical and structural studies. Through the use of defined substrates, we investigate the kinetic properties of the enzyme. This analysis was complemented by homology-based molecular modeling studies that sought to rationalize the substrate specificity of the enzyme and mode of action through an analysis of the active-site topology of the enzyme including identifying key enzyme-substrate interactions and assigning key amino acids within the active site of the enzyme. Taken together, our structural and biochemical studies indicate that 6-O-sulfatase is a predominantly exolytic enzyme that specifically acts on N-sulfated or N-acetylated 6-O-sulfated glucosamines present at the non-reducing end of HSGAG oligosaccharide substrates. This requirement for the N-acetyl or N-sulfo groups on the glucosamine substrate can be explained through eliciting favorable interactions with key residues within the active site of the enzyme. These findings provide a framework that enables the use of 6-O-sulfatase as a tool for HSGAG structure-activity studies as well as expand our biochemical and structural understanding of this important class of enzymes.

The trimethylammonium headgroup of choline is a major determinant for substrate binding and specificity in choline oxidase.

PubMed

Gadda, Giovanni; Powell, Nichole L N; Menon, Prashanthi

2004-10-15

Choline oxidase catalyzes the oxidation of choline to glycine betaine via two sequential flavin-linked transfers of hydride equivalents to molecular oxygen and formation of a betaine aldehyde intermediate. In the present study, choline and glycine betaine analogs were used as substrates and inhibitors for the enzyme to investigate the structural determinants that are relevant for substrate recognition and specificity. Competitive inhibition patterns with respect to choline were determined for a number of substituted amines at pH 6.5 and 25 degrees C. The Kis values for the carboxylate-containing ligands glycine betaine, N,N-dimethylglycine, and N-methylglycine increased monotonically with decreasing number of methyl groups, consistent with the trimethylammonium portion of the ligand being important for binding. In contrast, the acetate portion of glycine betaine did not contribute to binding, as suggested by lack of changes in the Kis values upon substituting glycine betaine with inhibitors containing methyl, ethyl, allyl, and 2-amino-ethyl side chains. In agreement with the inhibition data, the specificity of the enzyme for the organic substrate (kcat/Km value) decreased when N,N-dimethylethanolamine, N-methylethanolamine, and the isosteric substrate 3,3-dimethyl-1-butanol were used as substrate instead of choline; a contribution of approximately 7 kcal mol(-1) toward substrate discrimination was estimated for the interaction of the trimethylammonium portion of the substrate with the active site of choline oxidase.

Treatment patterns from 647 patients with Gaucher disease: An analysis from the Gaucher Outcome Survey.

PubMed

Deegan, P; Fernandez-Sasso, D; Giraldo, P; Lau, H; Panahloo, Z; Zimran, A

2018-02-01

The Gaucher Outcome Survey (GOS) is an international disease-specific registry established in 2010 for patients with a confirmed diagnosis of Gaucher disease (GD), regardless of GD type or treatment status. For insight into how GD management varies among countries, we analyzed treatment patterns in GOS. As of October 30, 2015, data on GD-specific treatment (enzyme replacement therapy, substrate reduction therapy, or chemical chaperone therapy) received at any time were available for 647 patients. At analysis, velaglucerase alfa (316/573, 55.1%) and imiglucerase (184/573, 32.1%) were the treatments most widely used. Of the 647 treated patients, 446 (68.9%) had been treated for >5years and 368 (56.9%) had received only one GD-specific drug therapy. There were 377 patients who received velaglucerase alfa. Velaglucerase alfa was most widely used at 60U/kg every other week (134/492 dose entries, 27.2%), but there were differences in dosing between the three highest-enrolling countries (defined as >100 GOS patients enrolled in each), with most patients in Israel receiving <20U/kg, most patients in the United Kingdom receiving 20 to <40U/kg, and most in the United States receiving 60U/kg. This analysis provides a foundation upon which to examine real-life outcomes data from different treatment regimens globally. Copyright © 2016 Shire Human Genetic Therapies, Inc. Published by Elsevier Inc. All rights reserved.

Biodegradation kinetics of the nitramine explosive CL-20 in soil and microbial cultures.

PubMed

Panikov, N S; Sizova, M V; Ros, D; Christodoulatos, C; Balas, W; Nicolich, S

2007-06-01

The cyclic nitramine explosive CL-20 (C(6)H(6)N(12)O(12), 2,4,6,8,10,12-hexanitro-2,4,6,8,10,12 -hexaazaisowurtzitane) is a relatively new energetic compound which could be a persistent organic pollutant. To follow its biodegradation dynamics, CL-20 was added to soil alone or together with organic co-substrates and N-source and incubated under oxic and anoxic conditions. Without co-substrates, the CL-20 degradation was detectable only under anoxic conditions. The highest degradation rate was found under aerobic conditions and with the addition of co-substrates, succinate and pyruvate being more efficient than acetate, glucose, starch or yeast extract. When added to intact soil, CL-20 degradation was not affected by the N content, but in soil serially diluted with N-free succinate-mineral medium, the process became N-limited. About 40% of randomly selected bacterial colonies grown on succinate agar medium were able to decompose CL-20. Based on 16S rDNA gene sequence and cell morphology, they were affiliated to Pseudomonas, Rhodococcus, Ochrobactrum, Mycobacterium and Ralstonia. In the pure culture of Pseudomonas sp. MS-P grown on the succinate-mineral N(+) medium, the degradation kinetics were first order with the same apparent kinetic constant throughout growth and decline phases of the batch culture. The observed kinetics agreed with the model that supposes co-metabolic transformation of CL-20 uncoupled from cell growth, which can be carried out by several constitutive cellular enzymes with wide substrate specificity.

Application of hybrid organic/inorganic polymers as coatings on metallic substrates

NASA Astrophysics Data System (ADS)

Augustinho, T. R.; Motz, G.; Ihlow, S.; Machado, R. A. F.

2016-09-01

Acrylic polymers, particularly poly (methyl methacrylate) (PMMA), have certain specific properties, such as good film formation, transparency, and good mechanical properties, which have been widely used in paints, coatings and adhesives. However, the limited chemical and physical stability of these pure polymers limits their applications when exposed to hostile conditions, as in ship hulls, for example. A suitable way to enhance PMMA properties is the addition of silicon polymers with very good protective characteristics. In this study, a PMMA and HTT 1800 (commercial silazane) copolymer were applied on metallic substrate and compared to pure PMMA and HTT 1800. All the materials were applied as coatings. They were applied on stainless steel via dip-coating to investigate the coating properties. Thermal cycling was employed to analyze coating durability at high temperatures (50 °C to 600 °C). Optical microscopy (OM) and scanning electron microscopy (SEM) were used to characterize the coated surfaces, and the adhesion of pure PMMA, pure HTT 1800 and PMMA/HTT 1800 coatings on metallic substrate was investigated by Cross-Cut-Test (ASTM D 3359). The sessile drop method was used to determine the contact angle. PMMA coatings presented complete degradation from 250 °C, while hybrid coatings of PMMA and HTT 1800 have good protection until 400 °C. The adherence of the coating on metallic substrate showed improvement in all synthesized materials when compared to pure PMMA, obtaining the best adherence possible. The contact angle test showed that the hydrophobicity of the hybrid coatings is higher than that of the pure coatings.

In vitro metabolism of brucine by human liver microsomes and its interactions with CYP substrates.

PubMed

Li, Xin; Wang, Kai; Wei, Wei; Liu, Yong-yu; Gong, Lu

2013-08-25

Brucine, one of the main active ingredients in semen Strychni, has been included in many oral prescriptions of traditional Chinese medicine. In this study, we investigated the in vitro metabolism of brucine by human liver microsomes (HLMs) and the metabolic interactions of brucine with the substrates of cytochrome P450 (CYP450). Brucine was incubated with HLMs or CYP3A4 and then analysed by Liquid chromatography/mass spectrometry. The Km and Vmax values for HLMs were 30.53±3.14μM and 0.08±0.0029nmol/mg protein/min, respectively, while the corresponding values for CYP3A4 were 20.12±3.05μM and 6.40±0.21nmol/nmol P450/min. CYP3A4 may be the major enzyme responsible for brucine metabolism in HLMs, other human isoforms of CYP showed minimal or no effect on brucine metabolism. The inhibitory action of brucine was observed in CYP3A4 for the 1'-hydroxylation of midazolam, with inhibitory concentration 50 (IC50) of 8.4-fold higher than specific inhibitors in HLMs. Furthermore, brucine significantly inhibited the CYP3A4-catalyzed midazolam 1'-hydroxylation (Ki=2.14μM) at a concentration lower than 10μM, but no obvious inhibitory effects were observed on other CYP substrates (IC50>50μM). These results suggest that brucine has the potential to interact with a wide range of xenobiotics and endogenous chemicals especially CYP3A4 substrates. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Radio frequency diodes and circuits fabricated via adhesion lithography (Conference Presentation)

NASA Astrophysics Data System (ADS)

Georgiadou, Dimitra G.; Semple, James; Wyatt-Moon, Gwenhivir; Anthopoulos, Thomas D.

2016-09-01

The commercial interest in Radio Frequency Identification (RFID) tags keeps growing, as new application sectors, spanning from healthcare to electronic article surveillance (EAS) and personal identification, are constantly emerging for these types of electronic devices. The increasing demand for the so-called "smart labels" necessitates their high throughput manufacturing, and indeed on thin flexible substrates, that will reduce the cost and render them competitive to the currently widely employed barcodes. Adhesion Lithography (a-Lith) is a novel patterning technique that allows the facile high yield fabrication of co-planar large aspect ratio (<100,000) metal electrodes separated by a sub-20 nm gap on large area substrates of any type. Deposition of high mobility semiconductors from their solution at low, compatible with plastic substrates, temperatures and application of specific processing protocols can dramatically improve the performance of the fabricated Schottky diodes. It will be shown that in this manner both organic and inorganic high speed diodes and rectifiers can be obtained, operating at frequencies much higher than the 13.56 MHz benchmark, currently employed in passive RFID tags and near filed communications (NFC). This showcases the universality of this method towards fabricating high speed p- and n-type diodes, irrespective of the substrate, simply based on the extreme downscaling of key device dimensions obtained in these nanoscale structures. The potential for scaling up this technique at low cost, combined with the significant performance optimisation and improved functionality that can be attained through intelligent material selection, render a-Lith unique within the field of plastic electronics.

Irradiation of Yarrowia lipolytica NRRL YB-567 creating novel strains with enhanced ammonia and oil production on protein and carbohydrate substrates

USDA-ARS?s Scientific Manuscript database

Increased interest in sustainable production of renewable diesel and other valuable bioproducts is redoubling efforts to improve economic feasibility of microbial-based oil production. The yeast Yarrowia lipolytica is capable of employing a wide variety of substrates to produce oil and valuable co-p...

Peptide microarray analysis of substrate specificity of the transmembrane Ser/Thr kinase KPI-2 reveals reactivity with cystic fibrosis transmembrane conductance regulator and phosphorylase.

PubMed

Wang, Hong; Brautigan, David L

2006-11-01

Human lemur (Lmr) kinases are predicted to be Tyr kinases based on sequences and are related to neurotrophin receptor Trk kinases. This study used homogeneous recombinant KPI-2 (Lmr2, LMTK2, Cprk, brain-enriched protein kinase) kinase domain and a library of 1,154 peptides on a microarray to analyze substrate specificity. We found that KPI-2 is strictly a Ser/Thr kinase that reacts with Ser either preceded by or followed by Pro residues but unlike other Pro-directed kinases does not strictly require an adjacent Pro residue. The most reactive peptide in the library corresponds to Ser-737 of cystic fibrosis transmembrane conductance regulator, and the recombinant R domain of cystic fibrosis transmembrane conductance regulator was a preferred substrate. Furthermore the KPI-2 kinase phosphorylated peptides corresponding to the single site in phosphorylase and purified phosphorylase b, making this only the second known phosphorylase b kinase. Phosphorylase was used as a specific substrate to show that KPI-2 is inhibited in living cells by addition of nerve growth factor or serum. The results demonstrate the utility of the peptide library to probe specificity and discover kinase substrates and offer a specific assay that reveals hormonal regulation of the activity of this unusual transmembrane kinase.

Ultra-low roughness magneto-rheological finishing for EUV mask substrates

NASA Astrophysics Data System (ADS)

Dumas, Paul; Jenkins, Richard; McFee, Chuck; Kadaksham, Arun J.; Balachandran, Dave K.; Teki, Ranganath

2013-09-01

EUV mask substrates, made of titania-doped fused silica, ideally require sub-Angstrom surface roughness, sub-30 nm flatness, and no bumps/pits larger than 1 nm in height/depth. To achieve the above specifications, substrates must undergo iterative global and local polishing processes. Magnetorheological finishing (MRF) is a local polishing technique which can accurately and deterministically correct substrate figure, but typically results in a higher surface roughness than the current requirements for EUV substrates. We describe a new super-fine MRF® polishing fluid whichis able to meet both flatness and roughness specifications for EUV mask blanks. This eases the burden on the subsequent global polishing process by decreasing the polishing time, and hence the defectivity and extent of figure distortion.

The modern era of research on language evolution: Moving forward. Comment on "Towards a computational comparative neuroprimatology: Framing the language-ready brain" by Michael A. Arbib

NASA Astrophysics Data System (ADS)

Stout, Dietrich

2016-03-01

Twenty-five years ago, Pinker and Bloom [1] helped reinvigorate research on language evolution by arguing that language ;shows signs of complex design for the communication of propositional structures, and the only explanation for the origin of organs with complex design is the process of natural selection.; Since then, empirical research has tested the assertions of (cross-cultural) universality, (cross-species) uniqueness, and (cross-domain) specificity underpinning this argument from design. Appearances aside, points of consensus have emerged. The existence of a core computational and neural substrate unique to language and/or humans is still debated, but it is widely agreed that: 1) human language performance overlaps with behaviors in other domains and species, and 2) such general, pre-existing capacities provided the context for language-specific evolution (e.g. [2]).

Isolation of Lysosomes from Mammalian Tissues and Cultured Cells.

PubMed

Aguado, Carmen; Pérez-Jiménez, Eva; Lahuerta, Marcos; Knecht, Erwin

2016-01-01

Lysosomes participate within the cells in the degradation of organelles, macromolecules, and a wide variety of substrates. In any study on specific roles of lysosomes, both under physiological and pathological conditions, it is advisable to include methods that allow their reproducible and reliable isolation. However, purification of lysosomes is a difficult task, particularly in the case of cultured cells. This is mainly because of the heterogeneity of these organelles, along with their low number and high fragility. Also, isolation methods, while disrupting plasma membranes, have to preserve the integrity of lysosomes, as the breakdown of their membranes releases enzymes that could damage all cell organelles, including themselves. The protocols described below have been routinely used in our laboratory for the specific isolation of lysosomes from rat liver, NIH/3T3, and other cultured cells, but can be adapted to other mammalian tissues or cell lines.

[Industrial application of lipases].

PubMed

Bancerz, Renata

2017-01-01

The ability of lipases to perform specific reactions of transformation (biotransformation) makes these enzymes a useful tool used in many syntheses, for example: in the production of detergents, cosmetics, biosurfactants, in the oil-chemical, paper, dairy, food or pharmaceutical industries. Lipases are ubiquitous enzymes but only lipases produced by microorganisms are important for industrial applications due to their wide variety of properties such as stability in organic solvents, action under mild conditions, high substrate specificity and region- and enantioselectivity, as well as the relatively simple methods of their production in fermentors and recovery from the culture medium. This paper reviews the latest achievements in the production of lipases in the submerged fermentation and solid state fermentation using waste products from the agricultural industry. In addition, new applications of lipases were described, including those for the synthesis of biopolymers and biodiesel and for the production of enantiomeric pharmaceuticals, agrochemicals and flavoring compounds.

fMRI Brain-Computer Interface: A Tool for Neuroscientific Research and Treatment

PubMed Central

Sitaram, Ranganatha; Caria, Andrea; Veit, Ralf; Gaber, Tilman; Rota, Giuseppina; Kuebler, Andrea; Birbaumer, Niels

2007-01-01

Brain-computer interfaces based on functional magnetic resonance imaging (fMRI-BCI) allow volitional control of anatomically specific regions of the brain. Technological advancement in higher field MRI scanners, fast data acquisition sequences, preprocessing algorithms, and robust statistical analysis are anticipated to make fMRI-BCI more widely available and applicable. This noninvasive technique could potentially complement the traditional neuroscientific experimental methods by varying the activity of the neural substrates of a region of interest as an independent variable to study its effects on behavior. If the neurobiological basis of a disorder (e.g., chronic pain, motor diseases, psychopathy, social phobia, depression) is known in terms of abnormal activity in certain regions of the brain, fMRI-BCI can be targeted to modify activity in those regions with high specificity for treatment. In this paper, we review recent results of the application of fMRI-BCI to neuroscientific research and psychophysiological treatment. PMID:18274615

Mixed - Lineage Protein kinases (MLKs) in inflammation, metabolism, and other disease states.

PubMed

Craige, Siobhan M; Reif, Michaella M; Kant, Shashi

2016-09-01

Mixed lineage kinases, or MLKs, are members of the MAP kinase kinase kinase (MAP3K) family, which were originally identified among the activators of the major stress-dependent mitogen activated protein kinases (MAPKs), JNK and p38. During stress, the activation of JNK and p38 kinases targets several essential downstream substrates that react in a specific manner to the unique stressor and thus determine the fate of the cell in response to a particular challenge. Recently, the MLK family was identified as a specific modulator of JNK and p38 signaling in metabolic syndrome. Moreover, the MLK family of kinases appears to be involved in a very wide spectrum of disorders. This review discusses the newly identified functions of MLKs in multiple diseases including metabolic disorders, inflammation, cancer, and neurological diseases. Copyright © 2016 Elsevier B.V. All rights reserved.

Biocatalysts: application and engineering for industrial purposes.

PubMed

Jemli, Sonia; Ayadi-Zouari, Dorra; Hlima, Hajer Ben; Bejar, Samir

2016-01-01

Enzymes are widely applied in various industrial applications and processes, including the food and beverage, animal feed, textile, detergent and medical industries. Enzymes screened from natural origins are often engineered before entering the market place because their native forms do not meet the requirements for industrial application. Protein engineering is concerned with the design and construction of novel enzymes with tailored functional properties, including stability, catalytic activity, reaction product inhibition and substrate specificity. Two broad approaches have been used for enzyme engineering, namely, rational design and directed evolution. The powerful and revolutionary techniques so far developed for protein engineering provide excellent opportunities for the design of industrial enzymes with specific properties and production of high-value products at lower production costs. The present review seeks to highlight the major fields of enzyme application and to provide an updated overview on previous protein engineering studies wherein natural enzymes were modified to meet the operational conditions required for industrial application.

Optogenetic Assessment of Horizontal Interactions in Primary Visual Cortex

PubMed Central

Huang, Xiaoying; Elyada, Yishai M.; Bosking, William H.; Walker, Theo

2014-01-01

Columnar organization of orientation selectivity and clustered horizontal connections linking orientation columns are two of the distinctive organizational features of primary visual cortex in many mammalian species. However, the functional role of these connections has been harder to characterize. Here we examine the extent and nature of horizontal interactions in V1 of the tree shrew using optical imaging of intrinsic signals, optogenetic stimulation, and multi-unit recording. Surprisingly, we find the effects of optogenetic stimulation depend primarily on distance and not on the specific orientation domains or axes in the cortex, which are stimulated. In addition, across a wide range of variation in both visual and optogenetic stimulation we find linear addition of the two inputs. These results emphasize that the cortex provides a rich substrate for functional interactions that are not limited to the orientation-specific interactions predicted by the monosynaptic distribution of horizontal connections. PMID:24695715

Growth of porous anodized alumina on the sputtered aluminum films with 2D-3D morphology for high specific surface area

NASA Astrophysics Data System (ADS)

Liao, M. W.; Chung, C. K.

2014-08-01

The porous anodic aluminum oxide (AAO) with high-aspect-ratio pore channels is widely used as a template for fabricating nanowires or other one-dimensional (1D) nanostructures. The high specific surface area of AAO can also be applied to the super capacitor and the supporting substrate for catalysis. The rough surface could be helpful to enhance specific surface area but it generally results in electrical field concentration even to ruin AAO. In this article, the aluminum (Al) films with the varied 2D-3D morphology on Si substrates were prepared using magnetron sputtering at a power of 50 W-185 W for 1 h at a working pressure of 2.5 × 10-1 Pa. Then, AAO was fabricated from the different Al films by means of one-step hybrid pulse anodizing (HPA) between the positive 40 V and the negative -2 V (1 s:1 s) for 3 min in 0.3 M oxalic acid at a room temperature. The microstructure and morphology of Al films were characterized by X-ray diffraction, scanning electron microscope and atomic force microscope, respectively. Some hillocks formed at the high target power could be attributed to the grain texture growth in the normal orientation of Al(1 1 1). The 3D porous AAO structure which is different from the conventional 2D planar one has been successfully demonstrated using HPA on the film with greatly rough hillock-surface formed at the highest power of 185 W. It offers a potential application of the new 3D AAO to high specific surface area devices.

Purification, characterization and specificity of chondroitin lyases and glycuronidase from Flavobacterium heparinum.

PubMed Central

Gu, K; Linhardt, R J; Laliberté, M; Gu, K; Zimmermann, J

1995-01-01

The chondroitin lyases from Flavobacterium heparinum (Cytophaga heparinia) have been widely used in depolymerization of glycosaminoglycan and proteoglycan chondroitin sulphates. Oligosaccharide products derived from chondroitin sulphate can be further degraded by glycuronidases and sulphatases obtained from the same organism. There has been no reported purification of these enzymes to homogeneity nor is there any information on their physical and kinetic characteristics. The absence of pure enzymes has resulted in a lack of understanding of the optimal conditions for their catalytic activity and their substrate specificity. This has limited the use of these enzymes as reagents for preparation of oligosaccharides for structure and activity studies. Reproducible schemes to purify a chondroitin AC lyase, a glycuronidase and chondroitin B lyase from Flavobacterium heparinum to apparent homogeneity are described. Chondroitin AC lyase (chondroitinase AC, EC 4.2.2.5), glycuronidase [chondro-(1-->3)-glycuronidase, no EC number] and chondroitin B lyase (chondroitinase B, no EC number) have M(r) values (assessed by SDS/PAGE) of 74,000, 41,800 and 55,200 respectively, and isoelectric points (determined by isoelectric focusing) of 8.85, 9.28 and 9.05 respectively. Chondroitin lyase AC and B contain pyroglutamic acid at their N-termini precluding their analysis by Edman degradation. Deblocking with pyroglutamate aminopeptidase facilitated the determination of their N-terminal sequences. The kinetic properties of these enzymes have been determined as well as the optimum conditions for their catalytic activity. The specificity of the glycouronidase, determined using 17 different disaccharide substrates, shows that it only acts on unsulphated or 6-O-sulphated 1-->3 linkages. The chondroitin lyases are both endolytic enzymes, and oligosaccharide mapping shows their expected specificity towards the chondroitin and dermatan sulphate polymers. Images Figure 2 Figure 4 PMID:8526872

Principles of motivation revealed by the diverse functions of neuropharmacological and neuroanatomical substrates underlying feeding behavior

PubMed Central

Baldo, Brian A.; Pratt, Wayne E.; Will, Matthew J.; Hanlon, Erin C.; Bakshi, Vaishali P.; Cador, Martine

2013-01-01

Circuits that participate in specific subcomponents of feeding (e.g., gustatory perception, peripheral feedback relevant to satiety and energy balance, reward coding, etc.) are found at all levels of the neural axis. Further complexity is conferred by the wide variety of feeding-modulatory neurotransmitters and neuropeptides that act within these circuits. An ongoing challenge has been to refine the understanding of the functional specificity of these neurotransmitters and circuits, and there have been exciting advances in recent years. We focus here on foundational work of Dr. Ann Kelley that identified distinguishable actions of striatal opioid peptide modulation and dopamine transmission in subcomponents of reward processing. We also discuss her work in overlaying these neuropharmacological effects upon anatomical pathways that link the telencephalon (cortex and basal ganglia) with feeding-control circuits in the hypothalamus. Using these seminal contributions as a starting point, we will discuss new findings that expand our understanding of (1) the specific, differentiable motivational processes that are governed by central dopamine and opioid transmission, (2) the manner in which other striatal neuromodulators, specifically acetylcholine, endocannabinoids and adenosine, modulate these motivational processes (including via interactions with opioid systems), and (3) the organization of the cortical-subcortical network that subserves opioid-driven feeding. The findings discussed here strengthen the view that incentive-motivational properties of food are coded by substrates and neural circuits that are distinguishable from those that mediate the acute hedonic experience of food reward. Striatal opioid transmission modulates reward processing by engaging frontotemporal circuits, possibly via a hypothalamic-thalamic axis, that ultimately impinges upon hypothalamic modules dedicated to autonomic function and motor pattern control. We will conclude by discussing implications for understanding disorders of “non-homeostatic” feeding. PMID:23466532

Principles of motivation revealed by the diverse functions of neuropharmacological and neuroanatomical substrates underlying feeding behavior.

PubMed

Baldo, Brian A; Pratt, Wayne E; Will, Matthew J; Hanlon, Erin C; Bakshi, Vaishali P; Cador, Martine

2013-11-01

Circuits that participate in specific subcomponents of feeding (e.g., gustatory perception, peripheral feedback relevant to satiety and energy balance, reward coding, etc.) are found at all levels of the neural axis. Further complexity is conferred by the wide variety of feeding-modulatory neurotransmitters and neuropeptides that act within these circuits. An ongoing challenge has been to refine the understanding of the functional specificity of these neurotransmitters and circuits, and there have been exciting advances in recent years. We focus here on foundational work of Dr. Ann Kelley that identified distinguishable actions of striatal opioid peptide modulation and dopamine transmission in subcomponents of reward processing. We also discuss her work in overlaying these neuropharmacological effects upon anatomical pathways that link the telencephalon (cortex and basal ganglia) with feeding-control circuits in the hypothalamus. Using these seminal contributions as a starting point, we will discuss new findings that expand our understanding of (1) the specific, differentiable motivational processes that are governed by central dopamine and opioid transmission, (2) the manner in which other striatal neuromodulators, specifically acetylcholine, endocannabinoids and adenosine, modulate these motivational processes (including via interactions with opioid systems), and (3) the organization of the cortical-subcortical network that subserves opioid-driven feeding. The findings discussed here strengthen the view that incentive-motivational properties of food are coded by substrates and neural circuits that are distinguishable from those that mediate the acute hedonic experience of food reward. Striatal opioid transmission modulates reward processing by engaging frontotemporal circuits, possibly via a hypothalamic-thalamic axis, that ultimately impinges upon hypothalamic modules dedicated to autonomic function and motor pattern control. We will conclude by discussing implications for understanding disorders of "non-homeostatic" feeding. Copyright © 2013 Elsevier Ltd. All rights reserved.

QM/MM Investigation of Substrate and Product Specificities of Suv4-20h2: How Does This Enzyme Generate Dimethylated H4K20 from Monomethylated Substrate?

PubMed

Qian, Ping; Guo, Haobo; Wang, Liang; Guo, Hong

2017-06-13

Protein lysine methyltransferases (PKMTs) catalyze the methylation of lysine residues on histone proteins in the regulation of chromatin structure and gene expression. In contrast to many other PKMTs for which unmodified lysine is the methylation target, the enzymes in the Suv4-20 family are able to generate dimethylated product (H4K20me2) based exclusively on the monomethylated H4K20 substrate (H4K20me1). The origin of such substrate/product specificity is still not clear. Here, molecular dynamics (MD) and free energy (potential of mean force) simulations are undertaken using quantum mechanical/molecular mechanical (QM/MM) potentials to understand the substrate/product specificities of Suv4-20h2, a member of the Suv4-20 family. The free energy barriers for mono-, di-, and trimethylation in Suv4-20h2 obtained from the simulations are found to be well correlated with the specificities observed experimentally with the allowed dimethylation based on the H4K20me1 substrate and prohibited monomethylation and trimethylation based on H4K20 and H4K20me2, respectively. It is demonstrated that the reason for the relatively efficient dimethylation is an effective transition state (TS) stabilization through strengthening the CH···O interactions as well as the presence of a cation-π interaction at the transition state. The simulations also show that the failures of Suv4-20h2 to catalyze monomethylation and trimethylation are due, respectively, to a less effective TS stabilization and inability of the reactant complex containing H4K20me2 to adopt a reactive (near attack) configuration for methyl transfer. The results suggest that care must be exercised in the prediction of the substrate specificity based only on the existence of near attack configurations in substrate complexes.

Heat and Mass Transfer Measurements for Tray-Fermented Fungal Products

NASA Astrophysics Data System (ADS)

Jou, R.-Y.; Lo, C.-T.

2011-01-01

In this study, heat and mass transfer in static tray fermentation, which is widely used in solid-state fermentation (SSF) to produce fungal products, such as enzymes or koji, is investigated. Specifically, kinetic models of transport phenomena in the whole-tray chamber are emphasized. The effects of temperature, moisture, and humidity on microbial growth in large-scale static tray fermentation are essential to scale-up SSF and achieve uniform fermentation. In addition, heat and mass transfer of static tray fermentation of Trichoderma fungi with two tray setups—traditional linen coverings and stacks in a temperature-humidity chamber is examined. In both these setups, the following factors of fermentation were measured: air velocity, air temperature, illumination, pH, carbon dioxide (CO2) concentration, and substrate temperature, and the effects of bed height, moisture of substrate, and relative humidity of air are studied. A thin (1 cm) bed at 28 °C and 95 % relative humidity is found to be optimum. Furthermore, mixing was essential for achieving uniform fermentation of Trichoderma fungi. This study has important applications in large-scale static tray fermentation of fungi.

Characterization of an atypical, thermostable, organic solvent- and acid-tolerant 2'-deoxyribosyltransferase from Chroococcidiopsis thermalis.

PubMed

Del Arco, Jon; Sánchez-Murcia, Pedro Alejandro; Mancheño, José Miguel; Gago, Federico; Fernández-Lucas, Jesús

2018-06-05

In our search for thermophilic and acid-tolerant nucleoside 2'-deoxyribosyltransferases (NDTs), we found a good candidate in an enzyme encoded by Chroococcidiopsis thermalis PCC 7203 (CtNDT). Biophysical and biochemical characterization revealed CtNDT as a homotetramer endowed with good activity and stability at both high temperatures (50-100 °C) and a wide range of pH values (from 3 to 7). CtNDT recognizes purine bases and their corresponding 2'-deoxynucleosides but is also proficient using cytosine and 2'-deoxycytidine as substrates. These unusual features preclude the strict classification of CtNDT as either a type I or a type II NDT and further suggest that this simple subdivision may need to be updated in the future. Our findings also hint at a possible link between oligomeric state and NDT's substrate specificity. Interestingly from a practical perspective, CtNDT displays high activity (80-100%) in the presence of several water-miscible co-solvents in a proportion of up to 20% and was successfully employed in the enzymatic production of several therapeutic nucleosides such as didanosine, vidarabine, and cytarabine.

Clustering molecular dynamics trajectories for optimizing docking experiments.

PubMed

De Paris, Renata; Quevedo, Christian V; Ruiz, Duncan D; Norberto de Souza, Osmar; Barros, Rodrigo C

2015-01-01

Molecular dynamics simulations of protein receptors have become an attractive tool for rational drug discovery. However, the high computational cost of employing molecular dynamics trajectories in virtual screening of large repositories threats the feasibility of this task. Computational intelligence techniques have been applied in this context, with the ultimate goal of reducing the overall computational cost so the task can become feasible. Particularly, clustering algorithms have been widely used as a means to reduce the dimensionality of molecular dynamics trajectories. In this paper, we develop a novel methodology for clustering entire trajectories using structural features from the substrate-binding cavity of the receptor in order to optimize docking experiments on a cloud-based environment. The resulting partition was selected based on three clustering validity criteria, and it was further validated by analyzing the interactions between 20 ligands and a fully flexible receptor (FFR) model containing a 20 ns molecular dynamics simulation trajectory. Our proposed methodology shows that taking into account features of the substrate-binding cavity as input for the k-means algorithm is a promising technique for accurately selecting ensembles of representative structures tailored to a specific ligand.

Cell-Imprinted Substrates Modulate Differentiation, Redifferentiation, and Transdifferentiation.

PubMed

Bonakdar, Shahin; Mahmoudi, Morteza; Montazeri, Leila; Taghipoor, Mojtaba; Bertsch, Arnaud; Shokrgozar, Mohammad Ali; Sharifi, Shahriar; Majidi, Mohammad; Mashinchian, Omid; Hamrang Sekachaei, Mohammad; Zolfaghari, Pegah; Renaud, Philippe

2016-06-08

Differentiation of stem cells into mature cells through the use of physical approaches is of great interest. Here, we prepared smart nanoenvironments by cell-imprinted substrates based on chondrocytes, tenocytes, and semifibroblasts as templates and demonstrated their potential for differentiation, redifferentiation, and transdifferentiation. Analysis of shape and upregulation/downregulation of specific genes of stem cells, which were seeded on these cell-imprinted substrates, confirmed that imprinted substrates have the capability to induce specific shapes and molecular characteristics of the cell types that were used as templates for cell-imprinting. Interestingly, immunofluorescent staining of a specific protein in chondrocytes (i.e., collagen type II) confirmed that adipose-derived stem cells, semifibroblasts, and tenocytes can acquire the chondrocyte phenotype after a 14 day culture on chondrocyte-imprinted substrates. In summary, we propose that common polystyrene tissue culture plates can be replaced by this imprinting technique as an effective and promising way to regulate any cell phenotype in vitro with significant potential applications in regenerative medicine and cell-based therapies.

Insights into the Specificity of Lysine Acetyltransferases

DOE PAGES

Tucker, Alex C.; Taylor, Keenan C.; Rank, Katherine C.; ...

2014-11-07

Reversible lysine acetylation by protein acetyltransferases is a conserved regulatory mechanism that controls diverse cellular pathways. Gcn5-related N-acetyltransferases (GNATs), named after their founding member, are found in all domains of life. GNATs are known for their role as histone acetyltransferases, but non-histone bacterial protein acetytransferases have been identified. Only structures of GNAT complexes with short histone peptide substrates are available in databases. Given the biological importance of this modification and the abundance of lysine in polypeptides, how specificity is attained for larger protein substrates is central to understanding acetyl-lysine-regulated networks. In this paper, we report the structure of a GNATmore » in complex with a globular protein substrate solved to 1.9 Å. GNAT binds the protein substrate with extensive surface interactions distinct from those reported for GNAT-peptide complexes. Finally, our data reveal determinants needed for the recognition of a protein substrate and provide insight into the specificity of GNATs.« less

Flexible organic light emitting diodes fabricated on biocompatible silk fibroin substrate

NASA Astrophysics Data System (ADS)

Liu, Yuqiang; Xie, Yuemin; Liu, Yuan; Song, Tao; Zhang, Ke-Qin; Liao, Liangsheng; Sun, Baoquan

2015-10-01

Flexible and biodegradable electronics are currently under extensive investigation for biocompatible and environmentally-friendly applications. Synthetic plastic foils are widely used as substrates for flexible electronics. But typical plastic substrates such as polyethylene naphthalate (PEN) could not be degraded in a natural bio-environment. A great demand still exists for a next-generation biocompatible and biodegradable substrate for future application. For example, electronic devices can be potentially integrated into the human body. In this work, we demonstrate that the biocompatible and biodegradable natural silk fibroin (SF) films embedded with silver nanowires (AgNWs) mesh could be employed as conductive transparent substrates to fabricate flexible organic light emitting diodes (OLEDs). Compared with commercial PEN substrates coated with indium tin oxide, the AgNWs/SF composite substrates exhibit a similar sheet resistance of 12 Ω sq-1, a lower surface roughness, as well as a broader light transmission range. Flexible OLEDs based on AgNWs/SF substrates achieve a current efficiency of 19 cd A-1, demonstrating the potential of the flexible AgNWs/SF films as conductive and transparent substrates for next-generation biodegradable devices.

Low energy electron catalyst: the electronic origin of catalytic strategies.

PubMed

Davis, Daly; Sajeev, Y

2016-10-12

Using a low energy electron (LEE) as a catalyst, the electronic origin of the catalytic strategies corresponding to substrate selectivity, reaction specificity and reaction rate enhancement is investigated for a reversible unimolecular elementary reaction. An electronic energy complementarity between the catalyst and the substrate molecule is the origin of substrate selectivity and reaction specificity. The electronic energy complementarity is induced by tuning the electronic energy of the catalyst. The energy complementarity maximizes the binding forces between the catalyst and the molecule. Consequently, a new electronically metastable high-energy reactant state and a corresponding new low barrier reaction path are resonantly created for a specific reaction of the substrate through the formation of a catalyst-substrate transient adduct. The LEE catalysis also reveals a fundamental structure-energy correspondence in the formation of the catalyst-substrate transient adduct. Since the energy complementarities corresponding to the substrate molecules of the forward and the backward steps of the reversible reactions are not the same due to their structural differences, the LEE catalyst exhibits a unique one-way catalytic strategy, i.e., the LEE catalyst favors the reversible reaction more effectively in one direction. A characteristic stronger binding of the catalyst to the transition state of the reaction than in the initial reactant state and the final product state is the molecular origin of barrier lowering.

Stereoselective oxidation of aliphatic diols and reduction of hydroxy-ketones with galactitol dehydrogenase from Rhodobacter sphaeroides D.

PubMed

Kohring, G W; Wiehr, P; Jeworski, M; Giffhorn, F

2003-01-01

From the Rhodobacter sphaeroides mutant D a galactitol dehydrogenase (GDH) was isolated and characterized in an earlier investigation (1). The enzyme expressed activity with a wide spread substrate spectrum, like sugars, sugar alcohols, secondary alcohols or the corresponding ketones and it can be used for the production of the rare sugar L-tagatose by regioselective oxidation of galactitol (2). This study focuses on the preparation of optically pure aliphatic diols by oxidation of one enantiomer or stereospecific reduction of keto-alcohols and diketones. The oxidation of 1,2-propanediol, 1,2-butanediol, 1,2-pentanediol and 1,2-hexanediol occurred highly specific with the S-enantiomer leaving the R-enantiomer of the diols in the reaction vessel. Also (S)-1,2,6-hexanetriol was oxidized by GDH to 1,6-dihydroxy-2-hexanone. The Km values of these reactions decreased with increasing length of the carbon chain. Reduction of hydroxyacetone or 1-hydroxy-2-butanone resulted in an excess of 93% (S)-1,2-propanediol and more than 98% of (S)-1,2-butanediol, respectively. The diketone 2,3-hexanedione was only reduced to (2R,3S)-2,3-hexanediol, one of the possible four configurations. The wide substrate spectrum on one hand and the selectivity in the reaction on the other hand make GDH a very interesting enzyme for the production of optically pure building blocks in the chemical synthesis of bioactive compounds.

Structural and kinetic studies of a novel nerol dehydrogenase from Persicaria minor, a nerol-specific enzyme for citral biosynthesis.

PubMed

Tan, Cheng Seng; Hassan, Maizom; Mohamed Hussein, Zeti Azura; Ismail, Ismanizan; Ho, Kok Lian; Ng, Chyan Leong; Zainal, Zamri

2018-02-01

Geraniol degradation pathway has long been elucidated in microorganisms through bioconversion studies, yet weakly characterised in plants; enzyme with specific nerol-oxidising activity has not been reported. A novel cDNA encodes nerol dehydrogenase (PmNeDH) was isolated from Persicaria minor. The recombinant PmNeDH (rPmNeDH) is a homodimeric enzyme that belongs to MDR (medium-chain dehydrogenases/reductases) superfamily that catalyses the first oxidative step of geraniol degradation pathway in citral biosynthesis. Kinetic analysis revealed that rPmNeDH has a high specificity for allylic primary alcohols with backbone ≤10 carbons. rPmNeDH has ∼3 fold higher affinity towards nerol (cis-3,7-dimethyl-2,6-octadien-1-ol) than its trans-isomer, geraniol. To our knowledge, this is the first alcohol dehydrogenase with higher preference towards nerol, suggesting that nerol can be effective substrate for citral biosynthesis in P. minor. The rPmNeDH crystal structure (1.54 Å) showed high similarity with enzyme structures from MDR superfamily. Structure guided mutation was conducted to describe the relationships between substrate specificity and residue substitutions in the active site. Kinetics analyses of wild-type rPmNeDH and several active site mutants demonstrated that the substrate specificity of rPmNeDH can be altered by changing any selected active site residues (Asp 280 , Leu 294 and Ala 303 ). Interestingly, the L294F, A303F and A303G mutants were able to revamp the substrate preference towards geraniol. Furthermore, mutant that exhibited a broader substrate range was also obtained. This study demonstrates that P. minor may have evolved to contain enzyme that optimally recognise cis-configured nerol as substrate. rPmNeDH structure provides new insights into the substrate specificity and active site plasticity in MDR superfamily. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jacob, Reed B.; Ding, Feng; Ye, Dongmei

Organophosphates are widely used for peaceful (agriculture) and military purposes (chemical warfare agents). The extraordinary toxicity of organophosphates and the risk of deployment, make it critical to develop means for their rapid and efficient deactivation. Organophosphate hydrolase (OPH) already plays an important role in organophosphate remediation, but is insufficient for therapeutic or prophylactic purposes primarily due to low substrate affinity. Current efforts focus on directly modifying the active site to differentiate substrate specificity and increase catalytic activity. Here, we present a novel strategy for enhancing the general catalytic efficiency of OPH through computational redesign of the residues that are allostericallymore » coupled to the active site and validated our design by mutagenesis. Specifically, we identify five such hot-spot residues for allosteric regulation and assay these mutants for hydrolysis activity against paraoxon, a chemical-weapons simulant. A high percentage of the predicted mutants exhibit enhanced activity over wild-type (k cat =16.63 s -1), such as T199I/T54I (899.5 s -1) and C227V/T199I/T54I (848 s -1), while the Km remains relatively unchanged in our high-throughput cell-free expression system. Further computational studies of protein dynamics reveal four distinct distal regions coupled to the active site that display significant changes in conformation dynamics upon these identified mutations. These results validate a computational design method that is both efficient and easily adapted as a general procedure for enzymatic enhancement.« less

Improving specific activity and thermostability of Escherichia coli phytase by structure-based rational design.

PubMed

Wu, Tzu-Hui; Chen, Chun-Chi; Cheng, Ya-Shan; Ko, Tzu-Ping; Lin, Cheng-Yen; Lai, Hui-Lin; Huang, Ting-Yung; Liu, Je-Ruei; Guo, Rey-Ting

2014-04-10

Escherichia coli phytase (EcAppA) which hydrolyzes phytate has been widely applied in the feed industry, but the need to improve the enzyme activity and thermostability remains. Here, we conduct rational design with two strategies to enhance the EcAppA performance. First, residues near the substrate binding pocket of EcAppA were modified according to the consensus sequence of two highly active Citrobacter phytases. One out of the eleven mutants, V89T, exhibited 17.5% increase in catalytic activity, which might be a result of stabilized protein folding. Second, the EcAppA glycosylation pattern was modified in accordance with the Citrobacter phytases. An N-glycosylation motif near the substrate binding site was disrupted to remove spatial hindrance for phytate entry and product departure. The de-glycosylated mutants showed 9.6% increase in specific activity. On the other hand, the EcAppA mutants that adopt N-glycosylation motifs from CbAppA showed improved thermostability that three mutants carrying single N-glycosylation motif exhibited 5.6-9.5% residual activity after treatment at 80°C (1.8% for wild type). Furthermore, the mutant carrying all three glycosylation motifs exhibited 27% residual activity. In conclusion, a successful rational design was performed to obtain several useful EcAppA mutants with better properties for further applications. Copyright © 2014 Elsevier B.V. All rights reserved.

Three-dimensional Structure of Saccharomyces Invertase

PubMed Central

Sainz-Polo, M. Angela; Ramírez-Escudero, Mercedes; Lafraya, Alvaro; González, Beatriz; Marín-Navarro, Julia; Polaina, Julio; Sanz-Aparicio, Julia

2013-01-01

Invertase is an enzyme that is widely distributed among plants and microorganisms and that catalyzes the hydrolysis of the disaccharide sucrose into glucose and fructose. Despite the important physiological role of Saccharomyces invertase (SInv) and the historical relevance of this enzyme as a model in early biochemical studies, its structure had not yet been solved. We report here the crystal structure of recombinant SInv at 3.3 Å resolution showing that the enzyme folds into the catalytic β-propeller and β-sandwich domains characteristic of GH32 enzymes. However, SInv displays an unusual quaternary structure. Monomers associate in two different kinds of dimers, which are in turn assembled into an octamer, best described as a tetramer of dimers. Dimerization plays a determinant role in substrate specificity because this assembly sets steric constraints that limit the access to the active site of oligosaccharides of more than four units. Comparative analysis of GH32 enzymes showed that formation of the SInv octamer occurs through a β-sheet extension that seems unique to this enzyme. Interaction between dimers is determined by a short amino acid sequence at the beginning of the β-sandwich domain. Our results highlight the role of the non-catalytic domain in fine-tuning substrate specificity and thus supplement our knowledge of the activity of this important family of enzymes. In turn, this gives a deeper insight into the structural features that rule modularity and protein-carbohydrate recognition. PMID:23430743

Pichia pastoris regulates its gene-specific response to different carbon sources at the transcriptional, rather than the translational, level.

PubMed

Prielhofer, Roland; Cartwright, Stephanie P; Graf, Alexandra B; Valli, Minoska; Bill, Roslyn M; Mattanovich, Diethard; Gasser, Brigitte

2015-03-11

The methylotrophic, Crabtree-negative yeast Pichia pastoris is widely used as a heterologous protein production host. Strong inducible promoters derived from methanol utilization genes or constitutive glycolytic promoters are typically used to drive gene expression. Notably, genes involved in methanol utilization are not only repressed by the presence of glucose, but also by glycerol. This unusual regulatory behavior prompted us to study the regulation of carbon substrate utilization in different bioprocess conditions on a genome wide scale. We performed microarray analysis on the total mRNA population as well as mRNA that had been fractionated according to ribosome occupancy. Translationally quiescent mRNAs were defined as being associated with single ribosomes (monosomes) and highly-translated mRNAs with multiple ribosomes (polysomes). We found that despite their lower growth rates, global translation was most active in methanol-grown P. pastoris cells, followed by excess glycerol- or glucose-grown cells. Transcript-specific translational responses were found to be minimal, while extensive transcriptional regulation was observed for cells grown on different carbon sources. Due to their respiratory metabolism, cells grown in excess glucose or glycerol had very similar expression profiles. Genes subject to glucose repression were mainly involved in the metabolism of alternative carbon sources including the control of glycerol uptake and metabolism. Peroxisomal and methanol utilization genes were confirmed to be subject to carbon substrate repression in excess glucose or glycerol, but were found to be strongly de-repressed in limiting glucose-conditions (as are often applied in fed batch cultivations) in addition to induction by methanol. P. pastoris cells grown in excess glycerol or glucose have similar transcript profiles in contrast to S. cerevisiae cells, in which the transcriptional response to these carbon sources is very different. The main response to different growth conditions in P. pastoris is transcriptional; translational regulation was not transcript-specific. The high proportion of mRNAs associated with polysomes in methanol-grown cells is a major finding of this study; it reveals that high productivity during methanol induction is directly linked to the growth condition and not only to promoter strength.

A universal small molecule, inorganic phosphate, restricts the substrate specificity of Dicer-2 in small RNA biogenesis

PubMed Central

Fukunaga, Ryuya; Zamore, Phillip D

2014-01-01

The enzyme Dicer is central to the production of small silencing RNAs such as microRNAs (miRNAs) and small interfering RNAs (siRNAs). Like other insects, Drosophila melanogaster uses different Dicers to make siRNAs and miRNAs: Dicer-1 produces miRNAs from pre-miRNAs, whereas Dicer-2 generates siRNAs from long double-stranded RNA (dsRNA). How do the 2 Dicers achieve their substrate specificity? Here, we review recent findings that inorganic phosphate restricts the substrate specificity of Dicer-2 to long dsRNA. Inorganic phosphate inhibits Dicer-2 from binding and cleaving pre-miRNAs, without affecting the processing of long dsRNA. Crystal structures of a fragment of human Dicer in complex with an RNA duplex identify a phosphate-binding pocket that recognizes both the 5′-monophosphate of a substrate RNA and inorganic phosphate. We propose that inorganic phosphate occupies the phosphate-binding pocket in the fly Dicer-2, blocking binding of pre-miRNA and restricting pre-miRNA processing to Dicer-1. Thus, a small molecule can alter the substrate specificity of a nucleic acid-processing enzyme. PMID:24787225

Mammalian Protein Arginine Methyltransferase 7 (PRMT7) Specifically Targets RXR Sites in Lysine- and Arginine-rich Regions*

PubMed Central

Feng, You; Maity, Ranjan; Whitelegge, Julian P.; Hadjikyriacou, Andrea; Li, Ziwei; Zurita-Lopez, Cecilia; Al-Hadid, Qais; Clark, Amander T.; Bedford, Mark T.; Masson, Jean-Yves; Clarke, Steven G.

2013-01-01

The mammalian protein arginine methyltransferase 7 (PRMT7) has been implicated in roles of transcriptional regulation, DNA damage repair, RNA splicing, cell differentiation, and metastasis. However, the type of reaction that it catalyzes and its substrate specificity remain controversial. In this study, we purified a recombinant mouse PRMT7 expressed in insect cells that demonstrates a robust methyltransferase activity. Using a variety of substrates, we demonstrate that the enzyme only catalyzes the formation of ω-monomethylarginine residues, and we confirm its activity as the prototype type III protein arginine methyltransferase. This enzyme is active on all recombinant human core histones, but histone H2B is a highly preferred substrate. Analysis of the specific methylation sites within intact histone H2B and within H2B and H4 peptides revealed novel post-translational modification sites and a unique specificity of PRMT7 for methylating arginine residues in lysine- and arginine-rich regions. We demonstrate that a prominent substrate recognition motif consists of a pair of arginine residues separated by one residue (RXR motif). These findings will significantly accelerate substrate profile analysis, biological function study, and inhibitor discovery for PRMT7. PMID:24247247

Mammalian protein arginine methyltransferase 7 (PRMT7) specifically targets RXR sites in lysine- and arginine-rich regions.

PubMed

Feng, You; Maity, Ranjan; Whitelegge, Julian P; Hadjikyriacou, Andrea; Li, Ziwei; Zurita-Lopez, Cecilia; Al-Hadid, Qais; Clark, Amander T; Bedford, Mark T; Masson, Jean-Yves; Clarke, Steven G

2013-12-27

The mammalian protein arginine methyltransferase 7 (PRMT7) has been implicated in roles of transcriptional regulation, DNA damage repair, RNA splicing, cell differentiation, and metastasis. However, the type of reaction that it catalyzes and its substrate specificity remain controversial. In this study, we purified a recombinant mouse PRMT7 expressed in insect cells that demonstrates a robust methyltransferase activity. Using a variety of substrates, we demonstrate that the enzyme only catalyzes the formation of ω-monomethylarginine residues, and we confirm its activity as the prototype type III protein arginine methyltransferase. This enzyme is active on all recombinant human core histones, but histone H2B is a highly preferred substrate. Analysis of the specific methylation sites within intact histone H2B and within H2B and H4 peptides revealed novel post-translational modification sites and a unique specificity of PRMT7 for methylating arginine residues in lysine- and arginine-rich regions. We demonstrate that a prominent substrate recognition motif consists of a pair of arginine residues separated by one residue (RXR motif). These findings will significantly accelerate substrate profile analysis, biological function study, and inhibitor discovery for PRMT7.

Using oriented peptide array libraries to evaluate methylarginine-specific antibodies and arginine methyltransferase substrate motifs

PubMed Central

Gayatri, Sitaram; Cowles, Martis W.; Vemulapalli, Vidyasiri; Cheng, Donghang; Sun, Zu-Wen; Bedford, Mark T.

2016-01-01

Signal transduction in response to stimuli relies on the generation of cascades of posttranslational modifications that promote protein-protein interactions and facilitate the assembly of distinct signaling complexes. Arginine methylation is one such modification, which is catalyzed by a family of nine protein arginine methyltransferases, or PRMTs. Elucidating the substrate specificity of each PRMT will promote a better understanding of which signaling networks these enzymes contribute to. Although many PRMT substrates have been identified, and their methylation sites mapped, the optimal target motif for each of the nine PRMTs has not been systematically addressed. Here we describe the use of Oriented Peptide Array Libraries (OPALs) to methodically dissect the preferred methylation motifs for three of these enzymes – PRMT1, CARM1 and PRMT9. In parallel, we show that an OPAL platform with a fixed methylarginine residue can be used to validate the methyl-specific and sequence-specific properties of antibodies that have been generated against different PRMT substrates, and can also be used to confirm the pan nature of some methylarginine-specific antibodies. PMID:27338245

Cloning and characterization of a galactitol 2-dehydrogenase from Rhizobium legumenosarum and its application in D-tagatose production.

PubMed

Jagtap, Sujit Sadashiv; Singh, Ranjitha; Kang, Yun Chan; Zhao, Huimin; Lee, Jung-Kul

2014-05-10

Galactitol 2-dehydrogenase (GDH) belongs to the protein subfamily of short-chain dehydrogenases/reductases and can be used to produce optically pure building blocks and for the bioconversion of bioactive compounds. An NAD(+)-dependent GDH from Rhizobium leguminosarum bv. viciae 3841 (RlGDH) was cloned and overexpressed in Escherichia coli. The RlGDH protein was purified as an active soluble form using His-tag affinity chromatography. The molecular mass of the purified enzyme was estimated to be 28kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 114kDa by gel filtration chromatography, suggesting that the enzyme is a homotetramer. The enzyme has an optimal pH and temperature of 9.5 and 35°C, respectively. The purified recombinant RlGDH catalyzed the oxidation of a wide range of substrates, including polyvalent aliphatic alcohols and polyols, to the corresponding ketones and ketoses. Among various polyols, galactitol was the preferred substrate of RlGDH with a Km of 8.8mM, kcat of 835min(-1) and a kcat/Km of 94.9min(-1)mM(-1). Although GDHs have been characterized from a few other sources, RlGDH is distinguished from other GDHs by its higher specific activity for galactitol and broad substrate spectrum, making RlGDH a good choice for practical applications. Copyright © 2014 Elsevier Inc. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhu Shanshan; Zhang Hong; Matunis, Michael J.

SUMOs (small ubiquitin-related modifiers) are eukaryotic proteins that are covalently conjugated to other proteins and thereby regulate a wide range of important cellular processes. The molecular mechanisms by which SUMO modification influences the functions of most target proteins and cellular processes, however, remain poorly defined. A major obstacle to investigating the effects of SUMO modification is the availability of a system for selectively inducing the modification or demodification of an individual protein. To address this problem, we have developed a procedure using the rapamycin heterodimerizer system. This procedure involves co-expression of rapamycin-binding domain fusion proteins of SUMO and candidate SUMOmore » substrates in living cells. Treating cells with rapamycin induces a tight association between SUMO and a single SUMO substrate, thereby allowing specific downstream effects to be analyzed. Using RanGAP1 as a model SUMO substrate, the heterodimerizer system was used to investigate the molecular mechanism by which SUMO modification targets RanGAP1 from the cytoplasm to nuclear pore complexes (NPCs). Our results revealed a dual role for Ubc9 in targeting RanGAP1 to NPCs: In addition to conjugating SUMO-1 to RanGAP1, Ubc9 is also required to form a stable ternary complex with SUMO-1 modified RanGAP1 and Nup358. As illustrated by our studies, the rapamycin heterodimerizer system represents a novel tool for studying the molecular effects of SUMO modification.« less

Design and isolation of ribozyme-substrate pairs using RNase P-based ribozymes containing altered substrate binding sites.

PubMed Central

Mobley, E M; Pan, T

1999-01-01

Substrate recognition and cleavage by the bacterial RNase P RNA requires two domains, a specificity domain, or S-domain, and a catalytic domain, or C-domain. The S-domain binds the T stem-loop region in a pre-tRNA substrate to confer specificity for tRNA substrates. In this work, the entire S-domain of the Bacillus subtilis RNase P RNA is replaced with an artificial substrate binding module. New RNA substrates are isolated by in vitro selection using two libraries containing random regions of 60 nt. At the end of the selection, the cleavage rates of the substrate library are approximately 0.7 min(-1)in 10 mM MgCl(2)at 37 degrees C, approximately 4-fold better than the cleavage of a pre-tRNA substrate by the wild-type RNase P RNA under the same conditions. The contribution of the S-domain replacement to the catalytic efficiency is from 6- to 22 000-fold. Chemical and nuclease mapping of two ribozyme-product complexes shows that this contribution correlates with direct interactions between the S-domain replacement and the selected substrate. These results demonstrate the feasibility of design and isolation of RNase P-based, matching ribozyme-substrate pairs without prior knowledge of the sequence or structure of the interactive modules in the ribozyme or substrate. PMID:10518624

Biochemistry students' ideas about how an enzyme interacts with a substrate.

PubMed

Linenberger, Kimberly J; Bretz, Stacey Lowery

2015-01-01

Enzyme-substrate interactions are a fundamental concept of biochemistry that is built upon throughout multiple biochemistry courses. Central to understanding enzyme-substrate interactions is specific knowledge of exactly how an enzyme and substrate interact. Within this narrower topic, students must understand the various binding sites on an enzyme and be able to reason from simplistic lock and key or induced fit models to the more complex energetics model of transition state theory. Learning to understand these many facets of enzyme-substrate interactions and reasoning from multiple models present challenges where students incorrectly make connections between concepts or make no connection at all. This study investigated biochemistry students' understanding of enzyme-substrate interactions through the use of clinical interviews and a national administration (N = 707) of the Enzyme-Substrate Interactions Concept Inventory. Findings include misconceptions regarding the nature of enzyme-substrate interactions, naïve ideas about the active site, a lack of energetically driven interactions, and an incomplete understanding of the specificity pocket. © 2015 by the International Union of Biochemistry and Molecular Biology.

Structural insights into the difference in substrate recognition of two mannoside phosphorylases from two GH130 subfamilies.

PubMed

Ye, Yuxin; Saburi, Wataru; Odaka, Rei; Kato, Koji; Sakurai, Naofumi; Komoda, Keisuke; Nishimoto, Mamoru; Kitaoka, Motomitsu; Mori, Haruhide; Yao, Min

2016-03-01

In Ruminococcus albus, 4-O-β-D-mannosyl-D-glucose phosphorylase (RaMP1) and β-(1,4)-mannooligosaccharide phosphorylase (RaMP2) belong to two subfamilies of glycoside hydrolase family 130. The two enzymes phosphorolyze β-mannosidic linkages at the nonreducing ends of their substrates, and have substantially diverse substrate specificity. The differences in their mechanism of substrate binding have not yet been fully clarified. In the present study, we report the crystal structures of RaMP1 with/without 4-O-β-D-mannosyl-d-glucose and RaMP2 with/without β-(1→4)-mannobiose. The structures of the two enzymes differ at the +1 subsite of the substrate-binding pocket. Three loops are proposed to determine the different substrate specificities. One of these loops is contributed from the adjacent molecule of the oligomer structure. In RaMP1, His245 of loop 3 forms a hydrogen-bond network with the substrate through a water molecule, and is indispensible for substrate binding. © 2016 Federation of European Biochemical Societies.

Synthesis of ceria based superhydrophobic coating on Ni20Cr substrate via cathodic electrodeposition.

PubMed

Pedraza, F; Mahadik, S A; Bouchaud, B

2015-12-21

In this work, superhydrophobic cerium oxide coating surface (111) with dual scale texture on Ni20Cr substrate is obtained by combination of electropolishing the substrate and subsequent cathodic electrodeposition and long-term UVH surface relaxation. To form hierarchical structures of CeO2 is controllable by varying the substrate roughness, and electropolishing period. The results indicated that at the optimal condition, the surface of the cerium oxide coating showed a superhydrophobicity with a great water contact angle (151.0 ± 1.4°) with Gecko state. An interface model for electropolishing of substrate surface in cerium nitrate medium is proposed. We expect that this facile process can be readily and widely adopted for the design of superhydrophobic coating on engineering materials.

Low-cost laser diode array

DOEpatents

Freitas, B.L.; Skidmore, J.A.

1999-06-01

A substrate is used to fabricate a low-cost laser diode array. A substrate is machined from an electrically insulative material that is thermally conductive, or two substrates can be bonded together in which the top substrate is electrically as well as thermally conductive. The substrate thickness is slightly longer than the cavity length, and the width of the groove is wide enough to contain a bar and spring (which secures the laser bar firmly along one face of the groove). The spring also provides electrical continuity from the backside of the bar to the adjacent metalization layer on the laser bar substrate. Arrays containing one or more bars can be formed by creating many grooves at various spacings. Along the groove, many bars can be adjoined at the edges to provide parallel electrical conduction. This architecture allows precise and predictable registration of an array of laser bars to a self-aligned microlens array at low cost. 19 figs.

Catalytic mechanism of phenylacetone monooxygenases for non-native linear substrates.

PubMed

Carvalho, Alexandra T P; Dourado, Daniel F A R; Skvortsov, Timofey; de Abreu, Miguel; Ferguson, Lyndsey J; Quinn, Derek J; Moody, Thomas S; Huang, Meilan

2017-10-11

Phenylacetone monooxygenase (PAMO) is the most stable and thermo-tolerant member of the Baeyer-Villiger monooxygenase family, and therefore it is an ideal candidate for the synthesis of industrially relevant compounds. However, its limited substrate scope has largely limited its industrial applications. In the present work, we provide, for the first time, the catalytic mechanism of PAMO for the native substrate phenylacetone as well as for a linear non-native substrate 2-octanone, using molecular dynamics simulations, quantum mechanics and quantum mechanics/molecular mechanics calculations. We provide a theoretical basis for the preference of the enzyme for the native aromatic substrate over non-native linear substrates. Our study provides fundamental atomic-level insights that can be employed in the rational engineering of PAMO for wide applications in industrial biocatalysis, in particular, in the biotransformation of long-chain aliphatic oils into potential biodiesels.

Low-cost laser diode array

DOEpatents

Freitas, Barry L.; Skidmore, Jay A.

1999-01-01

A substrate is used to fabricate a low-cost laser diode array. A substrate is machined from an electrically insulative material that is thermally conductive, or two substrates can be bonded together in which the top substrate is electrically as well as thermally conductive. The substrate thickness is slightly longer than the cavity length, and the width of the groove is wide enough to contain a bar and spring (which secures the laser bar firmly along one face of the groove). The spring also provides electrical continuity from the backside of the bar to the adjacent metalization layer on the laser bar substrate. Arrays containing one or more bars can be formed by creating many grooves at various spacings. Along the groove, many bars can be adjoined at the edges to provide parallel electrical conduction. This architecture allows precise and predictable registration of an array of laser bars to a self-aligned microlens array at low cost.

Identification and substrate prediction of new Fragaria x ananassa aquaporins and expression in different tissues and during strawberry fruit development.

PubMed

Merlaen, Britt; De Keyser, Ellen; Van Labeke, Marie-Christine

2018-01-01

The newly identified aquaporin coding sequences presented here pave the way for further insights into the plant-water relations in the commercial strawberry ( Fragaria x ananassa ). Aquaporins are water channel proteins that allow water to cross (intra)cellular membranes. In Fragaria x ananassa , few of them have been identified hitherto, hampering the exploration of the water transport regulation at cellular level. Here, we present new aquaporin coding sequences belonging to different subclasses: plasma membrane intrinsic proteins subtype 1 and subtype 2 (PIP1 and PIP2) and tonoplast intrinsic proteins (TIP). The classification is based on phylogenetic analysis and is confirmed by the presence of conserved residues. Substrate-specific signature sequences (SSSSs) and specificity-determining positions (SDPs) predict the substrate specificity of each new aquaporin. Expression profiling in leaves, petioles and developing fruits reveals distinct patterns, even within the same (sub)class. Expression profiles range from leaf-specific expression over constitutive expression to fruit-specific expression. Both upregulation and downregulation during fruit ripening occur. Substrate specificity and expression profiles suggest that functional specialization exists among aquaporins belonging to a different but also to the same (sub)class.

The Structure of Lombricine Kinase

PubMed Central

Bush, D. Jeffrey; Kirillova, Olga; Clark, Shawn A.; Davulcu, Omar; Fabiola, Felcy; Xie, Qing; Somasundaram, Thayumanasamy; Ellington, W. Ross; Chapman, Michael S.

2011-01-01

Lombricine kinase is a member of the phosphagen kinase family and a homolog of creatine and arginine kinases, enzymes responsible for buffering cellular ATP levels. Structures of lombricine kinase from the marine worm Urechis caupo were determined by x-ray crystallography. One form was crystallized as a nucleotide complex, and the other was substrate-free. The two structures are similar to each other and more similar to the substrate-free forms of homologs than to the substrate-bound forms of the other phosphagen kinases. Active site specificity loop 309–317, which is disordered in substrate-free structures of homologs and is known from the NMR of arginine kinase to be inherently dynamic, is resolved in both lombricine kinase structures, providing an improved basis for understanding the loop dynamics. Phosphagen kinases undergo a segmented closing on substrate binding, but the lombricine kinase ADP complex is in the open form more typical of substrate-free homologs. Through a comparison with prior complexes of intermediate structure, a correlation was revealed between the overall enzyme conformation and the substrate interactions of His178. Comparative modeling provides a rationale for the more relaxed specificity of these kinases, of which the natural substrates are among the largest of the phosphagen substrates. PMID:21212263

Larval development sites of the main Culicoides species (Diptera: Ceratopogonidae) in northern Europe and distribution of coprophilic species larvae in Belgian pastures.

PubMed

Zimmer, Jean-Yves; Brostaux, Yves; Haubruge, Eric; Francis, Frédéric

2014-10-15

Some Culicoides species of biting midges (Diptera: Ceratopogonidae) are biological virus vectors worldwide and have indeed been associated with outbreaks of important epizoonoses in recent years, such as bluetongue and Schmallenberg disease in northern Europe. These diseases, which affect domestic and wild ruminants, have caused considerable economic losses. Knowledge of substrates suitable for Culicoides larval development is important, particularly for the main vector temperate species. This study, realized during two years, aimed to highlight the larval development sites of these biting midge species in the immediate surroundings of ten Belgian cattle farms. Moreover, spatial distribution of the coprophilic Culicoides larvae (C. chiopterus and C. dewulfi) within pastures was studied with increasing distance from farms along linear transects (farm-pasture-woodland). A total of 4347 adult specimens belonging to 13 Culicoides species were obtained by incubation of 2131 soil samples belonging to 102 different substrates; 18 of these substrates were suitable for larval development. The Obsoletus complex (formed by two species) was observed in a wide range of substrates, including silage residues, components of a chicken coop, dung adhering to walls inside stables, leftover feed along the feed bunk, a compost pile of sugar beet residues, soil of a livestock trampling area, and decaying wood, while the following served as substrates for the other specimens: C. chiopterus, mainly cow dung; C. dewulfi, cow dung and molehill soil; C. circumscriptus, algae; C. festivipennis, algae and soil in stagnant water; C. nubeculosus, algae and silt specifically from the edge of a pond; C. punctatus, mainly wet soil between silage reserves; C. salinarius, algae; and C. stigma, algae and wet soil between silage reserves. We also recorded significantly higher densities of coprophilic larvae within pastures in cow dung located near forests, which is likely due to the localization of potential hosts; the presence of these larvae within cow dung is, however, uninfluenced by relative distance from farms. A better knowledge of the microhabitats of Culicoides biting midges and their spatial distribution may allow the development of targeted species-specific vector control strategies, and may help to prevent the creation of new larval development sites. Copyright © 2014 Elsevier B.V. All rights reserved.

Low-cost and highly efficient DNA biosensor for heavy metal ion using specific DNAzyme-modified microplate and portable glucometer-based detection mode.

PubMed

Zhang, Jin; Tang, Ying; Teng, Liumei; Lu, Minghua; Tang, Dianping

2015-06-15

A simple and low-cost DNA sensing platform based on Pb(2+)-specific DNAzyme-modified microplate was successfully developed for highly sensitive monitoring of lead ion (Pb(2+), one kind of toxic heavy metal ion) in the environmental samples coupling with a portable personal glucometer (PGM)-based detection mode. The detection cell was first prepared simply by means of immobilizing the DNAzyme on the streptavidin-modified microplate. Gold nanoparticle labeled with single-stranded DNA and invertase (Enz-AuNP-DNA) was utilized as the signal-transduction tag to produce PGM substrate (glucose). Upon addition of lead ion into the microplate, the substrate strand of the immobilized DNAzyme was catalytically cleaved by target Pb(2+), and the newly generated single-strand DNA in the microplate could hybridize again with the single-stranded DNA on the Enz-AuNP-DNA. Accompanying with the Enz-AuNP-DNA, the carried invertase could convert sucrose into glucose. The as-produced glucose could be monitored by using a widely accessible PGM for in situ amplified digital readout. Based on Enz-AuNP-DNA amplification strategy, as low as 1.0 pM Pb(2+) could be detected under the optimal conditions. Moreover, the methodology also showed good reproducibility and high selectivity toward target Pb(2+) against other metal ions because of highly specific Pb(2+)-dependent DNAzyme, and was applicable for monitoring Pb(2+) in the naturally contaminated sewage and spiked drinking water samples. Copyright © 2015 Elsevier B.V. All rights reserved.

Quantitative iTRAQ-based secretome analysis reveals species-specific and temporal shifts in carbon utilization strategies among manganese(II)-oxidizing Ascomycete fungi

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zeiner, Carolyn A.; Purvine, Samuel O.; Zink, Erika M.

Fungi generate a wide range of extracellular hydrolytic and oxidative enzymes and reactive metabolites, collectively known as the secretome, that synergistically drive plant litter decomposition in the environment. While secretome studies of model organisms have greatly expanded our knowledge of these enzymes, few have extended secretome characterization to environmental isolates or directly compared temporal patterns of enzyme utilization among diverse species. Thus, the mechanisms of carbon (C) degradation by many ubiquitous soil fungi remain poorly understood. Here we use a combination of iTRAQ proteomics and custom bioinformatic analyses to compare the protein composition of the secretomes of four manganese(II)-oxidizing Ascomycetemore » fungi over a three-week time course. We demonstrate that although the fungi produce a similar suite of extracellular enzymes, they exhibit striking differences in the regulation of these enzymes among species and over time, revealing species-specific and temporal shifts in C utilization strategies as they degrade the same substrate. Specifically, our findings suggest that Paraconiothyrium sporulosum AP3s5-JAC2a and Alternaria alternata SRC1lrK2f employ sequential enzyme secretion patterns concomitant with decreasing resource availability, Stagonospora sp. SRC1lsM3a preferentially degrades proteinaceous substrate before switching to carbohydrates, and Pyrenochaeta sp. DS3sAY3a utilizes primarily peptidases to aggressively attack carbon sources in a concentrated burst. This work highlights the diversity of operative metabolic strategies among cellulose-degrading Ascomycetes and enhances our understanding of their role in C turnover in the environment.« less

Quantitative iTRAQ-based secretome analysis reveals species-specific and temporal shifts in carbon utilization strategies among manganese(II)-oxidizing Ascomycete fungi.

PubMed

Zeiner, Carolyn A; Purvine, Samuel O; Zink, Erika M; Paša-Tolić, Ljiljana; Chaput, Dominique L; Wu, Si; Santelli, Cara M; Hansel, Colleen M

2017-09-01

Fungi generate a wide range of extracellular hydrolytic and oxidative enzymes and reactive metabolites, collectively known as the secretome, that synergistically drive plant litter decomposition in the environment. While secretome studies of model organisms have greatly expanded our knowledge of these enzymes, few have extended secretome characterization to environmental isolates, particularly filamentous Ascomycetes, or directly compared temporal patterns of enzyme utilization among diverse species. Thus, the mechanisms of carbon (C) degradation by many ubiquitous soil fungi remain poorly understood. Here we use a combination of iTRAQ proteomics and extracellular enzyme activity assays to compare the protein composition of the secretomes of four manganese(II)-oxidizing Ascomycete fungi over a three-week time course. We demonstrate that the fungi exhibit striking differences in the regulation of extracellular lignocellulose-degrading enzymes among species and over time, revealing species-specific and temporal shifts in C utilization strategies as they degrade the same substrate. Specifically, our findings suggest that Alternaria alternata SRC1lrK2f and Paraconiothyrium sporulosum AP3s5-JAC2a employ sequential enzyme secretion patterns concomitant with decreasing resource availability. Stagonospora sp. SRC1lsM3a preferentially degrades proteinaceous substrate before switching to carbohydrates, and Pyrenochaeta sp. DS3sAY3a utilizes primarily peptidases to aggressively attack carbon sources in a concentrated burst. This work highlights the diversity of operative metabolic strategies among understudied yet ubiquitous cellulose-degrading Ascomycetes, enhancing our understanding of their contribution to C turnover in the environment. Copyright © 2017. Published by Elsevier Inc.

Biochemical profiling in silico--predicting substrate specificities of large enzyme families.

PubMed

Tyagi, Sadhna; Pleiss, Juergen

2006-06-25

A general high-throughput method for in silico biochemical profiling of enzyme families has been developed based on covalent docking of potential substrates into the binding sites of target enzymes. The method has been tested by systematically docking transition state--analogous intermediates of 12 substrates into the binding sites of 20 alpha/beta hydrolases from 15 homologous families. To evaluate the effect of side chain orientations to the docking results, 137 crystal structures were included in the analysis. A good substrate must fulfil two criteria: it must bind in a productive geometry with four hydrogen bonds between the substrate and the catalytic histidine and the oxyanion hole, and a high affinity of the enzyme-substrate complex as predicted by a high docking score. The modelling results in general reproduce experimental data on substrate specificity and stereoselectivity: the differences in substrate specificity of cholinesterases toward acetyl- and butyrylcholine, the changes of activity of lipases and esterases upon the size of the acid moieties, activity of lipases and esterases toward tertiary alcohols, and the stereopreference of lipases and esterases toward chiral secondary alcohols. Rigidity of the docking procedure was the major reason for false positive and false negative predictions, as the geometry of the complex and docking score may sensitively depend on the orientation of individual side chains. Therefore, appropriate structures have to be identified. In silico biochemical profiling provides a time efficient and cost saving protocol for virtual screening to identify the potential substrates of the members of large enzyme family from a library of molecules.

Wideband Low Side Lobe Aperture Coupled Patch Phased Array Antennas

NASA Astrophysics Data System (ADS)

Poduval, Dhruva

Low profile printed antenna arrays with wide bandwidth, high gain, and low Side Lobe Level (SLL) are in great demand for current and future commercial and military communication systems and radar. Aperture coupled patch antennas have been proposed to obtain wide impedance bandwidths in the past. Aperture coupling is preferred particularly for phased arrays because of their advantage of integration to other active devices and circuits, e.g. phase shifters, power amplifiers, low noise amplifiers, mixers etc. However, when designing such arrays, the interplay between array performance characteristics, such as gain, side lobe level, back lobe level, mutual coupling etc. must be understood and optimized under multiple design constraints, e.g. substrate material properties and thicknesses, element to element spacing, and feed lines and their orientation and arrangements with respect to the antenna elements. The focus of this thesis is to investigate, design, and develop an aperture coupled patch array with wide operating bandwidth (30%), high gain (17.5 dBi), low side lobe level (20 dB), and high Forward to Backward (F/B) ratio (21.8 dB). The target frequency range is 2.4 to 3 GHz given its wide application in WLAN, LTE (Long Term Evolution) and other communication systems. Notwithstanding that the design concept can very well be adapted at other frequencies. Specifically, a 16 element, 4 by 4 planar microstrip patch array is designed using HFSS and experimentally developed and tested. Starting from mutual coupling minimization a corporate feeding scheme is designed to achieve the needed performance. To reduce the SLL the corporate feeding network is redesigned to obtain a specific amplitude taper. Studies are conducted to determine the optimum location for a metallic reflector under the feed line to improve the F/B. An experimental prototype of the antenna was built and tested validating and demonstrating the performance levels expected from simulation predictions. Finally, simulated beam scanning in several angles of the array is shown considering specific phases for each antenna element in the array.

Oxidation of Wine Polyphenols by Secretomes of Wild Botrytis cinerea Strains from White and Red Grape Varieties and Determination of Their Specific Laccase Activity.

PubMed

Zimdars, Sabrina; Hitschler, Julia; Schieber, Andreas; Weber, Fabian

2017-12-06

Processing of Botrytis cinerea-infected grapes leads to enhanced enzymatic browning reactions mainly caused by the enzyme laccase which is able to oxidize a wide range of phenolic compounds. The extent of color deterioration depends on the activity of the enzymes secreted by the fungus. The present study revealed significant differences in the oxidative properties of secretomes of several B. cinerea strains isolated from five grape varieties. The presumed laccase-containing secretomes varied in their catalytic activity toward six phenolic compounds present in grapes. All strains led to identical product profiles for five of six substrates, but two strains showed deviating product profiles during gallic acid oxidation. Fast oxidation of caffeic acid, ferulic acid, and malvidin 3-O-glucoside was observed. Product formation rates and relative product concentrations were determined. The results reflect the wide range of enzyme activity and the corresponding different impact on color deterioration by B. cinerea.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nuzzo, Ralph G.; Rogers, John A.; Menard, Etienne

The invention provides methods and devices for fabricating printable semiconductor elements and assembling printable semiconductor elements onto substrate surfaces. Methods, devices and device components of the present invention are capable of generating a wide range of flexible electronic and optoelectronic devices and arrays of devices on substrates comprising polymeric materials. The present invention also provides stretchable semiconductor structures and stretchable electronic devices capable of good performance in stretched configurations.

Pd/V.sub.2O.sub.5 device for colorimetric H.sub.2 detection

DOEpatents

Liu, Ping [San Diego, CA; Tracy, C Edwin [Golden, CO; Pitts, J Roland [Lakewood, CO; Smith, II, R. Davis; Lee, Se-Hee [Lakewood, CO

2008-09-02

A sensor structure for chemochromic optical detection of hydrogen gas over a wide response range, that exhibits stability during repeated coloring/bleaching cycles upon exposure and removal of hydrogen gas, comprising: a glass substrate (20); a vanadium oxide layer (21) coated on the glass substrate; and a palladium layer (22) coated on the vanadium oxide layer.

Recent progress in flexible OLED displays

NASA Astrophysics Data System (ADS)

Hack, Michael G.; Weaver, Michael S.; Mahon, Janice K.; Brown, Julie J.

2001-09-01

Organic light emitting device (OLED) technology has recently been shown to demonstrate excellent performance and cost characteristics for use in numerous flat panel display (FPD) applications. OLED displays emit bright, colorful light with excellent power efficiency, wide viewing angle and video response rates. OLEDs are also demonstrating the requisite environmental robustness for a wide variety of applications. OLED technology is also the first FPD technology with the potential to be highly functional and durable in a flexible format. The use of plastic and other flexible substrate materials offers numerous advantages over commonly used glass substrates, including impact resistance, light weight, thinness and conformability. Currently, OLED displays are being fabricated on rigid substrates, such as glass or silicon wafers. At Universal Display Corporation (UDC), we are developing a new class of flexible OLED displays (FOLEDs). These displays also have extremely low power consumption through the use of electrophosphorescent doped OLEDs. To commercialize FOLED technology, a number of technical issues related to packaging and display processing on flexible substrates need to be addressed. In this paper, we report on our recent results to demonstrate the key technologies that enable the manufacture of power efficient, long-life flexible OLED displays for commercial and military applications.

Cell-Bound Lipase and Esterase of Brevibacterium linens

PubMed Central

Sørhaug, Terje; Ordal, Z. John

1974-01-01

The activities of glycerol ester hydrolase, lipase (EC 3.1.1.3) and carboxylesterase, and esterase (EC 3.1.1.1) were determined for whole cell preparations of Brevibacterium linens by using the pH-stat assay. The culture growth liquors were inactive against the three substrates, tributyrin emulsion, triacetin, and methyl butyrate. Cells washed in water had less activity than cells washed in 5% NaCl; the ratio of activities was close to 1:2 for all strains using tributyrin emulsion as the substrate. For the esterase substrates, this relationship varied widely and was strain dependent. The ability to hydrolyze the two esterase substrates varied independently of the level of lipase activity. PMID:4824883

Sensitive kinase assay linked with phosphoproteomics for identifying direct kinase substrates

PubMed Central

Xue, Liang; Wang, Wen-Horng; Iliuk, Anton; Hu, Lianghai; Galan, Jacob A.; Yu, Shuai; Hans, Michael; Geahlen, Robert L.; Tao, W. Andy

2012-01-01

Our understanding of the molecular control of many disease pathologies requires the identification of direct substrates targeted by specific protein kinases. Here we describe an integrated proteomic strategy, termed kinase assay linked with phosphoproteomics, which combines a sensitive kinase reaction with endogenous kinase-dependent phosphoproteomics to identify direct substrates of protein kinases. The unique in vitro kinase reaction is carried out in a highly efficient manner using a pool of peptides derived directly from cellular kinase substrates and then dephosphorylated as substrate candidates. The resulting newly phosphorylated peptides are then isolated and identified by mass spectrometry. A further comparison of these in vitro phosphorylated peptides with phosphopeptides derived from endogenous proteins isolated from cells in which the kinase is either active or inhibited reveals new candidate protein substrates. The kinase assay linked with phosphoproteomics strategy was applied to identify unique substrates of spleen tyrosine kinase (Syk), a protein-tyrosine kinase with duel properties of an oncogene and a tumor suppressor in distinctive cell types. We identified 64 and 23 direct substrates of Syk specific to B cells and breast cancer cells, respectively. Both known and unique substrates, including multiple centrosomal substrates for Syk, were identified, supporting a unique mechanism that Syk negatively affects cell division through its centrosomal kinase activity. PMID:22451900

Interactions between Casein Kinase Iε (CKIε) and Two Substrates from Disparate Signaling Pathways Reveal Mechanisms for Substrate-Kinase Specificity

PubMed Central

Dahlberg, Caroline Lund; Nguyen, Elizabeth Z.; Goodlett, David; Kimelman, David

2009-01-01

Background Members of the Casein Kinase I (CKI) family of serine/threonine kinases regulate diverse biological pathways. The seven mammalian CKI isoforms contain a highly conserved kinase domain and divergent amino- and carboxy-termini. Although they share a preferred target recognition sequence and have overlapping expression patterns, individual isoforms often have specific substrates. In an effort to determine how substrates recognize differences between CKI isoforms, we have examined the interaction between CKIε and two substrates from different signaling pathways. Methodology/Principal Findings CKIε, but not CKIα, binds to and phosphorylates two proteins: Period, a transcriptional regulator of the circadian rhythms pathway, and Disheveled, an activator of the planar cell polarity pathway. We use GST-pull-down assays data to show that two key residues in CKIα's kinase domain prevent Disheveled and Period from binding. We also show that the unique C-terminus of CKIε does not determine Dishevelled's and Period's preference for CKIε nor is it essential for binding, but instead plays an auxillary role in stabilizing the interactions of CKIε with its substrates. We demonstrate that autophosphorylation of CKIε's C-terminal tail prevents substrate binding, and use mass spectrometry and chemical crosslinking to reveal how a phosphorylation-dependent interaction between the C-terminal tail and the kinase domain prevents substrate phosphorylation and binding. Conclusions/Significance The biochemical interactions between CKIε and Disheveled, Period, and its own C-terminus lead to models that explain CKIε's specificity and regulation. PMID:19274088

Effects of substrate concentrations on the growth of heterotrophic bacteria and algae in secondary facultative ponds.

PubMed

Kayombo, S; Mbwette, T S A; Katima, J H Y; Jorgensen, S E

2003-07-01

This paper presents the effect of substrate concentration on the growth of a mixed culture of algae and heterotrophic bacteria in secondary facultative ponds (SFPs) utilizing settled domestic sewage as a sole source of organic carbon. The growth of the mixed culture was studied at the concentrations ranging between 200 and 800 mg COD/l in a series of batch chemostat reactors. From the laboratory data, the specific growth rate (micro) was determined using the modified Gompertz model. The maximum specific growth rate ( micro(max)) and half saturation coefficients (K(s)) were calculated using the Monod kinetic equation. The maximum observed growth rate ( micro(max)) for heterotrophic bacteria was 3.8 day(-1) with K(s) of 200 mg COD/l. The micro(max) for algal biomass based on suspended volatile solids was 2.7 day(-1) with K(s) of 110 mg COD/l. The micro(max) of algae based on the chlorophyll-a was 3.5 day(-1) at K(s) of 50mg COD/l. The observed specific substrate removal by heterotrophic bacteria varied between the concentrations of substrate used and the average value was 0.82 (mg COD/mg biomass). The specific substrate utilization rate in the bioreactors was direct proportional to the specific growth rate. Hence, the determined Monod kinetic parameters are useful for the definition of the operation of SFPs.

A Haloalkane Dehalogenase from a Marine Microbial Consortium Possessing Exceptionally Broad Substrate Specificity.

PubMed

Buryska, Tomas; Babkova, Petra; Vavra, Ondrej; Damborsky, Jiri; Prokop, Zbynek

2018-01-15

The haloalkane dehalogenase enzyme DmmA was identified by marine metagenomic screening. Determination of its crystal structure revealed an unusually large active site compared to those of previously characterized haloalkane dehalogenases. Here we present a biochemical characterization of this interesting enzyme with emphasis on its structure-function relationships. DmmA exhibited an exceptionally broad substrate specificity and degraded several halogenated environmental pollutants that are resistant to other members of this enzyme family. In addition to having this unique substrate specificity, the enzyme was highly tolerant to organic cosolvents such as dimethyl sulfoxide, methanol, and acetone. Its broad substrate specificity, high overexpression yield (200 mg of protein per liter of cultivation medium; 50% of total protein), good tolerance to organic cosolvents, and a broad pH range make DmmA an attractive biocatalyst for various biotechnological applications. IMPORTANCE We present a thorough biochemical characterization of the haloalkane dehalogenase DmmA from a marine metagenome. This enzyme with an unusually large active site shows remarkably broad substrate specificity, high overexpression, significant tolerance to organic cosolvents, and activity under a broad range of pH conditions. DmmA is an attractive catalyst for sustainable biotechnology applications, e.g., biocatalysis, biosensing, and biodegradation of halogenated pollutants. We also report its ability to convert multiple halogenated compounds to corresponding polyalcohols. Copyright © 2018 American Society for Microbiology.

UFD4 lacking the proteasome-binding region catalyses ubiquitination but is impaired in proteolysis.

PubMed

Xie, Youming; Varshavsky, Alexander

2002-12-01

The ubiquitin system recognizes degradation signals of protein substrates through E3-E2 ubiquitin ligases, which produce a substrate-linked multi-ubiquitin chain. Ubiquitinated substrates are degraded by the 26S proteasome, which consists of the 20S protease and two 19S particles. We previously showed that UBR1 and UFD4, two E3 ligases of the yeast Saccharomyces cerevisiae, interact with specific proteasomal subunits. Here we advance this analysis for UFD4 and show that it interacts with RPT4 and RPT6, two subunits of the 19S particle. The 201-residue amino-terminal region of UFD4 is essential for its binding to RPT4 and RPT6. UFD4(DeltaN), which lacks this N-terminal region, adds ubiquitin to test substrates with apparently wild-type activity, but is impaired in conferring short half-lives on these substrates. We propose that interaction of a targeted substrate with the 26S proteasome involves contacts of specific proteasomal subunits with the substrate-bound ubiquitin ligase, with the substrate-linked multi-ubiquitin chain and with the substrate itself. This multiple-site binding may function to slow down dissociation of the substrate from the proteasome and to facilitate the unfolding of substrate through ATP-dependent movements of the chaperone subunits of the 19S particle.

Exploration of CIGAS Alloy System for Thin-Film Photovoltaics on Novel Lightweight and Flexible Substrates

NASA Technical Reports Server (NTRS)

Woods, Lawrence M.; Kalla, Ajay; Ribelin, Rosine

2007-01-01

Thin-film photovoltaics (TFPV) on lightweight and flexible substrates offer the potential for very high solar array specific power (W/kg). ITN Energy Systems, Inc. (ITN) is developing flexible TFPV blanket technology that has potential for specific power greater than 2000 W/kg (including space coatings) that could result in solar array specific power between 150 and 500 W/kg, depending on array size, when mated with mechanical support structures specifically designed to take advantage of the lightweight and flexible substrates.(1) This level of specific power would far exceed the current state of the art for spacecraft PV power generation, and meet the needs for future spacecraft missions.(2) Furthermore the high specific power would also enable unmanned aircraft applications and balloon or high-altitude airship (HAA) applications, in addition to modular and quick deploying tents for surface assets or lunar base power, as a result of the high power density (W/sq m) and ability to be integrated into the balloon, HAA or tent fabric. ITN plans to achieve the high specific power by developing single-junction and two-terminal monolithic tandem-junction PV cells using thin-films of high-efficiency and radiation resistant CuInSe2 (CIS) partnered with bandgap-tunable CIS-alloys with Ga (CIGS) or Al (CIAS) on novel lightweight and flexible substrates. Of the various thin-film technologies, single-junction and radiation resistant CIS and associated alloys with gallium, aluminum and sulfur have achieved the highest levels of TFPV device performance, with the best efficiency reaching 19.5% under AM1.5 illumination conditions and on thick glass substrates.(3) Thus, it is anticipated that single- and tandem-junction devices with flexible substrates and based on CIS and related alloys will achieve the highest levels of thin-film space and HAA solar array performance.

Germanium-Assisted Direct Growth of Graphene on Arbitrary Dielectric Substrates for Heating Devices

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Ziwen; Xue, Zhongying; Zhang, Miao

Direct growth of graphene on dielectric substrates is a prerequsite for the development of graphene-based electronic and optoelectronic devices. However, the current graphene synthesis directly on dielectric substrates always involves metal contamination problem, and the direct production of graphene patterns still remains unattainable and challenging. We propose herein a semiconducting Ge-assisted chemical vapor deposition approach to directly grow monolayer graphene on arbitrary dielectric substrates. By pre-patterning of catalytic Ge layer, the graphene with desired pattern can be achieved with extreme ease. Due to the catalysis of Ge, monolayer graphene is able to form on Ge covered dielectric substrates including SiOmore » 2/Si, quartz glass and sapphire substrates. Optimization of the process parameters leads to the complete sublimation of catalytic Ge layer during or immediately after monolayer graphene formation, thus resulting in direct deposition of large-area continuous graphene on dielectric substrates. The large-area, highly conductive graphene synthesized on transparent dielectric substrate using the proposed approach has exhibited wide applications, e.g., in defogger and in thermochromic displays, with both devices possessing excellent performances.« less

Germanium-Assisted Direct Growth of Graphene on Arbitrary Dielectric Substrates for Heating Devices

DOE PAGES

Wang, Ziwen; Xue, Zhongying; Zhang, Miao; ...

2017-05-31

Direct growth of graphene on dielectric substrates is a prerequsite for the development of graphene-based electronic and optoelectronic devices. However, the current graphene synthesis directly on dielectric substrates always involves metal contamination problem, and the direct production of graphene patterns still remains unattainable and challenging. We propose herein a semiconducting Ge-assisted chemical vapor deposition approach to directly grow monolayer graphene on arbitrary dielectric substrates. By pre-patterning of catalytic Ge layer, the graphene with desired pattern can be achieved with extreme ease. Due to the catalysis of Ge, monolayer graphene is able to form on Ge covered dielectric substrates including SiOmore » 2/Si, quartz glass and sapphire substrates. Optimization of the process parameters leads to the complete sublimation of catalytic Ge layer during or immediately after monolayer graphene formation, thus resulting in direct deposition of large-area continuous graphene on dielectric substrates. The large-area, highly conductive graphene synthesized on transparent dielectric substrate using the proposed approach has exhibited wide applications, e.g., in defogger and in thermochromic displays, with both devices possessing excellent performances.« less

Tea waste: an effective and economic substrate for oyster mushroom cultivation.

PubMed

Yang, Doudou; Liang, Jin; Wang, Yunsheng; Sun, Feng; Tao, Hong; Xu, Qiang; Zhang, Liang; Zhang, Zhengzhu; Ho, Chi-Tang; Wan, Xiaochun

2016-01-30

Tea waste is the residue that remains after tea leaves have been extracted by hot water to obtain water-soluble components. The waste contains a re-usable energy substrate and nutrients which may pollute the environment if they are not dealt with appropriately. Other agricultural wastes have been widely studied as substrates for cultivating mushrooms. In the present study, we cultivated oyster mushroom using tea waste as substrate. To study the feasibility of re-using it, tea waste was added to the substrate at different ratios in different experimental groups. Three mushroom strains (39, 71 and YOU) were compared and evaluated. Mycelia growth rate, yield, biological efficiency and growth duration were measured. Substrates with different tea waste ratios showed different growth and yield performance. The substrate containing 40-60% of tea waste resulted in the highest yield. Tea waste could be used as an effective and economic substrate for oyster mushroom cultivation. This study also provided a useful way of dealing with massive amounts of tea waste. © 2015 Society of Chemical Industry.

Engineering Nucleotide Specificity of Succinyl-CoA Synthetase in Blastocystis: The Emerging Role of Gatekeeper Residues.

PubMed

Vashisht, Kapil; Verma, Sonia; Gupta, Sunita; Lynn, Andrew M; Dixit, Rajnikant; Mishra, Neelima; Valecha, Neena; Hamblin, Karleigh A; Maytum, Robin; Pandey, Kailash C; van der Giezen, Mark

2017-01-24

Charged, solvent-exposed residues at the entrance to the substrate binding site (gatekeeper residues) produce electrostatic dipole interactions with approaching substrates, and control their access by a novel mechanism called "electrostatic gatekeeper effect". This proof-of-concept study demonstrates that the nucleotide specificity can be engineered by altering the electrostatic properties of the gatekeeper residues outside the binding site. Using Blastocystis succinyl-CoA synthetase (SCS, EC 6.2.1.5), we demonstrated that the gatekeeper mutant (ED) resulted in ATP-specific SCS to show high GTP specificity. Moreover, nucleotide binding site mutant (LF) had no effect on GTP specificity and remained ATP-specific. However, via combination of the gatekeeper mutant with the nucleotide binding site mutant (ED+LF), a complete reversal of nucleotide specificity was obtained with GTP, but no detectable activity was obtained with ATP. This striking result of the combined mutant (ED+LF) was due to two changes; negatively charged gatekeeper residues (ED) favored GTP access, and nucleotide binding site residues (LF) altered ATP binding, which was consistent with the hypothesis of the "electrostatic gatekeeper effect". These results were further supported by molecular modeling and simulation studies. Hence, it is imperative to extend the strategy of the gatekeeper effect in a different range of crucial enzymes (synthetases, kinases, and transferases) to engineer substrate specificity for various industrial applications and substrate-based drug design.

Kinetic modeling of lactic acid production from batch submerged fermentation of cheese whey

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tango, M.S.A.; Ghaly, A.E.

1999-12-01

A kinetic model for the production of lactic acid through batch submerged fermentation of cheese whey using Lactobacillus helveticus was developed. The model accounts for the effect of substrate limitation, substrate inhibition, lactic acid inhibition, maintenance energy and cell death on the cell growth, substrate utilization, and lactic acid production during the fermentation process. The model was evaluated using experimental data from Tango and Ghaly (1999). The predicted results obtained from the model compared well with experimental (R{sup 2} = 0.92--0.98). The model was also used to investigate the effect of the initial substrate concentration on the lag period, fermentationmore » time, specific growth rate, and cell productivity during batch fermentation. The maximum specific growth rate ({micro}{sub m}), the saturation constant (K{sub S}), the substrate inhibition constant (K{sub IS}), and the lactic acid inhibition constant (K{sub IP}) were found to be 0.25h{sup {minus}1}, 0.9 g/L, 250.0 g/L, and 60.0 g/L, respectively. High initial lactose concentration in cheese whey reduced both the specific growth rate and substrate utilization rate due to the substrate inhibition phenomenon. The maximum lactic acid production occurred at about 100 g/L initial lactose concentration after 40 h of fermentation. The maximum lactic acid concentration above which Lactobacillus helveticus did not grow was found to be 80.0 g/L.« less

Molecular Basis for the Relative Substrate Specificity of Human Immunodeficiency Virus Type 1 and Feline Immunodeficiency Virus Proteases

PubMed Central

Beck, Zachary Q.; Lin, Ying-Chuan; Elder, John H.

2001-01-01

We have used a random hexamer phage library to delineate similarities and differences between the substrate specificities of human immunodeficiency virus type 1 (HIV-1) and feline immunodeficiency virus (FIV) proteases (PRs). Peptide sequences were identified that were specifically cleaved by each protease, as well as sequences cleaved equally well by both enzymes. Based on amino acid distinctions within the P3-P3′ region of substrates that appeared to correlate with these cleavage specificities, we prepared a series of synthetic peptides within the framework of a peptide sequence cleaved with essentially the same efficiency by both HIV-1 and FIV PRs, Ac-KSGVF↓VVNGLVK-NH2 (arrow denotes cleavage site). We used the resultant peptide set to assess the influence of specific amino acid substitutions on the cleavage characteristics of the two proteases. The findings show that when Asn is substituted for Val at the P2 position, HIV-1 PR cleaves the substrate at a much greater rate than does FIV PR. Likewise, Glu or Gln substituted for Val at the P2′ position also yields peptides specifically susceptible to HIV-1 PR. In contrast, when Ser is substituted for Val at P1′, FIV PR cleaves the substrate at a much higher rate than does HIV-1 PR. In addition, Asn or Gln at the P1 position, in combination with an appropriate P3 amino acid, Arg, also strongly favors cleavage by FIV PR over HIV PR. Structural analysis identified several protease residues likely to dictate the observed specificity differences. Interestingly, HIV PR Asp30 (Ile-35 in FIV PR), which influences specificity at the S2 and S2′ subsites, and HIV-1 PR Pro-81 and Val-82 (Ile-98 and Gln-99 in FIV PR), which influence specificity at the S1 and S1′ subsites, are residues which are often involved in development of drug resistance in HIV-1 protease. The peptide substrate KSGVF↓VVNGK, cleaved by both PRs, was used as a template for the design of a reduced amide inhibitor, Ac-GSGVFΨ(CH2NH)VVNGL-NH2. This compound inhibited both FIV and HIV-1 PRs with approximately equal efficiency. These findings establish a molecular basis for distinctions in substrate specificity between human and feline lentivirus PRs and offer a framework for development of efficient broad-based inhibitors. PMID:11533208

Purification and substrate specificities of a fructanase from Kluyveromyces marxianus isolated from the fermentation process of Mezcal.

PubMed

Arrizon, Javier; Morel, Sandrine; Gschaedler, Anne; Monsan, Pierre

2011-02-01

A fructanase, produced by a Kluyveromyces marxianus strain isolated during the fermentation step of the elaboration process of "Mezcal de Guerrero" was purified and biochemically characterized. The active protein was a glycosylated dimer with a molecular weight of approximately 250 kDa. The specific enzymatic activity of the protein was determined for different substrates: sucrose, inulin, Agave tequilana fructan, levan and Actilight® and compared with the activity of Fructozyme®. The hydrolysis profile of the different substrates analyzed by HPAEC-PAD showed that the enzyme has different affinities over the substrates tested with a sucrose/inulin enzymatic activity ratio (S/I) of 125. For the hydrolysis of Agave tequilana fructans, the enzyme also showed a higher enzymatic activity and specificity than Fructozyme®, which is important for its potential application in the tequila industry. Copyright © 2010 Elsevier Ltd. All rights reserved.

Chemical probing of the human sirtuin 5 active site reveals its substrate acyl specificity and peptide-based inhibitors.

PubMed

Roessler, Claudia; Nowak, Theresa; Pannek, Martin; Gertz, Melanie; Nguyen, Giang T T; Scharfe, Michael; Born, Ilona; Sippl, Wolfgang; Steegborn, Clemens; Schutkowski, Mike

2014-09-26

Sirtuins are NAD(+)-dependent deacetylases acting as sensors in metabolic pathways and stress response. In mammals there are seven isoforms. The mitochondrial sirtuin 5 is a weak deacetylase but a very efficient demalonylase and desuccinylase; however, its substrate acyl specificity has not been systematically analyzed. Herein, we investigated a carbamoyl phosphate synthetase 1 derived peptide substrate and modified the lysine side chain systematically to determine the acyl specificity of Sirt5. From that point we designed six potent peptide-based inhibitors that interact with the NAD(+) binding pocket. To characterize the interaction details causing the different substrate and inhibition properties we report several X-ray crystal structures of Sirt5 complexed with these peptides. Our results reveal the Sirt5 acyl selectivity and its molecular basis and enable the design of inhibitors for Sirt5. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

ATP-independent reversal of a membrane protein aggregate by a chloroplast SRP

PubMed Central

Jaru-Ampornpan, Peera; Shen, Kuang; Lam, Vinh Q.; Ali, Mona; Doniach, Sebastian; Jia, Tony Z.; Shan, Shu-ou

2010-01-01

Membrane proteins impose enormous challenges to cellular protein homeostasis during their post-translational targeting, and require chaperones to keep them soluble and translocation-competent. Here we show that a novel targeting factor in the chloroplast Signal Recognition Particle (cpSRP), cpSRP43, is a highly specific molecular chaperone that efficiently reverses the aggregation of its substrate proteins. In contrast to AAA+-chaperones, cpSRP43 utilizes specific binding interactions with its substrate to mediate its disaggregase activity. This ‘disaggregase’ capability can allow targeting machineries to more effectively capture their protein substrates, and emphasizes a close connection between protein folding and trafficking processes. Moreover, cpSRP43 provides the first example of an ATP-independent disaggregase, and demonstrates that efficient reversal of protein aggregation can be attained by specific binding interactions between a chaperone and its substrate. PMID:20424608

Computational Redesign of Acyl-ACP Thioesterase with Improved Selectivity toward Medium-Chain-Length Fatty Acids

DOE Office of Scientific and Technical Information (OSTI.GOV)

Grisewood, Matthew J.; Hernández-Lozada, Néstor J.; Thoden, James B.

Enzyme and metabolic engineering offer the potential to develop biocatalysts for converting natural resources to a wide range of chemicals. To broaden the scope of potential products beyond natural metabolites, methods of engineering enzymes to accept alternative substrates and/or perform novel chemistries must be developed. DNA synthesis can create large libraries of enzyme-coding sequences, but most biochemistries lack a simple assay to screen for promising enzyme variants. Our solution to this challenge is structure-guided mutagenesis, in which optimization algorithms select the best sequences from libraries based on specified criteria (i.e., binding selectivity). We demonstrate this approach by identifying medium-chain (C8–C12)more » acyl-ACP thioesterases through structure-guided mutagenesis. Medium-chain fatty acids, which are products of thioesterase-catalyzed hydrolysis, are limited in natural abundance, compared to long-chain fatty acids; the limited supply leads to high costs of C6–C10 oleochemicals such as fatty alcohols, amines, and esters. Here, we applied computational tools to tune substrate binding of the highly active ‘TesA thioesterase in Escherichia coli. We used the IPRO algorithm to design thioesterase variants with enhanced C12 or C8 specificity, while maintaining high activity. After four rounds of structure-guided mutagenesis, we identified 3 variants with enhanced production of dodecanoic acid (C12) and 27 variants with enhanced production of octanoic acid (C8). The top variants reached up to 49% C12 and 50% C8 while exceeding native levels of total free fatty acids. A comparably sized library created by random mutagenesis failed to identify promising mutants. The chain length-preference of ‘TesA and the best mutant were confirmed in vitro using acyl-CoA substrates. Molecular dynamics simulations, confirmed by resolved crystal structures, of ‘TesA variants suggest that hydrophobic forces govern ‘TesA substrate specificity. Finally, we expect the design rules that we uncovered and the thioesterase variants that we identified will be useful to metabolic engineering projects aimed at sustainable production of medium-chain-length oleochemicals.« less

Computational Redesign of Acyl-ACP Thioesterase with Improved Selectivity toward Medium-Chain-Length Fatty Acids

DOE PAGES

Grisewood, Matthew J.; Hernández-Lozada, Néstor J.; Thoden, James B.; ...

2017-04-20

Enzyme and metabolic engineering offer the potential to develop biocatalysts for converting natural resources to a wide range of chemicals. To broaden the scope of potential products beyond natural metabolites, methods of engineering enzymes to accept alternative substrates and/or perform novel chemistries must be developed. DNA synthesis can create large libraries of enzyme-coding sequences, but most biochemistries lack a simple assay to screen for promising enzyme variants. Our solution to this challenge is structure-guided mutagenesis, in which optimization algorithms select the best sequences from libraries based on specified criteria (i.e., binding selectivity). We demonstrate this approach by identifying medium-chain (C8–C12)more » acyl-ACP thioesterases through structure-guided mutagenesis. Medium-chain fatty acids, which are products of thioesterase-catalyzed hydrolysis, are limited in natural abundance, compared to long-chain fatty acids; the limited supply leads to high costs of C6–C10 oleochemicals such as fatty alcohols, amines, and esters. Here, we applied computational tools to tune substrate binding of the highly active ‘TesA thioesterase in Escherichia coli. We used the IPRO algorithm to design thioesterase variants with enhanced C12 or C8 specificity, while maintaining high activity. After four rounds of structure-guided mutagenesis, we identified 3 variants with enhanced production of dodecanoic acid (C12) and 27 variants with enhanced production of octanoic acid (C8). The top variants reached up to 49% C12 and 50% C8 while exceeding native levels of total free fatty acids. A comparably sized library created by random mutagenesis failed to identify promising mutants. The chain length-preference of ‘TesA and the best mutant were confirmed in vitro using acyl-CoA substrates. Molecular dynamics simulations, confirmed by resolved crystal structures, of ‘TesA variants suggest that hydrophobic forces govern ‘TesA substrate specificity. Finally, we expect the design rules that we uncovered and the thioesterase variants that we identified will be useful to metabolic engineering projects aimed at sustainable production of medium-chain-length oleochemicals.« less

Freestanding ultrathin single-crystalline SiC substrate by MeV H ion-slicing

NASA Astrophysics Data System (ADS)

Jia, Qi; Huang, Kai; You, Tiangui; Yi, Ailun; Lin, Jiajie; Zhang, Shibin; Zhou, Min; Zhang, Bin; Zhang, Bo; Yu, Wenjie; Ou, Xin; Wang, Xi

2018-05-01

SiC is a widely used wide-bandgap semiconductor, and the freestanding ultrathin single-crystalline SiC substrate provides the material platform for advanced devices. Here, we demonstrate the fabrication of a freestanding ultrathin single-crystalline SiC substrate with a thickness of 22 μm by ion slicing using 1.6 MeV H ion implantation. The ion-slicing process performed in the MeV energy range was compared to the conventional case using low-energy H ion implantation in the keV energy range. The blistering behavior of the implanted SiC surface layer depends on both the implantation temperature and the annealing temperature. Due to the different straggling parameter for two implant energies, the distribution of implantation-induced damage is significantly different. The impact of implantation temperature on the high-energy and low-energy slicing was opposite, and the ion-slicing SiC in the MeV range initiates at a much higher temperature.

In situ differential reflectance spectroscopy of thin crystalline films of PTCDA on different substrates

NASA Astrophysics Data System (ADS)

Proehl, Holger; Nitsche, Robert; Dienel, Thomas; Leo, Karl; Fritz, Torsten

2005-04-01

We report an investigation of the excitonic properties of thin crystalline films of the archetypal organic semiconductor PTCDA (3,4,9,10-perylenetetracarboxylic dianhydride) grown on poly- and single crystalline surfaces. A sensitive setup capable of measuring the optical properties of ultrathin organic molecular crystals via differential reflectance spectroscopy (DRS) is presented. This tool allows to carry out measurements in situ, i.e., during the actual film growth, and over a wide spectral range, even on single crystalline surfaces with high symmetry or metallic surfaces, where widely used techniques like reflection anisotropy spectroscopy (RAS) or fluorescence excitation spectroscopy fail. The spectra obtained by DRS resemble mainly the absorption of the films if transparent substrates are used, which simplifies the analysis. In the case of mono- to multilayer films of PTCDA on single crystalline muscovite mica(0001) and Au(111) substrates, the formation of the solid state absorption from monomer to dimer and further to crystal-like absorption spectra can be monitored.

Genome-wide localization of exosome components to active promoters and chromatin insulators in Drosophila

PubMed Central

Lim, Su Jun; Boyle, Patrick J.; Chinen, Madoka; Dale, Ryan K.; Lei, Elissa P.

2013-01-01

Chromatin insulators are functionally conserved DNA–protein complexes situated throughout the genome that organize independent transcriptional domains. Previous work implicated RNA as an important cofactor in chromatin insulator activity, although the precise mechanisms are not yet understood. Here we identify the exosome, the highly conserved major cellular 3′ to 5′ RNA degradation machinery, as a physical interactor of CP190-dependent chromatin insulator complexes in Drosophila. Genome-wide profiling of exosome by ChIP-seq in two different embryonic cell lines reveals extensive and specific overlap with the CP190, BEAF-32 and CTCF insulator proteins. Colocalization occurs mainly at promoters but also boundary elements such as Mcp, Fab-8, scs and scs′, which overlaps with a promoter. Surprisingly, exosome associates primarily with promoters but not gene bodies of active genes, arguing against simple cotranscriptional recruitment to RNA substrates. Similar to insulator proteins, exosome is also significantly enriched at divergently transcribed promoters. Directed ChIP of exosome in cell lines depleted of insulator proteins shows that CTCF is required specifically for exosome association at Mcp and Fab-8 but not other sites, suggesting that alternate mechanisms must also contribute to exosome chromatin recruitment. Taken together, our results reveal a novel positive relationship between exosome and chromatin insulators throughout the genome. PMID:23358822

Characterization of the Serratia marcescens SdeCDE multidrug efflux pump studied via gene knockout mutagenesis.

PubMed

Begic, Sanela; Worobec, Elizabeth A

2008-05-01

Serratia marcescens is an important nosocomial agent having high antibiotic resistance. A major mechanism for S. marcescens antibiotic resistance is active efflux. To ascertain the substrate specificity of the S. marcescens SdeCDE efflux pump, we constructed pump gene deletion mutants. sdeCDE knockout strains showed no change in antibiotic susceptibility in comparison with the parental strains for any of the substrates, with the exception of novobiocin. In addition, novobiocin was the only antibiotic to be accumulated by sdeCDE-deficient strains. Based on the substrates used in our study, we conclude that SdeCDE is a Resistance-Nodulation-Cell Division family pump with limited substrate specificity.

Pharmaceutical-grade oral films as substrates for printed medicine.

PubMed

Wimmer-Teubenbacher, M; Planchette, C; Pichler, H; Markl, D; Hsiao, W K; Paudel, A; Stegemann, S

2018-05-18

In contact-less printing, such as piezo-electric drop on demand printing used in the study, the drop formation process is independent of the substrate. This means that having developed a printable formulation, printed pharmaceutical dosage forms can be obtained on any pharmaceutical grade substrate, such as polymer-based films. In this work we evaluated eight different oral films based on their suitability as printing substrates for sodium picosulfate. The different polymer films were compared regarding printed spot morphology, chemical stability and dissolution profile. The morphology of printed sodium picosulfate was investigated with scanning electron microscopy and optical coherence tomography. The spreading of the deposited drops was found to be governed by the contact angle of the ink with the substrate. The form of the sodium picosulfate drops changed on microcrystalline cellulose films at ambient conditions over 8 weeks and stayed unchanged on other tested substrates. Sodium picosulfate remained amorphous on all substrates according to small and wide angle X-ray scattering, differential scanning calorimetry and polarized light microscopy measurements. The absence of chemical interactions between the drug and substrates, as indicated by infrared spectroscopy, makes all tested substrates suitable for printing sodium picosulfate onto them. Copyright © 2018 Elsevier B.V. All rights reserved.

Improving the efficiency and directivity of THz photoconductive antennas by using a defective photonic crystal substrate

NASA Astrophysics Data System (ADS)

Rahmati, Ehsan; Ahmadi-Boroujeni, Mehdi

2018-04-01

One of the shortcomings of photoconductive (PC) antennas in terahertz (THz) generation is low effective radiated power in the desirable direction. In this paper, we propose a defective photonic crystal (DPC) substrate consisting of a customized 2D array of air holes drilled into a solid substrate in order to improve the radiation characteristics of THz PC antennas. The effect of the proposed structure on the performance of a conventional THz PC antenna has been examined from several aspects including radiation efficiency, directivity, and field distribution. By comparing the radiation performance of the THz antenna on the proposed DPC substrate to that of the conventional solid substrate, it is shown that the proposed technique can significantly improve the efficiency and directivity of the THz PC antenna over a wide frequency range. It is achieved by reducing the amount of power coupled to the substrate surface waves and limiting the radiation in undesirable directions. In addition, it is found that the sensitivity of directivity to the substrate thickness is considerably decreased and the adverse Fabry-Perot effects of the thick substrate are reduced by the application of the proposed DPC substrate.

Directions for Optimization of Photosynthetic Carbon Fixation: RuBisCO's Efficiency May Not Be So Constrained After All.

PubMed

Cummins, Peter L; Kannappan, Babu; Gready, Jill E

2018-01-01

The ubiquitous enzyme Ribulose 1,5-bisphosphate carboxylase-oxygenase (RuBisCO) fixes atmospheric carbon dioxide within the Calvin-Benson cycle that is utilized by most photosynthetic organisms. Despite this central role, RuBisCO's efficiency surprisingly struggles, with both a very slow turnover rate to products and also impaired substrate specificity, features that have long been an enigma as it would be assumed that its efficiency was under strong evolutionary pressure. RuBisCO's substrate specificity is compromised as it catalyzes a side-fixation reaction with atmospheric oxygen; empirical kinetic results show a trend to tradeoff between relative specificity and low catalytic turnover rate. Although the dominant hypothesis has been that the active-site chemistry constrains the enzyme's evolution, a more recent study on RuBisCO stability and adaptability has implicated competing selection pressures. Elucidating these constraints is crucial for directing future research on improving photosynthesis, as the current literature casts doubt on the potential effectiveness of site-directed mutagenesis to improve RuBisCO's efficiency. Here we use regression analysis to quantify the relationships between kinetic parameters obtained from empirical data sets spanning a wide evolutionary range of RuBisCOs. Most significantly we found that the rate constant for dissociation of CO 2 from the enzyme complex was much higher than previous estimates and comparable with the corresponding catalytic rate constant. Observed trends between relative specificity and turnover rate can be expressed as the product of negative and positive correlation factors. This provides an explanation in simple kinetic terms of both the natural variation of relative specificity as well as that obtained by reported site-directed mutagenesis results. We demonstrate that the kinetic behaviour shows a lesser rather than more constrained RuBisCO, consistent with growing empirical evidence of higher variability in relative specificity. In summary our analysis supports an explanation for the origin of the tradeoff between specificity and turnover as due to competition between protein stability and activity, rather than constraints between rate constants imposed by the underlying chemistry. Our analysis suggests that simultaneous improvement in both specificity and turnover rate of RuBisCO is possible.

Identification of Residues Involved in Substrate Specificity and Cytotoxicity of Two Closely Related Cutinases from Mycobacterium tuberculosis

PubMed Central

Dedieu, Luc; Serveau-Avesque, Carole; Canaan, Stéphane

2013-01-01

The enzymes belonging to the cutinase family are serine enzymes active on a large panel of substrates such as cutin, triacylglycerols, and phospholipids. In the M. tuberculosis H37Rv genome, seven genes coding for cutinase-like proteins have been identified with strong immunogenic properties suggesting a potential role as vaccine candidates. Two of these enzymes which are secreted and highly homologous, possess distinct substrates specificities. Cfp21 is a lipase and Cut4 is a phospholipase A2, which has cytotoxic effects on macrophages. Structural overlay of their three-dimensional models allowed us to identify three areas involved in the substrate binding process and to shed light on this substrate specificity. By site-directed mutagenesis, residues present in these Cfp21 areas were replaced by residues occurring in Cut4 at the same location. Three mutants acquired phospholipase A1 and A2 activities and the lipase activities of two mutants were 3 and 15 fold greater than the Cfp21 wild type enzyme. In addition, contrary to mutants with enhanced lipase activity, mutants that acquired phospholipase B activities induced macrophage lysis as efficiently as Cut4 which emphasizes the relationship between apparent phospholipase A2 activity and cytotoxicity. Modification of areas involved in substrate specificity, generate recombinant enzymes with higher activity, which may be more immunogenic than the wild type enzymes and could therefore constitute promising candidates for antituberculous vaccine production. PMID:23843969

Identification of residues involved in substrate specificity and cytotoxicity of two closely related cutinases from Mycobacterium tuberculosis.

PubMed

Dedieu, Luc; Serveau-Avesque, Carole; Canaan, Stéphane

2013-01-01

The enzymes belonging to the cutinase family are serine enzymes active on a large panel of substrates such as cutin, triacylglycerols, and phospholipids. In the M. tuberculosis H37Rv genome, seven genes coding for cutinase-like proteins have been identified with strong immunogenic properties suggesting a potential role as vaccine candidates. Two of these enzymes which are secreted and highly homologous, possess distinct substrates specificities. Cfp21 is a lipase and Cut4 is a phospholipase A2, which has cytotoxic effects on macrophages. Structural overlay of their three-dimensional models allowed us to identify three areas involved in the substrate binding process and to shed light on this substrate specificity. By site-directed mutagenesis, residues present in these Cfp21 areas were replaced by residues occurring in Cut4 at the same location. Three mutants acquired phospholipase A1 and A2 activities and the lipase activities of two mutants were 3 and 15 fold greater than the Cfp21 wild type enzyme. In addition, contrary to mutants with enhanced lipase activity, mutants that acquired phospholipase B activities induced macrophage lysis as efficiently as Cut4 which emphasizes the relationship between apparent phospholipase A2 activity and cytotoxicity. Modification of areas involved in substrate specificity, generate recombinant enzymes with higher activity, which may be more immunogenic than the wild type enzymes and could therefore constitute promising candidates for antituberculous vaccine production.

[Substrate-inhibitory analysis of monoamine oxidase from hepatopancreas of the octopus Bathypolypus arcticus].

PubMed

Basova, I N; Iagodina, O V

2012-01-01

Study of the substrate-inhibitory specificity of mitochondrial monoamine oxidase (MAO) of hepatopancreas of the octopus Bathypolypus arcticus revealed distinctive peculiarities of catalytic properties of this enzyme. The studied enzyme, on one hand, like the classic MAO of homoiothermal animals, is able to deaminate tyramine, serotonin, benzylamine, tryptamine, beta-phenylethylamine, while, on the other hand, deaminates histamine and does not deaminate putrescine--classic substrates of diamine oxidase (DAO). Results of the substrate-inhibitory analysis with use of chlorgiline and deprenyl are indirect proofs of the existence in the octopus hepatopancreas of one molecular MAO form. Semicarbazide and pyronine G turned out to be weak irreversible inhibitors, four derivatives of acridine--irreversible inhibitors of the intermediate effectiveness with respect to the octopus hepatopancreas MAO; specificity of action of inhibitors at deamination of different substrates was equal.

Substrates and materials used for nesting by North American Osmia bees (Hymenoptera: Apiformes: Megachilidae)

Treesearch

James H. Cane; Terry L. Griswold; Frank D. Parker

2007-01-01

Nesting substrates and construction materials are compared for 65 of North America's 139 described native species of Osmia bees. Most accounts report Osmia bees nesting in preexisting cavities in dead wood or pithy stems such as elderberry (Sambucus spp.), with cell partitions and plugs made from a pulp of finely masticated leaf tissue. Mud is widely used by...

Electron Beam "Writes" Silicon On Sapphire

NASA Technical Reports Server (NTRS)

Heinemann, Klaus

1988-01-01

Method of growing silicon on sapphire substrate uses beam of electrons to aid growth of semiconductor material. Silicon forms as epitaxial film in precisely localized areas in micron-wide lines. Promising fabrication method for fast, densely-packed integrated circuits. Silicon deposited preferentially in contaminated substrate zones and in clean zone irradiated by electron beam. Electron beam, like surface contamination, appears to stimulate decomposition of silane atmosphere.

The Neural Substrates Associated with Attentional Resources and Difficulty of Concurrent Processing of the Two Verbal Tasks

ERIC Educational Resources Information Center

Mizuno, Kei; Tanaka, Masaaki; Tanabe, Hiroki C.; Sadato, Norihiro; Watanabe, Yasuyoshi

2012-01-01

The kana pick-out test has been widely used in Japan to evaluate the ability to divide attention in both adult and pediatric patients. However, the neural substrates underlying the ability to divide attention using the kana pick-out test, which requires participants to pick out individual letters (vowels) in a story while also reading for…

Beyond D-luciferin: Expanding the Scope of Bioluminescence Imaging in vivo

PubMed Central

Adams, Spencer T.; Miller, Stephen C.

2014-01-01

The light-emitting chemical reaction catalyzed by the enzyme firefly luciferase is widely used for noninvasive imaging in live mice. However, photon emission from the luciferase is critically dependent on the chemical properties of its substrate, D-luciferin. In this review, we describe recent work to replace the natural luciferase substrate with synthetic analogs that extend the scope of bioluminescence imaging. PMID:25078002

DOE Office of Scientific and Technical Information (OSTI.GOV)

Deniz, O.; Sánchez-Sánchez, C.; Jaafar, R.

Electronic and thermal properties of chevron-type graphene nanoribbons can be widely tuned, making them interesting candidates for electronic and thermoelectric applications. In this paper, we use post-growth silicon intercalation to unambiguously access nanoribbons’ energy position of their electronic frontier states. These are otherwise obscured by substrate effects when investigated directly on the growth substrate. Finally, in agreement with first-principles calculations we find a band gap of 2.4 eV.

Structure and Activity Analyses of Escherichia coli K-12 NagD Provide Insight into the Evolution of Biochemical Function in the Haloakanoic Acid Dehlogenase Superfamily

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tremblay,L.; Dunaway-Mariano, D.; Allen, K.

2006-01-01

The HAD superfamily is a large superfamily of proteins which share a conserved core domain that provides those active site residues responsible for the chemistry common to all family members. The superfamily is further divided into the four subfamilies I, IIA, IIB, and III, based on the topology and insertion site of a cap domain that provides substrate specificity. This structural and functional division implies that members of a given HAD structural subclass may target substrates that have similar structural characteristics. To understand the structure/function relationships in all of the subfamilies, a type IIA subfamily member, NagD from Escherichia colimore » K-12, was selected (type I, IIB, and III members have been more extensively studied). The structure of the NagD protein was solved to 1.80 Angstroms with R{sub work} = 19.8% and R{sub free} = 21.8%. Substrate screening and kinetic analysis showed NagD to have high specificity for nucleotide monophosphates with kcat/Km = 3.12 x 10{sup 4} and 1.28 x 10{sup 4} {micro}M{sup -1} s{sup -1} for UMP and GMP, respectively. This specificity is consistent with the presence of analogues of NagD that exist as fusion proteins with a nucleotide pyrophosphatase from the Nudix family. Docking of the nucleoside substrate in the active site brings it in contact with conserved residues from the cap domain that can act as a substrate specificity loop (NagD residues 144-149) in the type IIA subfamily. NagD and other subfamily IIA and IIB members show the common trait that substrate specificity and catalytic efficiencies (k{sub cat}/K{sub m}) are low (1 x 10{sup 4} M{sup -1} s{sup -1}) and the boundaries defining physiological substrates are somewhat overlapping. The ability to catabolize other related secondary metabolites indicates that there is regulation at the genetic level.« less

DbPTM 3.0: an informative resource for investigating substrate site specificity and functional association of protein post-translational modifications.

PubMed

Lu, Cheng-Tsung; Huang, Kai-Yao; Su, Min-Gang; Lee, Tzong-Yi; Bretaña, Neil Arvin; Chang, Wen-Chi; Chen, Yi-Ju; Chen, Yu-Ju; Huang, Hsien-Da

2013-01-01

Protein modification is an extremely important post-translational regulation that adjusts the physical and chemical properties, conformation, stability and activity of a protein; thus altering protein function. Due to the high throughput of mass spectrometry (MS)-based methods in identifying site-specific post-translational modifications (PTMs), dbPTM (http://dbPTM.mbc.nctu.edu.tw/) is updated to integrate experimental PTMs obtained from public resources as well as manually curated MS/MS peptides associated with PTMs from research articles. Version 3.0 of dbPTM aims to be an informative resource for investigating the substrate specificity of PTM sites and functional association of PTMs between substrates and their interacting proteins. In order to investigate the substrate specificity for modification sites, a newly developed statistical method has been applied to identify the significant substrate motifs for each type of PTMs containing sufficient experimental data. According to the data statistics in dbPTM, >60% of PTM sites are located in the functional domains of proteins. It is known that most PTMs can create binding sites for specific protein-interaction domains that work together for cellular function. Thus, this update integrates protein-protein interaction and domain-domain interaction to determine the functional association of PTM sites located in protein-interacting domains. Additionally, the information of structural topologies on transmembrane (TM) proteins is integrated in dbPTM in order to delineate the structural correlation between the reported PTM sites and TM topologies. To facilitate the investigation of PTMs on TM proteins, the PTM substrate sites and the structural topology are graphically represented. Also, literature information related to PTMs, orthologous conservations and substrate motifs of PTMs are also provided in the resource. Finally, this version features an improved web interface to facilitate convenient access to the resource.

Design of N-acyl homoserine lactonase with high substrate specificity by a rational approach.

PubMed

Kyeong, Hyun-Ho; Kim, Jin-Hyun; Kim, Hak-Sung

2015-06-01

N-Acyl homoserine lactone (AHL) is a major quorum-sensing signaling molecule in many bacterial species. Quorum-quenching (QQ) enzymes, which degrade such signaling molecules, have attracted much attention as an approach to controlling and preventing bacterial virulence and pathogenesis. However, naturally occurring QQ enzymes show a broad substrate spectrum, raising the concern of unintentionally attenuating beneficial effects by symbiotic bacteria. Here we report the rational design of acyl homoserine lactonase with high substrate specificity. Through docking analysis, we identified three key residues which play a key role in the substrate preference of the enzyme. The key residues were changed in a way that increases hydrophobic contact with a substrate having a short acyl chain (C4-AHL) while generating steric clashes with that containing a long acyl chain (C12-AHL). The resulting mutants exhibited a significantly shifted preference toward a substrate with a short acyl chain. Molecular dynamics simulations suggested that the mutations affect the behavior of a flexible loop, allowing tighter binding of a substrate with a short acyl chain.

The Structure of Lombricine Kinase: Implications for Phosphagen Conformational Changes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bush, D. Jeffrey; Kirillova, Olga; Clark, Shawn A.

2012-05-29

Lombricine kinase is a member of the phosphagen kinase family and a homolog of creatine and arginine kinases, enzymes responsible for buffering cellular ATP levels. Structures of lombricine kinase from the marine worm Urechis caupo were determined by x-ray crystallography. One form was crystallized as a nucleotide complex, and the other was substrate-free. The two structures are similar to each other and more similar to the substrate-free forms of homologs than to the substrate-bound forms of the other phosphagen kinases. Active site specificity loop 309-317, which is disordered in substrate-free structures of homologs and is known from the NMR ofmore » arginine kinase to be inherently dynamic, is resolved in both lombricine kinase structures, providing an improved basis for understanding the loop dynamics. Phosphagen kinases undergo a segmented closing on substrate binding, but the lombricine kinase ADP complex is in the open form more typical of substrate-free homologs. Through a comparison with prior complexes of intermediate structure, a correlation was revealed between the overall enzyme conformation and the substrate interactions of His{sup 178}. Comparative modeling provides a rationale for the more relaxed specificity of these kinases, of which the natural substrates are among the largest of the phosphagen substrates.« less

QM/MM Free Energy Simulations of Salicylic Acid Methyltransferase: Effects of Stabilization of TS-like Structures on Substrate Specificity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yao, Jianzhuang; Xu, Qin; Chen, Feng

2010-01-01

Salicylic acid methyltransferases (SAMTs) synthesize methyl salicylate (MeSA) using salicylate as the substrate. MeSA synthesized in plants may function as an airborne signal to activate the expression of defense-related genes and could also be a critical mobile signaling molecule that travels from the site of plant infection to establish systemic immunity in the induction of disease resistance. Here the results of QM/MM free energy simulations for the methyl transfer process in Clarkia breweri SAMT (CbSAMT) are reported to determine the origin of the substrate specificity of SAMTs. The free energy barrier for the methyl transfer from S-adenosyl-l-methionine (AdoMet) to 4-hydroxybenzoatemore » in CbSAMT is found to be about 5 kcal/mol higher than that from AdoMet to salicylate, consistent with the experimental observations. It is suggested that the relatively high efficiency for the methylation of salicylate compared to 4-hydroxybenzoate is due, at least in part, to the reason that a part of the stabilization of the transition state (TS) configuration is already reflected in the reactant complex, presumably, through the binding. The results seem to indicate that the creation of the substrate complex (e.g., through mutagenesis and substrate modifications) with its structure closely resembling TS might be fruitful for improving the catalytic efficiency for some enzymes. The results show that the computer simulations may provide important insights into the origin of the substrate specificity for the SABATH family and could be used to help experimental efforts in generating engineered enzymes with altered substrate specificity.« less

Reconstructed ancestral enzymes reveal that negative selection drove the evolution of substrate specificity in ADP-dependent kinases.

PubMed

Castro-Fernandez, Víctor; Herrera-Morande, Alejandra; Zamora, Ricardo; Merino, Felipe; Gonzalez-Ordenes, Felipe; Padilla-Salinas, Felipe; Pereira, Humberto M; Brandão-Neto, Jose; Garratt, Richard C; Guixe, Victoria

2017-09-22

One central goal in molecular evolution is to pinpoint the mechanisms and evolutionary forces that cause an enzyme to change its substrate specificity; however, these processes remain largely unexplored. Using the glycolytic ADP-dependent kinases of archaea, including the orders Thermococcales , Methanosarcinales , and Methanococcales , as a model and employing an approach involving paleoenzymology, evolutionary statistics, and protein structural analysis, we could track changes in substrate specificity during ADP-dependent kinase evolution along with the structural determinants of these changes. To do so, we studied five key resurrected ancestral enzymes as well as their extant counterparts. We found that a major shift in function from a bifunctional ancestor that could phosphorylate either glucose or fructose 6-phosphate (fructose-6-P) as a substrate to a fructose 6-P-specific enzyme was started by a single amino acid substitution resulting in negative selection with a ground-state mode against glucose and a subsequent 1,600-fold change in specificity of the ancestral protein. This change rendered the residual phosphorylation of glucose a promiscuous and physiologically irrelevant activity, highlighting how promiscuity may be an evolutionary vestige of ancestral enzyme activities, which have been eliminated over time. We also could reconstruct the evolutionary history of substrate utilization by using an evolutionary model of discrete binary characters, indicating that substrate uses can be discretely lost or acquired during enzyme evolution. These findings exemplify how negative selection and subtle enzyme changes can lead to major evolutionary shifts in function, which can subsequently generate important adaptive advantages, for example, in improving glycolytic efficiency in Thermococcales . © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

[Substrate specifity in Amoeba proteus].

PubMed

Sopina, V A

2006-01-01

Three different phosphatases ("slow", "middle" and "fast") were found in Amoeba proteus (strain B) after PAGE and a subsequent gel staining in 1-naphthyl phosphate containing incubation mixture (pH 9.0). Substrate specificity of these phosphatases was determined in supernatants of homogenates using inhibitors of phosphatase activity. All phosphatases showed a broad substrate specificity. Of 10 tested compounds, p-nitrophenyl phosphate was a preferable substrate for all 3 phosphatases. All phosphatases were able to hydrolyse bis-p-nitrophenyl phosphate and, hence, displayed phosphodiesterase activity. All phosphatases hydrolysed O-phospho-L-tyrosine to a greater or lesser degree. Only little differences in substrate specificity of phosphatases were noticed: 1) "fast" and "middle" phosphatases hydrolysed naphthyl phosphates and O-phospho-L-tyrosine less efficiently than did "slow" phosphatase; 2) "fast" and "middle" phosphatases hydrolysed 2- naphthyl phosphate to a lesser degree than 1-naphthyl phosphate 3) "fast" and "middle" phosphatases hydrolysed O-phospho-L-serine and O-phospho-L-threonine with lower intensity as compared with "slow" phosphatase; 4) as distinct from "middle" and "slow" phosphatases, the "fast" phosphatase hydrolysed glucose-6-phosphate very poorly. The revealed broad substrate specificity of "slow" phosphatase together with data of inhibitory analysis and results of experiments with reactivation of this phosphatase by Zn2+-ions after its inactivation by EDTA strongly suggest that only the "slow" phosphatase is a true alkaline phosphatase (EC 3.1.3.1). The alkaline phosphatase of A. proteus is secreted into culture medium where its activity is low. The enzyme displays both phosphomono- and phosphodiesterase activities, in addition to supposed protein phosphatase activity. It still remains unknown, to which particular phosphatase class the amoeban "middle" and "fast" phosphatases (pH 9.0) may be assigned.

MnO2-Based Electrochemical Supercapacitors on Flexible Carbon Substrates

NASA Astrophysics Data System (ADS)

Tadjer, Marko J.; Mastro, Michael A.; Rojo, José M.; Mojena, Alberto Boscá; Calle, Fernando; Kub, Francis J.; Eddy, Charles R.

2014-04-01

Manganese dioxide films were grown on large area flexible carbon aerogel substrates. Characterization by x-ray diffraction confirmed α-MnO2 growth. Three types of films were compared as a function of hexamethylenetetramine (HMTA) concentration during growth. The highest concentration of HM TA produced MnO2 flower-like films, as observed by scanning electron microscopy, whose thickness and surface coverage lead to both a higher specific capacitance and higher series resistance. Specific capacitance was measured to be 64 F/g using a galvanostatic setup, compared to the 47 F/g-specific capacitance of the carbon aerogel substrate. Such supercapacitor devices can be fabricated on large area sheets of carbon aerogel to achieve high total capacitance.

Generation of a Mutant Mucor hiemalis Endoglycosidase That Acts on Core-fucosylated N-Glycans.

PubMed

Katoh, Toshihiko; Katayama, Takane; Tomabechi, Yusuke; Nishikawa, Yoshihide; Kumada, Jyunichi; Matsuzaki, Yuji; Yamamoto, Kenji

2016-10-28

Endo-β-N-acetylglucosaminidase M (Endo-M), an endoglycosidase from the fungus Mucor hiemalis, is a useful tool for chemoenzymatic synthesis of glycoconjugates, including glycoprotein-based therapeutics having a precisely defined glycoform, by virtue of its transglycosylation activity. Although Endo-M has been known to act on various N-glycans, it does not act on core-fucosylated N-glycans, which exist widely in mammalian glycoproteins, thus limiting its application. Therefore, we performed site-directed mutagenesis on Endo-M to isolate mutant enzymes that are able to act on mammalian-type core-α1,6-fucosylated glycans. Among the Endo-M mutant enzymes generated, those in which the tryptophan at position 251 was substituted with alanine or asparagine showed altered substrate specificities. Such mutant enzymes exhibited increased hydrolysis of a synthetic α1,6-fucosylated trimannosyl core structure, whereas their activity on the afucosylated form decreased. In addition, among the Trp-251 mutants, the W251N mutant was most efficient in hydrolyzing the core-fucosylated substrate. W251N mutants could act on the immunoglobulin G-derived core-fucosylated glycopeptides and human lactoferrin glycoproteins. This mutant was also capable of transferring the sialyl glycan from an activated substrate intermediate (sialyl glyco-oxazoline) onto an α1,6-fucosyl-N-acetylglucosaminyl biotin. Furthermore, the W251N mutant gained a glycosynthase-like activity when a N175Q substitution was introduced and it caused accumulation of the transglycosylation products. These findings not only give insights into the substrate recognition mechanism of glycoside hydrolase family 85 enzymes but also widen their scope of application in preparing homogeneous glycoforms of core-fucosylated glycoproteins for the production of potent glycoprotein-based therapeutics. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Defining efficient enzyme-cofactor pairs for bioorthogonal profiling of protein methylation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Islam, Kabirul; Chen, Yuling; Wu, Hong

2013-11-18

Protein methyltransferase (PMT)-mediated posttranslational modification of histone and nonhistone substrates modulates stability, localization, and interacting partners of target proteins in diverse cellular contexts. These events play critical roles in normal biological processes and are frequently deregulated in human diseases. In the course of identifying substrates of individual PMTs, bioorthogonal profiling of protein methylation (BPPM) has demonstrated its merits. In this approach, specific PMTs are engineered to process S-adenosyl-L-methionine (SAM) analogs as cofactor surrogates and label their substrates with distinct chemical modifications for target elucidation. Despite the proof-of-concept advancement of BPPM, few efforts have been made to explore its generality. Withmore » two cancer-relevant PMTs, EuHMT1 (GLP1/KMT1D) and EuHMT2 (G9a/KMT1C), as models, we defined the key structural features of engineered PMTs and matched SAM analogs that can render the orthogonal enzyme–cofactor pairs for efficient catalysis. Here we have demonstrated that the presence of sulfonium-β-sp 2 carbon and flexible, medium-sized sulfonium-δ-substituents are crucial for SAM analogs as BPPM reagents. The bulky cofactors can be accommodated by tailoring the conserved Y1211/Y1154 residues and nearby hydrophobic cavities of EuHMT1/2. Profiling proteome-wide substrates with BPPM allowed identification of >500 targets of EuHMT1/2 with representative targets validated using native EuHMT1/2 and SAM. This finding indicates that EuHMT1/2 may regulate many cellular events previously unrecognized to be modulated by methylation. The present work, therefore, paves the way to a broader application of the BPPM technology to profile methylomes of diverse PMTs and elucidate their downstream functions.« less

Phage & phosphatase: a novel phage-based probe for rapid, multi-platform detection of bacteria.

PubMed

Alcaine, S D; Pacitto, D; Sela, D A; Nugen, S R

2015-11-21

Genetic engineering of bacteriophages allows for the development of rapid, highly specific, and easily manufactured probes for the detection of bacterial pathogens. A challenge for novel probes is the ease of their adoption in real world laboratories. We have engineered the bacteriophage T7, which targets Escherichia coli, to carry the alkaline phosphatase gene, phoA. This inclusion results in phoA overexpression following phage infection of E. coli. Alkaline phosphatase is commonly used in a wide range of diagnostics, and thus a signal produced by our phage-based probe could be detected using common laboratory equipment. Our work demonstrates the successful: (i) modification of T7 phage to carry phoA; (ii) overexpression of alkaline phosphatase in E. coli; and (iii) detection of this T7-induced alkaline phosphatase activity using commercially available colorimetric and chemilumiscent methods. Furthermore, we demonstrate the application of our phage-based probe to rapidly detect low levels of bacteria and discern the antibiotic resistance of E. coli isolates. Using our bioengineered phage-based probe we were able to detect 10(3) CFU per mL of E. coli in 6 hours using a chemiluminescent substrate and 10(4) CFU per mL within 7.5 hours using a colorimetric substrate. We also show the application of this phage-based probe for antibiotic resistance testing. We were able to determine whether an E. coli isolate was resistant to ampicillin within 4.5 hours using chemiluminescent substrate and within 6 hours using a colorimetric substrate. This phage-based scheme could be readily adopted in labs without significant capital investments and can be translated to other phage-bacteria pairs for further detection.

Crystal structure of the NADP+ and tartrate-bound complex of L-serine 3-dehydrogenase from the hyperthermophilic archaeon Pyrobaculum calidifontis.

PubMed

Yoneda, Kazunari; Sakuraba, Haruhiko; Araki, Tomohiro; Ohshima, Toshihisa

2018-05-01

A gene encoding L-serine dehydrogenase (L-SerDH) that exhibits extremely low sequence identity to the Agrobacterium tumefaciens L-SerDH was identified in the hyperthermophilic archaeon Pyrobaculum calidifontis. The predicted amino acid sequence showed 36% identity with that of Pseudomonas aeruginosa L-SerDH, suggesting that P. calidifontis L-SerDH is a novel type of L-SerDH, like Ps. aeruginosa L-SerDH. The overexpressed enzyme appears to be the most thermostable L-SerDH described to date, and no loss of activity was observed by incubation for 30 min at temperatures up to 100 °C. The enzyme showed substantial reactivity towards D-serine, in addition to L-serine. Two different crystal structures of P. calidifontis L-SerDH were determined using the Se-MAD and MR method: the structure in complex with NADP + /sulfate ion at 1.18 Å and the structure in complex with NADP + /L-tartrate (substrate analog) at 1.57 Å. The fold of the catalytic domain showed similarity with that of Ps. aeruginosa L-SerDH. However, the active site structure significantly differed between the two enzymes. Based on the structure of the tartrate, L- and D-serine and 3-hydroxypropionate molecules were modeled into the active site and the substrate binding modes were estimated. A structural comparison suggests that the wide cavity at the substrate binding site is likely responsible for the high reactivity of the enzyme toward both L- and D-serine enantiomers. This is the first description of the structure of the novel type of L-SerDH with bound NADP + and substrate analog, and it provides new insight into the substrate binding mechanism of L-SerDH. The results obtained here may be very informative for the creation of L- or D-serine-specific SerDH by protein engineering.

Investigating the interplay between substrate stiffness and ligand chemistry in directing mesenchymal stem cell differentiation within 3D macro-porous substrates.

PubMed

Haugh, Matthew G; Vaughan, Ted J; Madl, Christopher M; Raftery, Rosanne M; McNamara, Laoise M; O'Brien, Fergal J; Heilshorn, Sarah C

2018-07-01

Dimensionality can have a profound impact on stiffness-mediated differentiation of mesenchymal stem cells (MSCs). However, while we have begun to understand cellular response when encapsulated within 3D substrates, the behavior of cells within macro-porous substrates is relatively underexplored. The goal of this study was to determine the influence of macro-porous topographies on stiffness-mediated differentiation of MSCs. We developed macro-porous recombinant elastin-like protein (ELP) substrates that allow independent control of mechanical properties and ligand chemistry. We then used computational modeling to probe the impact of pore topography on the mechanical stimulus that cells are exposed to within these substrates, and finally we investigated stiffness induced biases towards adipogenic and osteogenic differentiation of MSCs within macro-porous substrates. Computational modeling revealed that there is significant heterogeneity in the mechanical stimuli that cells are exposed to within porous substrates and that this heterogeneity is predominantly due to the wide range of possible cellular orientations within the pores. Surprisingly, MSCs grown within 3D porous substrates respond to increasing substrate stiffness by up-regulating both osteogenesis and adipogenesis. These results demonstrate that within porous substrates the behavior of MSCs diverges from previously observed responses to substrate stiffness, emphasizing the importance of topography as a determinant of cellular behavior. Copyright © 2018 Elsevier Ltd. All rights reserved.

Hydrolytic properties and substrate specificity of the foot-and-mouth disease leader protease.

PubMed

Santos, Jorge A N; Gouvea, Iuri E; Júdice, Wagner A S; Izidoro, Mario A; Alves, Fabiana M; Melo, Robson L; Juliano, Maria A; Skern, Tim; Juliano, Luiz

2009-08-25

Foot-and-mouth disease virus, a global animal pathogen, possesses a single-stranded RNA genome that, on release into the infected cell, is immediately translated into a single polyprotein. This polyprotein product is cleaved during synthesis by proteinases contained within it into the mature viral proteins. The first cleavage is performed by the leader protease (Lb(pro)) between its own C-terminus and the N-terminus of VP4. Lb(pro) also specifically cleaves the two homologues of cellular eukaryotic initiation factor (eIF) 4G, preventing translation of capped mRNA. Viral protein synthesis is initiated internally and is thus unaffected. We used a panel of specifically designed FRET peptides to examine the effects of pH and ionic strength on Lb(pro) activity and investigate the size of the substrate binding site and substrate specificity. Compared to the class prototypes, papain and the cathepsins, Lb(pro) possesses several unusual characteristics, including a high sensitivity to salt and a very specific substrate binding site extending up to P(7). Indeed, almost all substitutions investigated were detrimental to Lb(pro) activity. Analysis of structural data showed that Lb(pro) binds residues P(1)-P(3) in an extended conformation, whereas residues P(4)-P(7) are bound in a short 3(10) helix. The specificity of Lb(pro) as revealed by the substituted peptides could be explained for all positions except P(5). Strikingly, Lb(pro) residues L178 and L143 contribute to the architecture of more than one substrate binding pocket. The diverse functions of these two Lb(pro) residues explain why Lb(pro) is one of the smallest, but simultaneously most specific, papain-like enzymes.

Substrate-binding specificity of chitinase and chitosanase as revealed by active-site architecture analysis.

PubMed

Liu, Shijia; Shao, Shangjin; Li, Linlin; Cheng, Zhi; Tian, Li; Gao, Peiji; Wang, Lushan

2015-12-11

Chitinases and chitosanases, referred to as chitinolytic enzymes, are two important categories of glycoside hydrolases (GH) that play a key role in degrading chitin and chitosan, two naturally abundant polysaccharides. Here, we investigate the active site architecture of the major chitosanase (GH8, GH46) and chitinase families (GH18, GH19). Both charged (Glu, His, Arg, Asp) and aromatic amino acids (Tyr, Trp, Phe) are observed with higher frequency within chitinolytic active sites as compared to elsewhere in the enzyme structure, indicating significant roles related to enzyme function. Hydrogen bonds between chitinolytic enzymes and the substrate C2 functional groups, i.e. amino groups and N-acetyl groups, drive substrate recognition, while non-specific CH-π interactions between aromatic residues and substrate mainly contribute to tighter binding and enhanced processivity evident in GH8 and GH18 enzymes. For different families of chitinolytic enzymes, the number, type, and position of substrate atoms bound in the active site vary, resulting in different substrate-binding specificities. The data presented here explain the synergistic action of multiple enzyme families at a molecular level and provide a more reasonable method for functional annotation, which can be further applied toward the practical engineering of chitinases and chitosanases. Copyright © 2015 Elsevier Ltd. All rights reserved.

Reactive ion etched substrates and methods of making and using

DOEpatents

Rucker, Victor C [San Francisco, CA; Shediac, Rene [Oakland, CA; Simmons, Blake A [San Francisco, CA; Havenstrite, Karen L [New York, NY

2007-08-07

Disclosed herein are substrates comprising reactive ion etched surfaces and specific binding agents immobilized thereon. The substrates may be used in methods and devices for assaying or isolating analytes in a sample. Also disclosed are methods of making the reactive ion etched surfaces.

Substrate Material for Holographic Emulsions Utilizing Fluorinated Polyimide Film

NASA Technical Reports Server (NTRS)

Gierow, Paul A. (Inventor); Clayton, William R. (Inventor); St.Clair, Anne K. (Inventor)

1999-01-01

A new holographic substrate utilizing flexible. optically transparent fluorinated polyimides. Said substrates have 0 extremely low birefringence which results in a high signal to noise ratio in subsequent holograms. Specific examples of said fluorinated polyimides include 6FDA+APB and 6FDA+4BDAF.

Growth and high rate reactive ion etching of epitaxially grown barium hexaferrite films on single crystal silicon carbide substrates

NASA Astrophysics Data System (ADS)

Chen, Zhaohui

Ferrites are an invaluable group of insulating magnetic materials used for high frequency microwave applications in such passive electronic devices as isolators, phase shifters, and circulators. Because of their high permeability, non-reciprocal electromagnetic properties, and low eddy current losses, there are no other materials that serve such a broad range of applications. Until recently, they have been widely employed in bulk form, with little success in thin film-based applications in commercial or military microwave technologies. In today's technology, emerging electronic systems, such as high frequency, high power wireless and satellite communications (GPS, Bluetooth, WLAN, commercial radar, etc) thin film materials are in high demand. It is widely recognized that as high frequency devices shift to microwave frequencies the integration of passive devices with semiconductor electronics holds significant advantages in the realization of miniaturization, broader bandwidths, higher performance, speed, power and lower production costs. Thus, the primary objective of this thesis is to explore the integration of ferrite films with wide band gap semiconductor substrates for the realization of monolithic integrated circuits (MICs). This thesis focuses on two key steps for the integration of barium hexaferrite (Ba M-type or BaM) devices on semiconductor substrates. First, the development of high crystal quality ferrite film growth via pulsed laser deposition on wide band gap silicon carbide semiconductor substrates, and second, the effective patterning of BaM films using dry etching techniques. To address part one, BaM films were deposited on 6H silicon carbide (0001) substrates by Pulsed Laser Deposition. X-ray diffraction showed strong crystallographic alignment while pole figures exhibited reflections consistent with epitaxial growth. After optimized annealing, BaM films have a perpendicular magnetic anisotropy field of 16,900 Oe, magnetization (4piMs) of 4.4 kG, and ferromagnetic resonance peak-to-peak derivative linewidth at 53 GHz of 96 Oe. This combination of properties qualifies these films for microwave device applications. This marks the first growth of a microwave ferrite on SiC substrates and offers a new approach in the design and development of mu-wave and mm-wave monolithic integrated circuits. In part two, high-rate reactive ion etching using CHF3/SF6 gas mixtures was successfully demonstrated on BaM films, resulting in high aspect profile features of less than 50 nm in lateral dimension. These demonstrations enable the future integration of ferrites into MIC devices and technologies.

The influence of the surrounding gas on drop impact onto a wet substrate

NASA Astrophysics Data System (ADS)

Deegan, Robert; Zhang, Li; Toole, Jameson

2011-11-01

The impact of a droplet with a wet or solid substrate creates a spray of secondary droplets. The effect of the surrounding gas on this process was widely neglected prior to the work of Xu, Zhang, & Nagel which showed that lowering the gas pressure suppresses splashing for impact with a dry solid substrate. Here we present the results of our experimental investigation of the effect of the surrounding gas on the evolution of splashes from a wet substrate. We varied the density and pressure of the surrounding gas. We find quantitative changes to the onset thresholds of splashing and on the size distribution of, but no qualitative changes. The effects are most pronounced on the evolution of the ejecta sheet.

Nanofabrication on unconventional substrates using transferred hard masks

DOE PAGES

Li, Luozhou; Bayn, Igal; Lu, Ming; ...

2015-01-15

Here, a major challenge in nanofabrication is to pattern unconventional substrates that cannot be processed for a variety of reasons, such as incompatibility with spin coating, electron beam lithography, optical lithography, or wet chemical steps. Here, we present a versatile nanofabrication method based on re-usable silicon membrane hard masks, patterned using standard lithography and mature silicon processing technology. These masks, transferred precisely onto targeted regions, can be in the millimetre scale. They allow for fabrication on a wide range of substrates, including rough, soft, and non-conductive materials, enabling feature linewidths down to 10 nm. Plasma etching, lift-off, and ion implantationmore » are realized without the need for scanning electron/ion beam processing, UV exposure, or wet etching on target substrates.« less

The Age of Enlightenment: Evolving Opportunities in Brain Research Through Optical Manipulation of Neuronal Activity

PubMed Central

Jerome, Jason; Heck, Detlef H.

2011-01-01

Optical manipulation of neuronal activity has rapidly developed into the most powerful and widely used approach to study mechanisms related to neuronal connectivity over a range of scales. Since the early use of single site uncaging to map network connectivity, rapid technological development of light modulation techniques has added important new options, such as fast scanning photostimulation, massively parallel control of light stimuli, holographic uncaging, and two-photon stimulation techniques. Exciting new developments in optogenetics complement neurotransmitter uncaging techniques by providing cell-type specificity and in vivo usability, providing optical access to the neural substrates of behavior. Here we review the rapid evolution of methods for the optical manipulation of neuronal activity, emphasizing crucial recent developments. PMID:22275886

Synthesis of a Comprehensive Polyprenol Library for Evaluation of Bacterial Enzyme Lipid Substrate Specificity.

PubMed

Wu, Baolin; Woodward, Robert; Wen, Liuqing; Wang, Xuan; Zhao, Guohui; Wang, Peng George

2013-12-01

Polyprenols, a type of universal glycan lipid carrier, play important roles for glycan bio-assembly in wide variety of living systems. Chemical synthesis of natural polyisoprenols such as undecaprenol and dolichols, but especially their homologs, could serves as a powerful molecular tool to dissect and define the functions of enzymes involved in glycan biosynthesis. In this paper, we report an efficient and reliable method to construct this type of hydrophoic molecule through a base-mediated iterative coupling approach using a key bifunctional ( Z , Z )-diisoprenyl building block. The ligation with N -acetyl-D-glactosamine (GalNAc) with a set of the synthesized lipid analogs forming polyprenol pyrophosphate linked GalNAc (GalNAc-PP-lipid) conjugates is also demonstrated.

The age of enlightenment: evolving opportunities in brain research through optical manipulation of neuronal activity.

PubMed

Jerome, Jason; Heck, Detlef H

2011-01-01

Optical manipulation of neuronal activity has rapidly developed into the most powerful and widely used approach to study mechanisms related to neuronal connectivity over a range of scales. Since the early use of single site uncaging to map network connectivity, rapid technological development of light modulation techniques has added important new options, such as fast scanning photostimulation, massively parallel control of light stimuli, holographic uncaging, and two-photon stimulation techniques. Exciting new developments in optogenetics complement neurotransmitter uncaging techniques by providing cell-type specificity and in vivo usability, providing optical access to the neural substrates of behavior. Here we review the rapid evolution of methods for the optical manipulation of neuronal activity, emphasizing crucial recent developments.

Temperature and Microstructural Effects on the Superconducting Properties of Niobium Thin Films

DOE PAGES

Beebe, Melissa R.; Valente-Feliciano, Anne -Marie; Beringer, Douglas B.; ...

2016-11-23

Here, superconducting thin films have a wide range of dc and RF applications, from detectors to superconducting radio frequency. Amongst the most used materials, niobium (Nb) has the highest critical temperature (TC) and highest lower critical field (HC1) of the elemental superconductors and can be deposited on a variety of substrates, making Nb thin films very appealing for such applications. Here, we present temperature-dependent dc studies on the critical temperature and critical fields of Nb thin films grown on copper and r-plane sapphire surfaces. Additionally, we correlate the dc superconducting properties of these films with their microstructure, which allows formore » the possibility of tailoring future films for a specific application.« less

Regulation of Proteolysis by Human Deubiquitinating Enzymes

PubMed Central

Eletr, Ziad M.; Wilkinson, Keith D.

2013-01-01

The post-translational attachment of one or several ubiquitin molecules to a protein generates a variety of targeting signals that are used in many different ways in the cell. Ubiquitination can alter the activity, localization, protein-protein interactions or stability of the targeted protein. Further, a very large number of proteins are subject to regulation by ubiquitin-dependent processes, meaning that virtually all cellular functions are impacted by these pathways. Nearly a hundred enzymes from five different gene families (the deubiquitinating enzymes or DUBs), reverse this modification by hydrolyzing the (iso)peptide bond tethering ubiquitin to itself or the target protein. Four of these families are thiol proteases and one is a metalloprotease. DUBs of the Ubiquitin C-terminal Hydrolase (UCH) family act on small molecule adducts of ubiquitin, process the ubiquitin proprotein, and trim ubiquitin from the distal end of a polyubiquitin chain. Ubiquitin Specific Proteases (USP) tend to recognize and encounter their substrates by interaction of the variable regions of their sequence with the substrate protein directly, or with scaffolds or substrate adapters in multiprotein complexes. Ovarian Tumor (OTU) domain DUBs show remarkable specificity for different Ub chain linkages and may have evolved to recognize substrates on the basis of those linkages. The Josephin family of DUBs may specialize in distinguishing between polyubiquitin chains of different lengths. Finally, the JAB1/MPN+/MOV34 (JAMM) domain metalloproteases cleave the isopeptide bond near the attachment point of polyubiquitin and substrate, as well as being highly specific for the K63 poly-Ub linkage. These DUBs regulate proteolysis by: directly interacting with and co-regulating E3 ligases; altering the level of substrate ubiquitination; hydrolyzing or remodeling ubiquitinated and poly-ubiquitinated substrates; acting in specific locations in the cell and altering the localization of the target protein; and acting on proteasome bound substrates to facilitate or inhibit proteolysis. Thus, the scope and regulation of the ubiquitin pathway is very similar to that of phosphorylation, with the DUBs serving the same functions as the phosphatase. PMID:23845989

Method of depositing wide bandgap amorphous semiconductor materials

DOEpatents

Ellis, Jr., Frank B.; Delahoy, Alan E.

1987-09-29

A method of depositing wide bandgap p type amorphous semiconductor materials on a substrate without photosensitization by the decomposition of one or more higher order gaseous silanes in the presence of a p-type catalytic dopant at a temperature of about 200.degree. C. and a pressure in the range from about 1-50 Torr.

Classical Swine Fever Virus Glycoprotein Erns Is an Endoribonuclease with an Unusual Base Specificity

PubMed Central

Hausmann, Yvonne; Roman-Sosa, Gleyder; Thiel, Heinz-Jürgen; Rümenapf, Till

2004-01-01

The glycoprotein Erns of pestiviruses is a virion-associated and -secreted RNase that is involved in virulence. The requirements at the cleavage site in heteropolymeric RNA substrates were studied for Erns. Limited digestion of heteropolymeric RNA substrates indicated a cleavage 5′ of uridine residues irrespective of the preceding nucleotide (Np/U). To further study specificity radiolabeled RNA, molecules of 45 to 56 nucleotides in length were synthesized that contained no or a single Np/U cleavage site. Cleavage was only observed in substrates containing an ApU, CpU, GpU, or UpU dinucleotide and occurred in two steps, an initial NpU-specific and a consecutive unspecific degradation. The NpU-specific cleavage was resistant to 7 M urea while the second-order cleavage was sensitive to denaturation. Kinetic analyses revealed that Erns is a highly active endoribonuclease (kcat/Km = 2 × 106 to 10 × 106 M−1 s−1) with a strong affinity to NpU containing single-stranded RNA substrates (Km = 85 to 260 nM). PMID:15113930

CNG site-specific and methyl-sensitive endonuclease WEN1 from wheat seedlings.

PubMed

Fedoreyeva, L I; Vanyushin, B F

2011-06-01

Endonuclease WEN1 with apparent molecular mass about 27 kDa isolated from cytoplasmic vesicular fraction of aging coleoptiles of wheat seedlings has expressed site specificity action. This is a first detection and isolation of a site-specific endonuclease from higher eukaryotes, in general, and higher plants, in particular. The enzyme hydrolyzes deoxyribooligonucleotides of different composition on CNG (N is G, A, C, or T) sites by splitting the phosphodiester bond between C and N nucleotide residues in CNG sequence independent from neighbor nucleotide context except for CCCG. WEN1 prefers to hydrolyze methylated λ phage DNA and double-stranded deoxyribooligonucleotides containing 5-methylcytosine sites (m(5)CAG, m(5)CTG) compared with unmethylated substrates. The enzyme is also able to hydrolyze single-stranded substrates, but in this case it splits unmethylated substrates predominantly. Detection in wheat seedlings of WEN1 endonuclease that is site specific, sensitive to the substrate methylation status, and modulated with S-adenosyl-L-methionine indicates that in higher plants restriction--modification systems or some of their elements, at least, may exist.

Mutant fatty acid desaturase

DOEpatents

Shanklin, John; Cahoon, Edgar B.

2004-02-03

The present invention relates to a method for producing mutants of a fatty acid desaturase having a substantially increased activity towards fatty acid substrates with chains containing fewer than 18 carbons relative to an unmutagenized precursor desaturase having an 18 carbon atom chain length substrate specificity. The method involves inducing one or more mutations in the nucleic acid sequence encoding the precursor desaturase, transforming the mutated sequence into an unsaturated fatty acid auxotroph cell such as MH13 E. coli, culturing the cells in the absence of supplemental unsaturated fatty acids, thereby selecting for recipient cells which have received and which express a mutant fatty acid desaturase with an elevated specificity for fatty acid substrates having chain lengths of less than 18 carbon atoms. A variety of mutants having 16 or fewer carbon atom chain length substrate specificities are produced by this method. Mutant desaturases produced by this method can be introduced via expression vectors into prokaryotic and eukaryotic cells and can also be used in the production of transgenic plants which may be used to produce specific fatty acid products.

The effect of substrate composition and storage time on urine specific gravity in dogs.

PubMed

Steinberg, E; Drobatz, K; Aronson, L

2009-10-01

The purpose of this study is to evaluate the effects of substrate composition and storage time on urine specific gravity in dogs. A descriptive cohort study of 15 dogs. The urine specific gravity of free catch urine samples was analysed during a 5-hour time period using three separate storage methods; a closed syringe, a diaper pad and non-absorbable cat litter. The urine specific gravity increased over time in all three substrates. The syringe sample had the least change from baseline and the diaper sample had the greatest change from baseline. The urine specific gravity for the litter and diaper samples had a statistically significant increase from the 1-hour to the 5-hour time point. The urine specific gravity from canine urine stored either on a diaper or in a non-absorbable litter increased over time. Although the change was found to be statistically significant over the 5-hour study period it is unlikely to be clinically significant.

Stepwise Evolution of Coral Biomineralization Revealed with Genome-Wide Proteomics and Transcriptomics

PubMed Central

Sawada, Hitoshi; Satoh, Noriyuki

2016-01-01

Despite the importance of stony corals in many research fields related to global issues, such as marine ecology, climate change, paleoclimatogy, and metazoan evolution, very little is known about the evolutionary origin of coral skeleton formation. In order to investigate the evolution of coral biomineralization, we have identified skeletal organic matrix proteins (SOMPs) in the skeletal proteome of the scleractinian coral, Acropora digitifera, for which large genomic and transcriptomic datasets are available. Scrupulous gene annotation was conducted based on comparisons of functional domain structures among metazoans. We found that SOMPs include not only coral-specific proteins, but also protein families that are widely conserved among cnidarians and other metazoans. We also identified several conserved transmembrane proteins in the skeletal proteome. Gene expression analysis revealed that expression of these conserved genes continues throughout development. Therefore, these genes are involved not only skeleton formation, but also in basic cellular functions, such as cell-cell interaction and signaling. On the other hand, genes encoding coral-specific proteins, including extracellular matrix domain-containing proteins, galaxins, and acidic proteins, were prominently expressed in post-settlement stages, indicating their role in skeleton formation. Taken together, the process of coral skeleton formation is hypothesized as: 1) formation of initial extracellular matrix between epithelial cells and substrate, employing pre-existing transmembrane proteins; 2) additional extracellular matrix formation using novel proteins that have emerged by domain shuffling and rapid molecular evolution and; 3) calcification controlled by coral-specific SOMPs. PMID:27253604

High-speed thin-film transistors on single-crystalline, unstrained- and strained-silicon-based nanomembranes

NASA Astrophysics Data System (ADS)

Yuan, Hao-Chih

This research focuses on developing high-performance single-crystal Si-based nanomembranes and high-frequency thin-film transistors (TFTs) using these nanomembranes on flexible plastic substrates. Unstrained Si or SiGe nanomembranes with thickness of several tens to a couple of hundred nanometers are derived from silicon-on-insulator (SOI) or silicon-germanium-on-insulator (SGOI) and are subsequently transferred and integrated with flexible plastic host substrates via a one-step dry printing technique. Biaxial tensile-strained Si membranes that utilize elastic strain-sharing between Si and additionally grown SiGe thin films are also successfully integrated with plastic host substrates and exhibit predicted strain status and negligible density of dislocations. Biaxial tensile strain enhances electron mobility and lowers Schottky contact resistance. As a result, flexible TFTs built on the strained Si-membranes demonstrate much higher electron effective mobility and higher drive current than the unstrained counterpart. The dependence of drive current and transconductance on uniaxial tensile strain introducing by mechanical bending is also discussed. A novel combined "hot-and-cold" TFT fabrication process is developed specifically for realizing a wide spectrum of micro-electronics that can exhibit RF performance and can be integrated on low-temperature plastic substrate. The "hot" process that consists of ion implant and high-temperature annealing for desired doping type, profile, and concentration is realized on the bulk SOI/SGOI substrates followed by the "cold" process that includes room-temperature silicon-monoxide (SiO) deposition as gate dielectric layer to ensure the process compatibility with low-temperature, low-cost plastics. With these developments flexible Si-membrane n-type RF TFTs for analog applications and complementary TFTs for digital applications are demonstrated for the first time. RF TFTs with 1.5-mum channel length have demonstrated record-high f T and fmax values of 2.04 and 7.8 GHz, respectively. A small-signal equivalent circuit model study on the RF TFTs reveals the physics of how device layout affects fT and f max, which paves the way for further performance optimization and realization of integrated circuit on flexible substrate in the future.

Insight into the role of substrate-binding residues in conferring substrate specificity for the multifunctional polysaccharide lyase Smlt1473.

PubMed

MacDonald, Logan C; Berger, Bryan W

2014-06-27

Anionic polysaccharides are of growing interest in the biotechnology industry due to their potential pharmaceutical applications in drug delivery and wound treatment. Chemical composition and polymer length strongly influence the physical and biological properties of the polysaccharide and thus its potential industrial and medical applications. One promising approach to determining monomer composition and controlling the degree of polymerization involves the use of polysaccharide lyases, which catalyze the depolymerization of anionic polysaccharides via a β-elimination mechanism. Utilization of these enzymes for the production of custom-made oligosaccharides requires a high degree of control over substrate specificity. Previously, we characterized a polysaccharide lyase (Smlt1473) from Stenotrophomonas maltophilia k279a, which exhibited significant activity against hyaluronan (HA), poly-β-d-glucuronic acid (poly-GlcUA), and poly-β-d-mannuronic acid (poly-ManA) in a pH-regulated manner. Here, we utilize a sequence structure guided approach based on a homology model of Smlt1473 to identify nine putative substrate-binding residues and examine their effect on substrate specificity via site-directed mutagenesis. Interestingly, single point mutations H221F and R312L resulted in increased activity and specificity toward poly-ManA and poly-GlcUA, respectively. Furthermore, a W171A mutant nearly eliminated HA activity, while increasing poly-ManA and poly-GlcUA activity by at least 35%. The effect of these mutations was analyzed by comparison with the high resolution structure of Sphingomonas sp. A1-III alginate lyase in complex with poly-ManA tetrasaccharide and by taking into account the structural differences between HA, poly-GlcUA, and poly-ManA. Overall, our results demonstrate that even minor changes in active site architecture have a significant effect on the substrate specificity of Smlt1473, whose structural plasticity could be applied to the design of highly active and specific polysaccharide lyases. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Characterization and identification of ubiquitin conjugation sites with E3 ligase recognition specificities.

PubMed

Nguyen, Van-Nui; Huang, Kai-Yao; Huang, Chien-Hsun; Chang, Tzu-Hao; Bretaña, Neil; Lai, K; Weng, Julia; Lee, Tzong-Yi

2015-01-01

In eukaryotes, ubiquitin-conjugation is an important mechanism underlying proteasome-mediated degradation of proteins, and as such, plays an essential role in the regulation of many cellular processes. In the ubiquitin-proteasome pathway, E3 ligases play important roles by recognizing a specific protein substrate and catalyzing the attachment of ubiquitin to a lysine (K) residue. As more and more experimental data on ubiquitin conjugation sites become available, it becomes possible to develop prediction models that can be scaled to big data. However, no development that focuses on the investigation of ubiquitinated substrate specificities has existed. Herein, we present an approach that exploits an iteratively statistical method to identify ubiquitin conjugation sites with substrate site specificities. In this investigation, totally 6259 experimentally validated ubiquitinated proteins were obtained from dbPTM. After having filtered out homologous fragments with 40% sequence identity, the training data set contained 2658 ubiquitination sites (positive data) and 5532 non-ubiquitinated sites (negative data). Due to the difficulty in characterizing the substrate site specificities of E3 ligases by conventional sequence logo analysis, a recursively statistical method has been applied to obtain significant conserved motifs. The profile hidden Markov model (profile HMM) was adopted to construct the predictive models learned from the identified substrate motifs. A five-fold cross validation was then used to evaluate the predictive model, achieving sensitivity, specificity, and accuracy of 73.07%, 65.46%, and 67.93%, respectively. Additionally, an independent testing set, completely blind to the training data of the predictive model, was used to demonstrate that the proposed method could provide a promising accuracy (76.13%) and outperform other ubiquitination site prediction tool. A case study demonstrated the effectiveness of the characterized substrate motifs for identifying ubiquitination sites. The proposed method presents a practical means of preliminary analysis and greatly diminishes the total number of potential targets required for further experimental confirmation. This method may help unravel their mechanisms and roles in E3 recognition and ubiquitin-mediated protein degradation.

Extracellular matrix-derived hydrogels for dental stem cell delivery.

PubMed

Viswanath, Aiswarya; Vanacker, Julie; Germain, Loïc; Leprince, Julian G; Diogenes, Anibal; Shakesheff, Kevin M; White, Lisa J; des Rieux, Anne

2017-01-01

Decellularized mammalian extracellular matrices (ECM) have been widely accepted as an ideal substrate for repair and remodelling of numerous tissues in clinical and pre-clinical studies. Recent studies have demonstrated the ability of ECM scaffolds derived from site-specific homologous tissues to direct cell differentiation. The present study investigated the suitability of hydrogels derived from different source tissues: bone, spinal cord and dentine, as suitable carriers to deliver human apical papilla derived mesenchymal stem cells (SCAP) for spinal cord regeneration. Bone, spinal cord, and dentine ECM hydrogels exhibited distinct structural, mechanical, and biological characteristics. All three hydrogels supported SCAP viability and proliferation. However, only spinal cord and bone derived hydrogels promoted the expression of neural lineage markers. The specific environment of ECM scaffolds significantly affected the differentiation of SCAP to a neural lineage, with stronger responses observed with spinal cord ECM hydrogels, suggesting that site-specific tissues are more likely to facilitate optimal stem cell behavior for constructive spinal cord regeneration. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 319-328, 2017. © 2016 Wiley Periodicals, Inc.

Dip coating process: Silicon sheet growth development for the large-area silicon sheet task of the low-cost silicon solar array project

NASA Technical Reports Server (NTRS)

Heaps, J. D.; Maciolek, R. B.; Harrison, W. B.; Wolner, H. A.; Hendrickson, G.; Nelson, L. D.

1976-01-01

To date, an experimental dip-coating facility was constructed. Using this facility, relatively thin (1 mm) mullite and alumina substrates were successfully dip-coated with 2.5 - 3.0 ohm-cm, p-type silicon with areas of approximately 20 sq cm. The thickness and grain size of these coatings are influenced by the temperature of the melt and the rate at which the substrate is pulled from the melt. One mullite substrate had dendrite-like crystallites of the order of 1 mm wide and 1 to 2 cm long. Their axes were aligned along the direction of pulling. A large variety of substrate materials were purchased or developed enabling the program to commence a substrate definition evaluation. Due to the insulating nature of the substrate, the bottom layer of the p-n junction may have to be made via the top surface. The feasibility of accomplishing this was demonstrated using single crystal wafers.

Computational imaging of defects in commercial substrates for electronic and photonic devices

NASA Astrophysics Data System (ADS)

Fukuzawa, Masayuki; Kashiwagi, Ryo; Yamada, Masayoshi

2012-03-01

Computational defect imaging has been performed in commercial substrates for electronic and photonic devices by combining the transmission profile acquired with an imaging type of linear polariscope and the computational algorithm to extract a small amount of birefringence. The computational images of phase retardation δ exhibited spatial inhomogeneity of defect-induced birefringence in GaP, LiNbO3, and SiC substrates, which were not detected by conventional 'visual inspection' based on simple optical refraction or transmission because of poor sensitivity. The typical imaging time was less than 30 seconds for 3-inch diameter substrate with the spatial resolution of 200 μm, while that by scanning polariscope was 2 hours to get the same spatial resolution. Since our proposed technique have been achieved high sensitivity, short imaging time, and wide coverage of substrate materials, which are practical advantages over the laboratory-scale apparatus such as X-ray topography and electron microscope, it is useful for nondestructive inspection of various commercial substrates in production of electronic and photonic devices.

Arabidopsis ATG4 cysteine proteases specificity toward ATG8 substrates

PubMed Central

Park, Eunsook; Woo, Jongchan; Dinesh-Kumar, SP

2014-01-01

Macroautophagy (hereafter autophagy) is a regulated intracellular process during which cytoplasmic cargo engulfed by double-membrane autophagosomes is delivered to the vacuole or lysosome for degradation and recycling. Atg8 that is conjugated to phosphatidylethanolamine (PE) during autophagy plays an important role not only in autophagosome biogenesis but also in cargo recruitment. Conjugation of PE to Atg8 requires processing of the C-terminal conserved glycine residue in Atg8 by the Atg4 cysteine protease. The Arabidopsis plant genome contains 9 Atg8 (AtATG8a to AtATG8i) and 2 Atg4 (AtATG4a and AtATG4b) family members. To understand AtATG4’s specificity toward different AtATG8 substrates, we generated a unique synthetic substrate C-AtATG8-ShR (citrine-AtATG8-Renilla luciferase SuperhRLUC). In vitro analyses indicated that AtATG4a is catalytically more active and has broad AtATG8 substrate specificity compared with AtATG4b. Arabidopsis transgenic plants expressing the synthetic substrate C-AtAtg8a-ShR is efficiently processed by endogenous AtATG4s and targeted to the vacuole during nitrogen starvation. These results indicate that the synthetic substrate mimics endogenous AtATG8, and its processing can be monitored in vivo by a bioluminescence resonance energy transfer (BRET) assay. The synthetic Atg8 substrates provide an easy and versatile method to study plant autophagy during different biological processes. PMID:24658121

Use of Activity-Based Probes to Develop High Throughput Screening Assays That Can Be Performed in Complex Cell Extracts

PubMed Central

Deu, Edgar; Yang, Zhimou; Wang, Flora; Klemba, Michael; Bogyo, Matthew

2010-01-01

Background High throughput screening (HTS) is one of the primary tools used to identify novel enzyme inhibitors. However, its applicability is generally restricted to targets that can either be expressed recombinantly or purified in large quantities. Methodology and Principal Findings Here, we described a method to use activity-based probes (ABPs) to identify substrates that are sufficiently selective to allow HTS in complex biological samples. Because ABPs label their target enzymes through the formation of a permanent covalent bond, we can correlate labeling of target enzymes in a complex mixture with inhibition of turnover of a substrate in that same mixture. Thus, substrate specificity can be determined and substrates with sufficiently high selectivity for HTS can be identified. In this study, we demonstrate this method by using an ABP for dipeptidyl aminopeptidases to identify (Pro-Arg)2-Rhodamine as a specific substrate for DPAP1 in Plasmodium falciparum lysates and Cathepsin C in rat liver extracts. We then used this substrate to develop highly sensitive HTS assays (Z’>0.8) that are suitable for use in screening large collections of small molecules (i.e >300,000) for inhibitors of these proteases. Finally, we demonstrate that it is possible to use broad-spectrum ABPs to identify target-specific substrates. Conclusions We believe that this approach will have value for many enzymatic systems where access to large amounts of active enzyme is problematic. PMID:20700487

Mismatch repair factor MSH2-MSH3 binds and alters the conformation of branched DNA structures predicted to form during genetic recombination.

PubMed

Surtees, Jennifer A; Alani, Eric

2006-07-14

Genetic studies in Saccharomyces cerevisiae predict that the mismatch repair (MMR) factor MSH2-MSH3 binds and stabilizes branched recombination intermediates that form during single strand annealing and gene conversion. To test this model, we constructed a series of DNA substrates that are predicted to form during these recombination events. We show in an electrophoretic mobility shift assay that S. cerevisiae MSH2-MSH3 specifically binds branched DNA substrates containing 3' single-stranded DNA and that ATP stimulates its release from these substrates. Chemical footprinting analyses indicate that MSH2-MSH3 specifically binds at the double-strand/single-strand junction of branched substrates, alters its conformation and opens up the junction. Therefore, MSH2-MSH3 binding to its substrates creates a unique nucleoprotein structure that may signal downstream steps in repair that include interactions with MMR and nucleotide excision repair factors.

Aerobic biological treatment of leachates from municipal solid waste landfill.

PubMed

Andrés, P; Gutierrez, F; Arrabal, C; Cortijo, M

2004-01-01

The main objective of the study was to improve chemical oxygen demand (COD) elimination by secondary biological treatment from leachate of municipal solid waste landfill. This effluent was a supernatant liquid obtained after physicochemical processes and coagulating with Al3+ followed by ammoniacal stripping. First, respirometric assays were carried out to determine the substrate biodegradability. Specific sludge respiration rate (R(s)) vs. concentration of substrate (S), showed an increasing specific rate of assimilation of substrate (Rs), which reached the highest value, when the substrate concentration (COD) was between 75 and 200 mg O2 L(-1). Second, continuous experiments were made in an aerobic digester to test the previous respirometric data and the results showed removal efficiency of COD between 83 and 69%, and a substrate assimilation rate between 1.3 and 3.1 g COD g(-1) volatile suspended solids d(-1).

Developing upconversion nanoparticle-based smart substrates for remote temperature sensing

NASA Astrophysics Data System (ADS)

Coker, Zachary; Marble, Kassie; Alkahtani, Masfer; Hemmer, Philip; Yakovlev, Vladislav V.

2018-02-01

Recent developments in understanding of nanomaterial behaviors and synthesis have led to their application across a wide range of commercial and scientific applications. Recent investigations span from applications in nanomedicine and the development of novel drug delivery systems to nanoelectronics and biosensors. In this study, we propose the application of a newly engineered temperature sensitive water-based bio-compatible core/shell up-conversion nanoparticle (UCNP) in the development of a smart substrate for remote temperature sensing. We developed this smart substrate by dispersing functionalized nanoparticles into a polymer solution and then spin-coating the solution onto one side of a microscope slide to form a thin film substrate layer of evenly dispersed nanoparticles. By using spin-coating to deposit the particle solution we both create a uniform surface for the substrate while simultaneously avoid undesired particle agglomeration. Through this investigation, we have determined the sensitivity and capabilities of this smart substrate and conclude that further development can lead to a greater range of applications for this type smart substrate and use in remote temperature sensing in conjunction with other microscopy and spectroscopy investigations.

Buckling of a stiff thin film on an elastic graded compliant substrate.

PubMed

Chen, Zhou; Chen, Weiqiu; Song, Jizhou

2017-12-01

The buckling of a stiff film on a compliant substrate has attracted much attention due to its wide applications such as thin-film metrology, surface patterning and stretchable electronics. An analytical model is established for the buckling of a stiff thin film on a semi-infinite elastic graded compliant substrate subjected to in-plane compression. The critical compressive strain and buckling wavelength for the sinusoidal mode are obtained analytically for the case with the substrate modulus decaying exponentially. The rigorous finite element analysis (FEA) is performed to validate the analytical model and investigate the postbuckling behaviour of the system. The critical buckling strain for the period-doubling mode is obtained numerically. The influences of various material parameters on the results are investigated. These results are helpful to provide physical insights on the buckling of elastic graded substrate-supported thin film.

Surface-enhanced Raman spectroscopy with Au-nanoparticle substrate fabricated by using femtosecond pulse

NASA Astrophysics Data System (ADS)

Zhang, Wending; Li, Cheng; Gao, Kun; Lu, Fanfan; Liu, Min; Li, Xin; Zhang, Lu; Mao, Dong; Gao, Feng; Huang, Ligang; Mei, Ting; Zhao, Jianlin

2018-05-01

Au-nanoparticle (Au-NP) substrates for surface-enhanced Raman spectroscopy (SERS) were fabricated by grid-like scanning a Au-film using a femtosecond pulse. The Au-NPs were directly deposited on the Au-film surface due to the scanning process. The experimentally obtained Au-NPs presented local surface plasmon resonance effect in the visible spectral range, as verified by finite difference time domain simulations and measured reflection spectrum. The SERS experiment using the Au-NP substrates exhibited high activity and excellent substrate reproducibility and stability, and a clearly present Raman spectra of target analytes, e.g. Rhodamine-6G, Rhodamine-B and Malachite green, with concentrations down to 10‑9 M. This work presents an effective approach to producing Au-NP SERS substrates with advantages in activity, reproducibility and stability, which could be used in a wide variety of practical applications for trace amount detection.

Highly sensitive surface-enhanced Raman scattering substrate made from superaligned carbon nanotubes.

PubMed

Sun, Yinghui; Liu, Kai; Miao, Jiao; Wang, Zheyao; Tian, Baozhong; Zhang, Lina; Li, Qunqing; Fan, Shoushan; Jiang, Kaili

2010-05-12

Surface-enhanced Raman scattering (SERS) has attracted wide attention because it can enhance normally weak Raman signal by several orders of magnitude and facilitate the sensitive detection of molecules. Conventional SERS substrates are constructed by placing metal nanoparticles on a planar surface. Here we show that, if the planar surface was substituted by a unique nanoporous surface, the enhancement effect can be dramatically improved. The nanoporous surface can be easily fabricated in batches and at low costs by cross stacking superaligned carbon nanotube films. The as-prepared transparent and freestanding SERS substrate is capable of detecting ambient trinitrotoluene vapor, showing much higher Raman enhancement than ordinary planar substrates because of the extremely large surface area and the unique zero-dimensional at one-dimensional nanostructure. These results not only provide a new approach to ultrasensitive SERS substrates, but also are helpful for improving the fundamental understanding of SERS phenomena.

Surface-enhanced Raman spectroscopy with Au-nanoparticle substrate fabricated by using femtosecond pulse.

PubMed

Zhang, Wending; Li, Cheng; Gao, Kun; Lu, Fanfan; Liu, Min; Li, Xin; Zhang, Lu; Mao, Dong; Gao, Feng; Huang, Ligang; Mei, Ting; Zhao, Jianlin

2018-05-18

Au-nanoparticle (Au-NP) substrates for surface-enhanced Raman spectroscopy (SERS) were fabricated by grid-like scanning a Au-film using a femtosecond pulse. The Au-NPs were directly deposited on the Au-film surface due to the scanning process. The experimentally obtained Au-NPs presented local surface plasmon resonance effect in the visible spectral range, as verified by finite difference time domain simulations and measured reflection spectrum. The SERS experiment using the Au-NP substrates exhibited high activity and excellent substrate reproducibility and stability, and a clearly present Raman spectra of target analytes, e.g. Rhodamine-6G, Rhodamine-B and Malachite green, with concentrations down to 10 -9 M. This work presents an effective approach to producing Au-NP SERS substrates with advantages in activity, reproducibility and stability, which could be used in a wide variety of practical applications for trace amount detection.

Buckling of a stiff thin film on an elastic graded compliant substrate

NASA Astrophysics Data System (ADS)

Chen, Zhou; Chen, Weiqiu; Song, Jizhou

2017-12-01

The buckling of a stiff film on a compliant substrate has attracted much attention due to its wide applications such as thin-film metrology, surface patterning and stretchable electronics. An analytical model is established for the buckling of a stiff thin film on a semi-infinite elastic graded compliant substrate subjected to in-plane compression. The critical compressive strain and buckling wavelength for the sinusoidal mode are obtained analytically for the case with the substrate modulus decaying exponentially. The rigorous finite element analysis (FEA) is performed to validate the analytical model and investigate the postbuckling behaviour of the system. The critical buckling strain for the period-doubling mode is obtained numerically. The influences of various material parameters on the results are investigated. These results are helpful to provide physical insights on the buckling of elastic graded substrate-supported thin film.

Methods and devices for fabricating and assembling printable semiconductor elements

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nuzzo, Ralph G.; Rogers, John A.; Menard, Etienne

The invention provides methods and devices for fabricating printable semiconductor elements and assembling printable semiconductor elements onto substrate surfaces. Methods, devices and device components of the present invention are capable of generating a wide range of flexible electronic and optoelectronic devices and arrays of devices on substrates comprising polymeric materials. The present invention also provides stretchable semiconductor structures and stretchable electronic devices capable of good performance in stretched configurations.

Methods and devices for fabricating and assembling printable semiconductor elements

DOEpatents

Nuzzo, Ralph G; Rogers, John A; Menard, Etienne; Lee, Keon Jae; Khang, Dahl-Young; Sun, Yugang; Meitl, Matthew; Zhu, Zhengtao

2014-03-04

The invention provides methods and devices for fabricating printable semiconductor elements and assembling printable semiconductor elements onto substrate surfaces. Methods, devices and device components of the present invention are capable of generating a wide range of flexible electronic and optoelectronic devices and arrays of devices on substrates comprising polymeric materials. The present invention also provides stretchable semiconductor structures and stretchable electronic devices capable of good performance in stretched configurations.

Quasi-steady-state voltammetry of rapid electron transfer reactions at the macroscopic substrate of the scanning electrochemical microscope.

PubMed

Nioradze, Nikoloz; Kim, Jiyeon; Amemiya, Shigeru

2011-02-01

We report on a novel theory and experiment for scanning electrochemical microscopy (SECM) to enable quasi-steady-state voltammetry of rapid electron transfer (ET) reactions at macroscopic substrates. With this powerful approach, the substrate potential is cycled widely across the formal potential of a redox couple while the reactant or product of a substrate reaction is amperometrically detected at the tip in the feedback or substrate generation/tip collection mode, respectively. The plot of tip current versus substrate potential features the retraceable sigmoidal shape of a quasi-steady-state voltammogram although a transient voltammogram is obtained at the macroscopic substrate. Finite element simulations reveal that a short tip-substrate distance and a reversible substrate reaction (except under the tip) are required for quasi-steady-state voltammetry. Advantageously, a pair of quasi-steady-state voltammograms is obtained by employing both operation modes to reliably determine all transport, thermodynamic, and kinetic parameters as confirmed experimentally for rapid ET reactions of ferrocenemethanol and 7,7,8,8-tetracyanoquinodimethane at a Pt substrate with ∼0.5 μm-radius Pt tips positioned at 90 nm-1 μm distances. Standard ET rate constants of ∼7 cm/s were obtained for the latter mediator as the largest determined for a substrate reaction by SECM. Various potential applications of quasi-steady-state voltammetry are also proposed.

Molecular evolution and functional divergence of the cytochrome P450 3 (CYP3) Family in Actinopterygii (ray-finned fish).

PubMed

Yan, Jun; Cai, Zhonghua

2010-12-10

The cytochrome P450 (CYP) superfamily is a multifunctional hemethiolate enzyme that is widely distributed from Bacteria to Eukarya. The CYP3 family contains mainly the four subfamilies CYP3A, CYP3B, CYP3C and CYP3D in vertebrates; however, only the Actinopterygii (ray-finned fish) have all four subfamilies and detailed understanding of the evolutionary relationship of Actinopterygii CYP3 family members would be valuable. Phylogenetic relationships were constructed to trace the evolutionary history of the Actinopterygii CYP3 family genes. Selection analysis, relative rate tests and functional divergence analysis were combined to interpret the relationship of the site-specific evolution and functional divergence in the Actinopterygii CYP3 family. The results showed that the four CYP3 subfamilies in Actinopterygii might be formed by gene duplication. The first gene duplication event was responsible for divergence of the CYP3B/C clusters from ancient CYP3 before the origin of the Actinopterygii, which corresponded to the fish-specific whole genome duplication (WGD). Tandem repeat duplication in each of the homologue clusters produced stable CYP3B, CYP3C, CYP3A and CYP3D subfamilies. Acceleration of asymmetric evolutionary rates and purifying selection together were the main force for the production of new subfamilies and functional divergence in the new subset after gene duplication, whereas positive selection was detected only in the retained CYP3A subfamily. Furthermore, nearly half of the functional divergence sites appear to be related to substrate recognition, which suggests that site-specific evolution is closely related with functional divergence in the Actinopterygii CYP3 family. The split of fish-specific CYP3 subfamilies was related to the fish-specific WGD, and site-specific acceleration of asymmetric evolutionary rates and purifying selection was the main force for the origin of the new subfamilies and functional divergence in the new subset after gene duplication. Site-specific evolution in substrate recognition was related to functional divergence in the Actinopterygii CYP3 family.

Effect of tacticity on the structure and glass transition temperature of polystyrene adsorbed onto solid surfaces

NASA Astrophysics Data System (ADS)

Negash, Solomon; Tatek, Yergou B.; Tsige, Mesfin

2018-04-01

We have carried out atomistic (all-atom) molecular dynamics simulations to investigate the effect of tacticity on the structure and glass transition temperature (Tg) of polystyrene (PS) thin films adsorbed on two distinct types of solid substrates. The systems consist of thin films made of atactic, isotactic, and syndiotactic PS chains supported by graphite or hydroxylated α-quartz substrates, which are known to be atomically flat but chemically and structurally different. We have observed a marked dependence of the film structure on substrate type as well as on tacticity. For instance, rings' orientation near substrate surfaces depends on substrate type for atactic PS and isotactic PS films, while no such dependence is observed for syndiotactic PS films whose interfacial structure seems to result from their propensity to adopt the trans conformation rather than their specific interaction with the substrates. Moreover, our results indicate that glass transition temperatures of substrate supported polystyrene films are higher compared to those of the corresponding free-standing films. More specifically, PS films on graphite exhibit larger Tg values than those on α-quartz, and we have noticed that syndiotactic PS has the largest Tg irrespective of the substrate type. Furthermore, the local Tg in the region of the film in contact with the substrates shows a strong tacticity and substrate dependence, whereas no dependencies were found for the local Tg in the middle of the film. Substrate-film interaction energy and chains' dynamics near substrate-film interfaces were subsequently investigated in order to substantiate the obtained Tgs, and it was found that films with higher Tgs are strongly adsorbed on the substrates and/or exhibit smaller interfacial chains' dynamics essentially due to steric hindrance.

Whole-animal imaging of bacterial infection using endoscopic excitation of β-lactamase (BlaC)-specific fluorogenic probe

NASA Astrophysics Data System (ADS)

Nooshabadi, Fatemeh; Yang, Hee-Jeong; Cheng, Yunfeng; Xie, Hexin; Rao, Jianghong; Cirillo, Jeffrey D.; Maitland, Kristen C.

2016-03-01

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains one of the most frequent causes of death worldwide. The slow growth rate of Mtb limits progress toward understanding tuberculosis including diagnosis of infections and evaluating therapeutic efficacy. Development of near-infrared (NIR) β-lactamase (BlaC)-specific fluorogenic substrate has made a significant breakthrough in the whole-animal imaging to detect Mtb infection. The reporter enzyme fluorescence (REF) system using a BlaC-specific fluorogenic substrate has improved the detection sensitivity in whole-animal optical imaging down to ~104 colony forming units (CFU) of bacteria, about 100-fold improvement over recombinant strains. However, improvement of detection sensitivity is strongly needed for clinical diagnosis of early stage infection at greater tissue depth. In order to improve detection sensitivity, we have integrated a fiber-based microendoscpe into a whole-animal imaging system to transmit the excitation light from the fiber bundle to the fluorescent target directly and measure fluorescent level using BlaC-specific REF substrate in the mouse lung. REF substrate, CNIR800, was delivered via aerosol route to the pulmonary infected mice with M. bovis BCG strain at 24 hours post-infection and groups of mice were imaged at 1-4 hours post-administration of the substrate using the integrated imaging system. In this study we evaluated the kinetics of CNIR800 substrate using REF technology using the integrated imaging system. Integration of these technologies has great promise for improved detection sensitivity allowing pre-clinical imaging for evaluation of new therapeutic agents.

Effects of geomorphology, habitat, and spatial location on fish assemblages in a watershed in Ohio, USA.

PubMed

D'Ambrosio, Jessica L; Williams, Lance R; Witter, Jonathan D; Ward, Andy

2009-01-01

In this paper, we evaluate relationships between in-stream habitat, water chemistry, spatial distribution within a predominantly agricultural Midwestern watershed and geomorphic features and fish assemblage attributes and abundances. Our specific objectives were to: (1) identify and quantify key environmental variables at reach and system wide (watershed) scales; and (2) evaluate the relative influence of those environmental factors in structuring and explaining fish assemblage attributes at reach scales to help prioritize stream monitoring efforts and better incorporate all factors that influence aquatic biology in watershed management programs. The original combined data set consisted of 31 variables measured at 32 sites, which was reduced to 9 variables through correlation and linear regression analysis: stream order, percent wooded riparian zone, drainage area, in-stream cover quality, substrate quality, gradient, cross-sectional area, width of the flood prone area, and average substrate size. Canonical correspondence analysis (CCA) and variance partitioning were used to relate environmental variables to fish species abundance and assemblage attributes. Fish assemblages and abundances were explained best by stream size, gradient, substrate size and quality, and percent wooded riparian zone. Further data are needed to investigate why water chemistry variables had insignificant relationships with IBI scores. Results suggest that more quantifiable variables and consideration of spatial location of a stream reach within a watershed system should be standard data incorporated into stream monitoring programs to identify impairments that, while biologically limiting, are not fully captured or elucidated using current bioassessment methods.

Plasmin substrate binding site cooperativity guides the design of potent peptide aldehyde inhibitors.

PubMed

Swedberg, Joakim E; Harris, Jonathan M

2011-10-04

Perioperative bleeding is a cause of major blood loss and is associated with increased rates of postoperative morbidity and mortality. To combat this, antifibrinolytic inhibitors of the serine protease plasmin are commonly used to reduce bleeding during surgery. The most effective and previously widely used of these is the broad range serine protease inhibitor aprotinin. However, adverse clinical outcomes have led to use of alternative serine lysine analogues to inhibit plasmin. These compounds suffer from low selectivity and binding affinity. Consequently, a concerted effort to discover potent and selective plasmin inhibitors has developed. This study used a noncombinatorial peptide library to define plasmin's extended substrate specificity and guide the design of potent transition state analogue inhibitors. The various substrate binding sites of plasmin were found to exhibit a higher degree of cooperativity than had previously been appreciated. Peptide sequences capitalizing on these features produced high-affinity inhibitors of plasmin. The most potent of these, Lys-Met(sulfone)-Tyr-Arg-H [KM(O(2))YR-H], inhibited plasmin with a K(i) of 3.1 nM while maintaining 25-fold selectivity over plasma kallikrein. Furthermore, 125 nM (0.16 μg/mL) KM(O(2))YR-H attenuated fibrinolysis in vitro with an efficacy similar to that of 15 nM (0.20 μg/mL) aprotinin. To date, this is the most potent peptide inhibitor of plasmin that exhibits selectivity against plasma kallikrein, making this compound an attractive candidate for further therapeutic development.

Hybrid 3D printing and electrodeposition approach for controllable 3D alginate hydrogel formation.

PubMed

Shang, Wanfeng; Liu, Yanting; Wan, Wenfeng; Hu, Chengzhi; Liu, Zeyang; Wong, Chin To; Fukuda, Toshio; Shen, Yajing

2017-06-07

Calcium alginate hydrogels are widely used as biocompatible materials in a substantial number of biomedical applications. This paper reports on a hybrid 3D printing and electrodeposition approach for forming 3D calcium alginate hydrogels in a controllable manner. Firstly, a specific 3D hydrogel printing system is developed by integrating a customized ejection syringe with a conventional 3D printer. Then, a mixed solution of sodium alginate and CaCO 3 nanoparticles is filled into the syringe and can be continuously ejected out of the syringe nozzle onto a conductive substrate. When applying a DC voltage (∼5 V) between the substrate (anode) and the nozzle (cathode), the Ca 2+ released from the CaCO 3 particles can crosslink the alginate to form calcium alginate hydrogel on the substrate. To elucidate the gel formation mechanism and better control the gel growth, we can further establish and verify a gel growth model by considering several key parameters, i.e., applied voltage and deposition time. The experimental results indicate that the alginate hydrogel of various 3D structures can be formed by controlling the movement of the 3D printer. A cell viability test is conducted and shows that the encapsulated cells in the gel can maintain a high survival rate (∼99% right after gel formation). This research establishes a reliable method for the controllable formation of 3D calcium alginate hydrogel, exhibiting great potential for use in basic biology and applied biomedical engineering.

Positive selection sites in tertiary structure of Leguminosae chalcone isomerase 1.

PubMed

Wang, R K; Zhan, S F; Zhao, T J; Zhou, X L; Wang, C E

2015-03-20

Isoflavonoids and the related synthesis enzyme, chalcone isomerase 1 (CHI1), are unique in the Leguminosae, with diverse biological functions. Among the Leguminosae, the soybean is an important oil, protein crop, and model plant. In this study, we aimed to detect the generation pattern of Leguminosae CHI1. Genome-wide sequence analysis of CHI in 3 Leguminosae and 3 other closely related model plants was performed; the expression levels of soybean chalcone isomerases were also analyzed. By comparing positively selected sites and their protein structures, we retrieved the evolution patterns for Leguminosae CHI1. A total of 28 CHI and 7 FAP3 (CHI4) genes were identified and separated into 4 clades: CHI1, CHI2, CHI3, and FAP3. Soybean genes belonging to the same chalcone isomerase subfamily had similar expression patterns. CHI1, the unique chalcone isomerase subfamily in Leguminosae, showed signs of significant positive selection as well as special expression characteristics, indicating an accelerated evolution throughout its divergence. Eight sites were identified as undergoing positive selection with high confidence. When mapped onto the tertiary structure of CHI1, these 8 sites were observed surrounding the enzyme substrate only; some of them connected to the catalytic core of CHI. Thus, we inferred that the generation of Leguminosae CHI1 is dependent on the positively selected amino acids surrounding its catalytic substrate. In other words, the evolution of CHI1 was driven by specific selection or processing conditions within the substrate.

Metformin Is a Substrate and Inhibitor of the Human Thiamine Transporter, THTR-2 (SLC19A3).

PubMed

Liang, Xiaomin; Chien, Huan-Chieh; Yee, Sook Wah; Giacomini, Marilyn M; Chen, Eugene C; Piao, Meiling; Hao, Jia; Twelves, Jolyn; Lepist, Eve-Irene; Ray, Adrian S; Giacomini, Kathleen M

2015-12-07

The biguanide metformin is widely used as first-line therapy for the treatment of type 2 diabetes. Predominately a cation at physiological pH's, metformin is transported by membrane transporters, which play major roles in its absorption and disposition. Recently, our laboratory demonstrated that organic cation transporter 1, OCT1, the major hepatic uptake transporter for metformin, was also the primary hepatic uptake transporter for thiamine, vitamin B1. In this study, we tested the reverse, i.e., that metformin is a substrate of thiamine transporters (THTR-1, SLC19A2, and THTR-2, SLC19A3). Our study demonstrated that human THTR-2 (hTHTR-2), SLC19A3, which is highly expressed in the small intestine, but not hTHTR-1, transports metformin (Km = 1.15 ± 0.2 mM) and other cationic compounds (MPP(+) and famotidine). The uptake mechanism for hTHTR-2 was pH and electrochemical gradient sensitive. Furthermore, metformin as well as other drugs including phenformin, chloroquine, verapamil, famotidine, and amprolium inhibited hTHTR-2 mediated uptake of both thiamine and metformin. Species differences in the substrate specificity of THTR-2 between human and mouse orthologues were observed. Taken together, our data suggest that hTHTR-2 may play a role in the intestinal absorption and tissue distribution of metformin and other organic cations and that the transporter may be a target for drug-drug and drug-nutrient interactions.

Crystal Structures of An F420-Dependent Glucose-6-Phosphate Dehydrogenase Fgd1 Involved in the Activation of the Anti-Tb Drug Candidate Pa-824 Reveal the Basis of Coenzyme And Substrate Binding

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bashiri, G.; Squire, C.J.; Moreland, N.J.

2009-05-11

The modified flavin coenzyme F{sub 420} is found in a restricted number of microorganisms. It is widely distributed in mycobacteria, however, where it is important in energy metabolism, and in Mycobacterium tuberculosis (Mtb) is implicated in redox processes related to non-replicating persistence. In Mtb, the F{sub 420}-dependent glucose-6-phosphate dehydrogenase FGD1 provides reduced F{sub 420} for the in vivo activation of the nitroimidazopyran prodrug PA-824, currently being developed for anti-tuberculosis therapy against both replicating and persistent bacteria. The structure of M. tuberculosis FGD1 has been determined by x-ray crystallography both in its apo state and in complex with F{sub 420} andmore » citrate at resolutions of 1.90 and 1.95{angstrom}, respectively. The structure reveals a highly specific F{sub 420} binding mode, which is shared with several other F{sub 420}-dependent enzymes. Citrate occupies the substrate binding pocket adjacent to F{sub 420} and is shown to be a competitive inhibitor (IC{sub 50} 43 {micro}m). Modeling of the binding of the glucose 6-phosphate (G6P) substrate identifies a positively charged phosphate binding pocket and shows that G6P, like citrate, packs against the isoalloxazine moiety of F{sub 420} and helps promote a butterfly bend conformation that facilitates F{sub 420} reduction and catalysis.« less

Identification of the hot spot residues for pyridine derivative inhibitor CCT251455 and ATP substrate binding on monopolar spindle 1 (MPS1) kinase by molecular dynamic simulation.

PubMed

Chen, Kai; Duan, Wenxiu; Han, Qianqian; Sun, Xuan; Li, Wenqian; Hu, Shuangyun; Wan, Jiajia; Wu, Jiang; Ge, Yushu; Liu, Dan

2018-03-08

Protein kinase monopolar spindle 1 plays an important role in spindle assembly checkpoint at the onset of mitosis. Over expression of MPS1 correlated with a wide range of human tumors makes it an attractive target for finding an effective and specific inhibitor. In this work, we performed molecular dynamics simulations of protein MPS1 itself as well as protein bound systems with the inhibitor and natural substrate based on crystal structures. The reported orally bioavailable 1 h-pyrrolo [3,2-c] pyridine inhibitors of MPS1 maintained stable binding in the catalytic site, while natural substrate ATP could not stay. Comparative study of stability and flexibility of three systems reveals position shifting of β-sheet region within the catalytic site, which indicates inhibition mechanism was through stabilizing the β-sheet region. Binding free energies calculated with MM-GB/PBSA method shows different binding affinity for inhibitor and ATP. Finally, interactions between protein and inhibitor during molecular dynamic simulations were measured and counted. Residue Gly605 and Leu654 were suggested as important hot spots for stable binding of inhibitor by molecular dynamic simulation. Our results reveal an important position shifting within catalytic site for non-inhibited proteins. Together with hot spots found by molecular dynamic simulation, the results provide important information of inhibition mechanism and will be referenced for designing novel inhibitors.

Black, green, and red abalones. Species profiles: life histories and environmental requirements of coastal fishes and invertebrates (Pacific Southwest. )

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ault, J.S.

1985-03-01

Black, green, and red abalones (Haliotis cracherodii, H. fulgens, and H. rufescens, respectivley) are of commercial and ecological importance and are distributed widely along the California coast. The abalones are morphologically similar; species are distinguished by particular shell sculpture, color, and body characteristics. Their latitudinal and bathymetric distribution is stratified and most closely related to temperature. Small juveniles eat mainly microflora; adults eat primarily drift macro-algae, preferring specific brown or red algae, when available. Spawning occurs during summer; gonad ripening depends on food quality and quantity and water temperature. Larvae are lecithotrophic and remain planktonic for periods of 5 tomore » 14 days after hatching; settling is substrate specific. Postlarvae and adults require hard substrate for attachment. Juveniles are cryptic, adults usually more exposed. Growth rates are similar, although maximum size varies with species. Increases in shell length and body weight correlate positively with food abundance and temperature. Below depths of 6 m, sea urchins are major competitors for food and space. Predation by invertebrates is low. Decreased abalone production from central California is associated with range expansion and increased predation by sea otters, the major source of abalone mortality. General declines in California landings are due to mortality from improper picking and replacement, habitat degradation, and perhaps overfishing. Commercial and sport diving efforts have increased sharply, whereas annual landings of abalones declined from 1965 to 1982.« less

Extending enzyme molecular recognition with an expanded amino acid alphabet

PubMed Central

Windle, Claire L.; Simmons, Katie J.; Ault, James R.; Trinh, Chi H.; Nelson, Adam

2017-01-01

Natural enzymes are constructed from the 20 proteogenic amino acids, which may then require posttranslational modification or the recruitment of coenzymes or metal ions to achieve catalytic function. Here, we demonstrate that expansion of the alphabet of amino acids can also enable the properties of enzymes to be extended. A chemical mutagenesis strategy allowed a wide range of noncanonical amino acids to be systematically incorporated throughout an active site to alter enzymic substrate specificity. Specifically, 13 different noncanonical side chains were incorporated at 12 different positions within the active site of N-acetylneuraminic acid lyase (NAL), and the resulting chemically modified enzymes were screened for activity with a range of aldehyde substrates. A modified enzyme containing a 2,3-dihydroxypropyl cysteine at position 190 was identified that had significantly increased activity for the aldol reaction of erythrose with pyruvate compared with the wild-type enzyme. Kinetic investigation of a saturation library of the canonical amino acids at the same position showed that this increased activity was not achievable with any of the 20 proteogenic amino acids. Structural and modeling studies revealed that the unique shape and functionality of the noncanonical side chain enabled the active site to be remodeled to enable more efficient stabilization of the transition state of the reaction. The ability to exploit an expanded amino acid alphabet can thus heighten the ambitions of protein engineers wishing to develop enzymes with new catalytic properties. PMID:28196894

Crystal Structure of the Ectoine Hydroxylase, a Snapshot of the Active Site*

PubMed Central

Höppner, Astrid; Widderich, Nils; Lenders, Michael; Bremer, Erhard; Smits, Sander H. J.

2014-01-01

Ectoine and its derivative 5-hydroxyectoine are compatible solutes that are widely synthesized by bacteria to cope physiologically with osmotic stress. They also serve as chemical chaperones and maintain the functionality of macromolecules. 5-Hydroxyectoine is produced from ectoine through a stereo-specific hydroxylation, an enzymatic reaction catalyzed by the ectoine hydroxylase (EctD). The EctD protein is a member of the non-heme-containing iron(II) and 2-oxoglutarate-dependent dioxygenase superfamily and is evolutionarily well conserved. We studied the ectoine hydroxylase from the cold-adapted marine ultra-microbacterium Sphingopyxis alaskensis (Sa) and found that the purified SaEctD protein is a homodimer in solution. We determined the SaEctD crystal structure in its apo-form, complexed with the iron catalyst, and in a form that contained iron, the co-substrate 2-oxoglutarate, and the reaction product of EctD, 5-hydroxyectoine. The iron and 2-oxoglutarate ligands are bound within the EctD active site in a fashion similar to that found in other members of the dioxygenase superfamily. 5-Hydroxyectoine, however, is coordinated by EctD in manner different from that found in high affinity solute receptor proteins operating in conjunction with microbial import systems for ectoines. Our crystallographic analysis provides a detailed view into the active site of the ectoine hydroxylase and exposes an intricate network of interactions between the enzyme and its ligands that collectively ensure the hydroxylation of the ectoine substrate in a position- and stereo-specific manner. PMID:25172507

Approaches to Optimizing Animal Cell Culture Process: Substrate Metabolism Regulation and Protein Expression Improvement

NASA Astrophysics Data System (ADS)

Zhang, Yuanxing

Some high value proteins and vaccines for medical and veterinary applications by animal cell culture have an increasing market in China. In order to meet the demands of large-scale productions of proteins and vaccines, animal cell culture technology has been widely developed. In general, an animal cell culture process can be divided into two stages in a batch culture. In cell growth stage a high specific growth rate is expected to achieve a high cell density. In production stage a high specific production rate is stressed for the expression and secretion of qualified protein or replication of virus. It is always critical to maintain high cell viability in fed-batch and perfusion cultures. More concern has been focused on two points by the researchers in China. First, the cell metabolism of substrates is analyzed and the accumulation of toxic by-products is decreased through regulating cell metabolism in the culture process. Second, some important factors effecting protein expression are understood at the molecular level and the production ability of protein is improved. In pace with the rapid development of large-scale cell culture for the production of vaccines, antibodies and other recombinant proteins in China, the medium design and process optimization based on cell metabolism regulation and protein expression improvement will play an important role. The chapter outlines the main advances in metabolic regulation of cell and expression improvement of protein in animal cell culture in recent years.

Specific surface to evaluate the efficiencies of milling and pretreatment of wood for enzymatic saccharification

Treesearch

Junyong Zhu; G.S. Wang; X.J. Pan; Roland Gleisner

2009-01-01

Sieving methods have been almost exclusively used for feedstock size-reduction characterization in the biomass refining literature. This study demonstrates a methodology to properly characterize specific surface of biomass substrates through two dimensional measurement of each fiber of the substrate using a wet imaging technique. The methodology provides more...

Phospholipid substrate-specificity of the L-serine base-exchange enzyme in rat liver microsomal fraction.

PubMed Central

Bjerve, K S

1984-01-01

The specificity of the L-serine base-exchange enzyme towards the fatty acid composition of the phospholipid substrate was investigated with a rat liver microsomal fraction. The relative rates of L-serine incorporation into saturated-hexaenoic, saturated-pentaenoic, saturated-tetraenoic, saturated-trienoic, dienoic-dienoic, monoenoic-dienoic, saturated-dienoic and saturated-monoenoic + saturated-saturated phosphatidylserine molecular species were 42, 5, 23, 4, 5, 4, 5 and 11% respectively. This is similar to, but not identical with, the relative mass abundance of these molecular species in total liver cell phosphatidylserines. The results indicate that the substrate-specificity of the L-serine base-exchange enzyme can at least in part explain the observed fatty acid composition of rat liver phosphatidylserines. PMID:6430274

Quantitative iTRAQ-based secretome analysis reveals species-specific and temporal shifts in carbon utilization strategies among manganese(II)-oxidizing Ascomycete fungi

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zeiner, Carolyn A.; Purvine, Samuel O.; Zink, Erika M.

Fungi generate a wide range of extracellular hydrolytic and oxidative enzymes and reactive metabolites, collectively known as the secretome, that synergistically drive plant litter decomposition in the environment. While secretome studies of model organisms have greatly expanded our knowledge of these enzymes, few have extended secretome characterization to environmental isolates, particularly filamentous Ascomycetes, or directly compared temporal patterns of enzyme utilization among diverse species. Thus, the mechanisms of carbon (C) degradation by many ubiquitous soil fungi remain poorly understood. Here in this study we use a combination of iTRAQ proteomics and extracellular enzyme activity assays to compare the protein compositionmore » of the secretomes of four manganese(II)-oxidizing Ascomycete fungi over a three-week time course. We demonstrate that the fungi exhibit striking differences in the regulation of extracellular lignocellulose-degrading enzymes among species and over time, revealing species-specific and temporal shifts in C utilization strategies as they degrade the same substrate. Specifically, our findings suggest that Alternaria alternata SRC1lrK2f and Paraconiothyrium sporulosum AP3s5-JAC2a employ sequential enzyme secretion patterns concomitant with decreasing resource availability. Stagonospora sp. SRC1lsM3a preferentially degrades proteinaceous substrate before switching to carbohydrates, and Pyrenochaeta sp. DS3sAY3a utilizes primarily peptidases to aggressively attack carbon sources in a concentrated burst. In conclusion, this work highlights the diversity of operative metabolic strategies among understudied yet ubiquitous cellulose-degrading Ascomycetes, enhancing our understanding of their contribution to C turnover in the environment.« less

DNA-Templated Polymerization of Side-Chain-Functionalized Peptide Nucleic Acid Aldehydes

PubMed Central

Kleiner, Ralph E.; Brudno, Yevgeny; Birnbaum, Michael E.; Liu, David R.

2009-01-01

The DNA-templated polymerization of synthetic building blocks provides a potential route to the laboratory evolution of sequence-defined polymers with structures and properties not necessarily limited to those of natural biopolymers. We previously reported the efficient and sequence-specific DNA-templated polymerization of peptide nucleic acid (PNA) aldehydes. Here, we report the enzyme-free, DNA-templated polymerization of side-chain-functionalized PNA tetramer and pentamer aldehydes. We observed that the polymerization of tetramer and pentamer PNA building blocks with a single lysine-based side chain at various positions in the building block could proceed efficiently and sequence-specifically. In addition, DNA-templated polymerization also proceeded efficiently and in a sequence-specific manner with pentamer PNA aldehydes containing two or three lysine side chains in a single building block to generate more densely functionalized polymers. To further our understanding of side-chain compatibility and expand the capabilities of this system, we also examined the polymerization efficiencies of 20 pentamer building blocks each containing one of five different side-chain groups and four different side-chain regio- and stereochemistries. Polymerization reactions were efficient for all five different side-chain groups and for three of the four combinations of side-chain regio- and stereochemistries. Differences in the efficiency and initial rate of polymerization correlate with the apparent melting temperature of each building block, which is dependent on side-chain regio- and stereochemistry, but relatively insensitive to side-chain structure among the substrates tested. Our findings represent a significant step towards the evolution of sequence-defined synthetic polymers and also demonstrate that enzyme-free nucleic acid-templated polymerization can occur efficiently using substrates with a wide range of side-chain structures, functionalization positions within each building block, and functionalization densities. PMID:18341334

Quantitative iTRAQ-based secretome analysis reveals species-specific and temporal shifts in carbon utilization strategies among manganese(II)-oxidizing Ascomycete fungi

DOE PAGES

Zeiner, Carolyn A.; Purvine, Samuel O.; Zink, Erika M.; ...

2017-07-01

Fungi generate a wide range of extracellular hydrolytic and oxidative enzymes and reactive metabolites, collectively known as the secretome, that synergistically drive plant litter decomposition in the environment. While secretome studies of model organisms have greatly expanded our knowledge of these enzymes, few have extended secretome characterization to environmental isolates, particularly filamentous Ascomycetes, or directly compared temporal patterns of enzyme utilization among diverse species. Thus, the mechanisms of carbon (C) degradation by many ubiquitous soil fungi remain poorly understood. Here in this study we use a combination of iTRAQ proteomics and extracellular enzyme activity assays to compare the protein compositionmore » of the secretomes of four manganese(II)-oxidizing Ascomycete fungi over a three-week time course. We demonstrate that the fungi exhibit striking differences in the regulation of extracellular lignocellulose-degrading enzymes among species and over time, revealing species-specific and temporal shifts in C utilization strategies as they degrade the same substrate. Specifically, our findings suggest that Alternaria alternata SRC1lrK2f and Paraconiothyrium sporulosum AP3s5-JAC2a employ sequential enzyme secretion patterns concomitant with decreasing resource availability. Stagonospora sp. SRC1lsM3a preferentially degrades proteinaceous substrate before switching to carbohydrates, and Pyrenochaeta sp. DS3sAY3a utilizes primarily peptidases to aggressively attack carbon sources in a concentrated burst. In conclusion, this work highlights the diversity of operative metabolic strategies among understudied yet ubiquitous cellulose-degrading Ascomycetes, enhancing our understanding of their contribution to C turnover in the environment.« less

Dental Enamel Development: Proteinases and Their Enamel Matrix Substrates

PubMed Central

Bartlett, John D.

2013-01-01

This review focuses on recent discoveries and delves in detail about what is known about each of the proteins (amelogenin, ameloblastin, and enamelin) and proteinases (matrix metalloproteinase-20 and kallikrein-related peptidase-4) that are secreted into the enamel matrix. After an overview of enamel development, this review focuses on these enamel proteins by describing their nomenclature, tissue expression, functions, proteinase activation, and proteinase substrate specificity. These proteins and their respective null mice and human mutations are also evaluated to shed light on the mechanisms that cause nonsyndromic enamel malformations termed amelogenesis imperfecta. Pertinent controversies are addressed. For example, do any of these proteins have a critical function in addition to their role in enamel development? Does amelogenin initiate crystallite growth, does it inhibit crystallite growth in width and thickness, or does it do neither? Detailed examination of the null mouse literature provides unmistakable clues and/or answers to these questions, and this data is thoroughly analyzed. Striking conclusions from this analysis reveal that widely held paradigms of enamel formation are inadequate. The final section of this review weaves the recent data into a plausible new mechanism by which these enamel matrix proteins support and promote enamel development. PMID:24159389

Human Viperin Causes Radical SAM-Dependent Elongation of Escherichia coli, Hinting at Its Physiological Role.

PubMed

Nelp, Micah T; Young, Anthony P; Stepanski, Branden M; Bandarian, Vahe

2017-08-01

Viperin (virus inhibitory protein, endoplasmic reticulum-associated, interferon-inducible) is a widely distributed protein that is expressed in response to infection and causes antiviral effects against a broad spectrum of viruses. Viperin is a member of the radical S-adenosyl-l-methionine (SAM) superfamily of enzymes, which typically employ a 4Fe-4S cluster to reductively cleave SAM to initiate chemistry. Though the specific reaction catalyzed by viperin remains unknown, it has been shown that expression of viperin causes an increase in the fluidity of lipid membranes, which impedes the budding of nascent viral particles from the membrane inhibiting propagation of the infection. Herein, we show that expression of the human viperin homologue induces a dramatically elongated morphology of the host Escherichia coli cells. Mutation of an essential cysteine that coordinates the radical SAM cluster abrogates this effect. Thus, the native radical SAM activity of viperin is likely occurring in the host bacteria, indicating the elusive substrate is shared between both bacteria and humans, significantly narrowing the range of potential candidate substrates and providing a convenient bacterial platform from which future studies can occur.

Molecular pathways: targeting p21-activated kinase 1 signaling in cancer--opportunities, challenges, and limitations.

PubMed

Eswaran, Jeyanthy; Li, Da-Qiang; Shah, Anil; Kumar, Rakesh

2012-07-15

The evolution of cancer cells involves deregulation of highly regulated fundamental pathways that are central to normal cellular architecture and functions. p21-activated kinase 1 (PAK1) was initially identified as a downstream effector of the GTPases Rac and Cdc42. Subsequent studies uncovered a variety of new functions for this kinase in growth factor and steroid receptor signaling, cytoskeleton remodeling, cell survival, oncogenic transformation, and gene transcription, largely through systematic discovery of its direct, physiologically relevant substrates. PAK1 is widely upregulated in several human cancers, such as hormone-dependent cancer, and is intimately linked to tumor progression and therapeutic resistance. These exciting developments combined with the kinase-independent role of PAK1-centered phenotypic signaling in cancer cells elevated PAK1 as an attractive drug target. Structural and biochemical studies revealed the precise mechanism of PAK1 activation, offering the possibility to develop PAK1-targeted cancer therapeutic approaches. In addition, emerging reports suggest the potential of PAK1 and its specific phosphorylated substrates as cancer prognostic markers. Here, we summarize recent findings about the PAK1 molecular pathways in human cancer and discuss the current status of PAK1-targeted anticancer therapies.

Correlation of live-cell imaging with volume scanning electron microscopy.

PubMed

Lucas, Miriam S; Günthert, Maja; Bittermann, Anne Greet; de Marco, Alex; Wepf, Roger

2017-01-01

Live-cell imaging is one of the most widely applied methods in live science. Here we describe two setups for live-cell imaging, which can easily be combined with volume SEM for correlative studies. The first procedure applies cell culture dishes with a gridded glass support, which can be used for any light microscopy modality. The second approach is a flow-chamber setup based on Ibidi μ-slides. Both live-cell imaging strategies can be followed up with serial blockface- or focused ion beam-scanning electron microscopy. Two types of resin embedding after heavy metal staining and dehydration are presented making best use of the particular advantages of each imaging modality: classical en-bloc embedding and thin-layer plastification. The latter can be used only for focused ion beam-scanning electron microscopy, but is advantageous for studying cell-interactions with specific substrates, or when the substrate cannot be removed. En-bloc embedding has diverse applications and can be applied for both described volume scanning electron microscopy techniques. Finally, strategies for relocating the cell of interest are discussed for both embedding approaches and in respect to the applied light and scanning electron microscopy methods. Copyright © 2017 Elsevier Inc. All rights reserved.

Engineering and Applications of fungal laccases for organic synthesis

PubMed Central

Kunamneni, Adinarayana; Camarero, Susana; García-Burgos, Carlos; Plou, Francisco J; Ballesteros, Antonio; Alcalde, Miguel

2008-01-01

Laccases are multi-copper containing oxidases (EC 1.10.3.2), widely distributed in fungi, higher plants and bacteria. Laccase catalyses the oxidation of phenols, polyphenols and anilines by one-electron abstraction, with the concomitant reduction of oxygen to water in a four-electron transfer process. In the presence of small redox mediators, laccase offers a broader repertory of oxidations including non-phenolic substrates. Hence, fungal laccases are considered as ideal green catalysts of great biotechnological impact due to their few requirements (they only require air, and they produce water as the only by-product) and their broad substrate specificity, including direct bioelectrocatalysis. Thus, laccases and/or laccase-mediator systems find potential applications in bioremediation, paper pulp bleaching, finishing of textiles, bio-fuel cells and more. Significantly, laccases can be used in organic synthesis, as they can perform exquisite transformations ranging from the oxidation of functional groups to the heteromolecular coupling for production of new antibiotics derivatives, or the catalysis of key steps in the synthesis of complex natural products. In this review, the application of fungal laccases and their engineering by rational design and directed evolution for organic synthesis purposes are discussed. PMID:19019256