scholarly journals Molecular characterization of CHAD domains as inorganic polyphosphate-binding modules

2019 ◽  
Vol 2 (3) ◽  
pp. e201900385 ◽  
Author(s):  
Laura Lorenzo-Orts ◽  
Ulrich Hohmann ◽  
Jinsheng Zhu ◽  
Michael Hothorn
Keyword(s):  
Mutational Analysis ◽  
Dissociation Constants ◽  
Ricinus Communis ◽  
Complex Structure ◽  
Linear Polymers ◽  
Inorganic Polyphosphate ◽  
Metabolizing Enzymes ◽  
Surface Patches ◽  
Central Pore

Inorganic polyphosphates (polyPs) are linear polymers of orthophosphate units linked by phosphoanhydride bonds. Here, we report that bacterial, archaeal, and eukaryotic conserved histidine α-helical (CHAD) domains are specific polyP-binding modules. Crystal structures reveal that CHAD domains are formed by two four-helix bundles, giving rise to a central pore surrounded by conserved basic surface patches. Different CHAD domains bind polyPs with dissociation constants ranging from the nano- to mid-micromolar range, but not nucleic acids. A CHAD—polyP complex structure reveals the phosphate polymer binding across the central pore and along the two basic patches. Mutational analysis of CHAD—polyP interface residues validates the complex structure. The presence of a CHAD domain in the polyPase ygiF enhances its enzymatic activity. The only known CHAD protein from the plant Ricinus communis localizes to the nucleus/nucleolus when expressed in Arabidopsis and tobacco, suggesting that plants may harbor polyPs in these compartments. We propose that CHAD domains may be used to engineer the properties of polyP-metabolizing enzymes and to specifically localize polyP stores in eukaryotic cells and tissues.

scholarly journals Molecular characterization of CHAD domains as inorganic polyphosphate binding modules

2019 ◽  
Author(s):  
Laura Lorenzo-Orts ◽  
Ulrich Hohmann ◽  
Jinsheng Zhu ◽  
Michael Hothorn
Keyword(s):  
Mutational Analysis ◽  
Dissociation Constants ◽  
Complex Structure ◽  
High Specificity ◽  
Eukaryotic Cells ◽  
Inorganic Polyphosphates ◽  
Inorganic Polyphosphate ◽  
Surface Patches ◽  
C Terminus

AbstractInorganic polyphosphates (polyPs) are long polymers of orthophosphate units (Pi), linked by energy-rich phosphoanhydride bonds. Conserved histidine α-helical (CHAD) domains of unknown biochemical function are often located at the C-terminus of polyP-metabolizing triphosphate tunnel metalloenzymes (TTMs), or can be found as stand-alone proteins in bacterial operons harboring polyP kinases or phosphatases. Here we report that bacterial, archaeal and eukaryotic CHAD domains are specific polyP binding modules. Crystal structures reveal that CHAD domains are formed by two four-helix bundles, giving rise to a central cavity surrounded by two conserved basic surface patches. Different CHAD domains bind polyPs with dissociation constants ranging from the nano-to mid-micromolar range, but not DNA or other Pi-containing ligands. A 2.1 Å CHAD - polyP complex structure reveals the phosphate polymer binding across a central pore and along the two basic patches. Mutational analysis of CHAD – polyP interface residues validates the complex structure and reveals that CHAD domains evolved to bind long-chain polyPs. The presence of a CHAD domain in the polyPase ygiF enhances its enzymatic activity. In plants, CHAD domains bind polyP in vivo and localize to the nucleus and nucleolus, suggesting that plants harbor polyP stores in these compartments. We propose that CHAD domains may be used to engineer the properties of polyP-metabolizing enzymes and to specifically localize polyP stores in eukaryotic cells and tissues.SignificanceA domain of unknown function termed CHAD, present in all kingdoms of life, is characterized as a specific inorganic polyphosphate binding domain. The small size of the domain and its high specificity for inorganic polyphosphates suggest that it could be used as a tool to locate inorganic polyphosphate stores in pro- and eukaryotic cells and tissues.

Characterization of the glycoconjugates of boar testis and epididymis

Reproduction ◽  
2000 ◽  
pp. 325-335 ◽  
Author(s):  
A Calvo ◽  
LM Pastor ◽  
S Bonet ◽  
E Pinart ◽  
M Ventura
Keyword(s):  
Ricinus Communis ◽  
Lectin Histochemistry ◽  
Apical Part ◽  
Seminiferous Tubules ◽  
In Situ Characterization ◽  
Intense Staining ◽  
Terminal Galactose ◽  
Boar Spermatozoa

Lectin histochemistry was used to perform in situ characterization of the glycoconjugates present in boar testis and epididymis. Thirteen horseradish peroxidase- or digoxigenin-labelled lectins were used in samples obtained from healthy fertile boars. The acrosomes of the spermatids were stained intensely by lectins with affinity for galactose and N-acetyl-galactosamine residues, these being soybean, peanut and Ricinus communis agglutinins. Sertoli cells were stained selectively by Maackia ammurensis agglutinin. The lamina propria of seminiferous tubules showed the most intense staining with fucose-binding lectins. The Golgi area and the apical part of the principal cells of the epididymis were stained intensely with many lectins and their distribution was similar in the three zones of the epididymis. On the basis of lectin affinity, both testis and epididymis appear to have N- and O-linked glycoconjugates. Spermatozoa from different epididymal regions showed different expression of terminal galactose and N-acetyl-galactosamine. Sialic acid (specifically alpha2,3 neuraminic-5 acid) was probably incorporated into spermatozoa along the extratesticular ducts. These findings indicate that the development and maturation of boar spermatozoa are accompanied by changes in glycoconjugates. As some lectins stain cellular or extracellular compartments specifically, these lectins could be useful markers in histopathological evaluation of diseases of boar testis and epididymis.

scholarly journals Biodiesel Production from Melia azedarach and Ricinus communis Oil by Transesterification Process

Catalysts ◽  
2020 ◽  
Vol 10 (4) ◽  
pp. 427 ◽  
Author(s):  
Muhammad Awais ◽  
Sa’ed A Musmar ◽  
Faryal Kabir ◽  
Iram Batool ◽  
Muhammad Asif Rasheed ◽  
...  
Keyword(s):  
Ricinus Communis ◽  
Cost Effective ◽  
Biodiesel Production ◽  
Molar Ratio ◽  
Melia Azedarach ◽  
Renewable Fuel ◽  
Animal Fats ◽  
Edible Crops ◽  
American Society

Biodiesel is a renewable fuel usually produced from vegetable oils and animal fats. This study investigates the extraction of oil and its conversion into biodiesel by base-catalyzed transesterification. Firstly, the effect of various solvents (methanol, n-hexane, chloroform, di-ethyl ether) on extraction of oil from non-edible crops, such as R. communis and M. azedarach, were examined. It was observed that a higher concentration of oil was obtained from R. communis (43.6%) as compared to M. azedarach (35.6%) by using methanol and n-hexane, respectively. The extracted oils were subjected to NaOH (1%) catalyzed transesterification by analyzing the effect of oil/methanol molar ratio (1:4, 1:6, 1:8 and 1:10) and varying temperature (20, 40, 60 and 80 °C) for 2.5 h of reaction time. M. azedarach yielded 88% and R. communis yielded 93% biodiesel in 1:6 and 1:8 molar concentrations at ambient temperature whereas, 60 °C was selected as an optimum temperature, giving 90% (M. azedarach) and 94% (R. communis) biodiesel. The extracted oil and biodiesel were characterized for various parameters and most of the properties fulfilled the American Society for Testing and Materials (ASTM) standard biodiesel. The further characterization of fatty acids was done by Gas Chromatography/Mass Spectrometer (GC/MS) and oleic acid was found to be dominant in M. azedarach (61.5%) and R. communis contained ricinoleic acid (75.53%). Furthermore, the functional groups were analyzed by Fourier Transform Infrared Spectroscopy. The results suggested that both of the oils are easily available and can be used for commercial biodiesel production at a cost-effective scale.

scholarly journals KCTD proteins as Cullin3 E3 Ligase adapters

2014 ◽  
Vol 70 (a1) ◽  
pp. C305-C305
Author(s):  
Alan Ji ◽  
Gilbert Privé
Keyword(s):  
Crystal Structure ◽  
Structural Characterization ◽  
Dissociation Constants ◽  
E3 Ligase ◽  
Terminal Region ◽  
Btb Domain ◽  
Substrate Protein ◽  
Ubiquitin E3 Ligase ◽  
Ubiquitin Moiety

Cullin3 (Cul3) is an ubiquitin E3 ligase responsible for catalyzing the transfer of an ubiquitin moiety from an E2 enzyme to a target substrate protein. The C-terminal region of Cul3 binds RBX1/E2-ubiquitin, while, the N-terminal region interacts with various BTB domain proteins which serve as substrate adaptors. Previously, our group determined the crystal structures of the homodimeric BTB proteins SPOP and KLHL3 in complex with the N-terminal domain of Cul3, revealing the determinants responsible for the BTB/Cul3 interaction [1, 2]. A second class of BTB-domain containing proteins, the KCTD proteins, are also Cul3 substrate adaptors but these do not share many of the previously determined features for Cul3 binding. Furthermore, KCTD proteins form homotetramers and homopentamers via BTB oligomerization rather than the previously described homodimers. Despite these differences, many KCTD proteins interact with Cul3 with dissociation constants of approximately 50 nM. While the target substrates for many of the KCTD/Cul3 E3 ligase complexes are unknown, recent studies have implicated the GABAβ2 receptor as an interactor of KCTD 8, 12, 12b and 16. Here, we report the pentameric crystal structure of the KCTD9 BTB domain and our progress on the structural characterization of Cul3/KCTD/substrate complexes.

Further specificity characterization of von Willebrand factor inhibitors developed in two patients with severe von Willebrand disease

Blood ◽  
1988 ◽  
Vol 72 (1) ◽  
pp. 116-120 ◽  
Author(s):  
MF Lopez-Fernandez ◽  
R Martin ◽  
C Lopez-Berges ◽  
F Ramos ◽  
N Bosch ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Complex Structure ◽  
Relative Proportion ◽  
Von Willebrand Disease ◽  
Rabbit Antibody ◽  
Human Factor Viii ◽  
Rabbit Polyclonal Antibody ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Circulating inhibitors against von Willebrand factor (vWF) that show the properties of heterologous IgG antibodies have been described in a few patients with severe von Willebrand disease (vWD). The present study provides further characterization of inhibitors from two patients with severe vWD. Inhibitors in both, like polyclonal rabbit antibody, detected all sizes of multimers and the complex structure of each multimer from platelets and plasma of normal individuals as well as from plasma of patients with IIA, IIB, and IIC vWD. Both inhibitors and the rabbit antibody reacted mainly with the intact 225-Kd vWF subunit and the 189-H and 140-Kd fragments in contrast to monoclonal antibodies specific for vWF fragments that detected a higher relative proportion of 176-Kd fragment. Furthermore, all these antibodies recognized fragment III, although one inhibitor and rabbit polyclonal antibody reacted poorly and the other inhibitor did not react at all with reduced fragment II of vWF digested with Staphylococcus aureus V-8 protease. These data suggest that although human inhibitors from severe vWD patients may behave, to some extent, as polyclonal heterologous antibodies against native vWF, the former show striking differences in their target specificity as well as a much broader specificity than that described for human factor VIII inhibitors.

Structure of Polybutadienes

1967 ◽  
Vol 40 (5) ◽  
pp. 1529-1543 ◽  
Author(s):  
W. S. Bahary ◽  
D. I. Sapper
Keyword(s):  
Light Scattering ◽  
Molecular Size ◽  
Structural Features ◽  
Melting Point Depression ◽  
Linear Polymers ◽  
Long Chain Branching ◽  
Molecular Weights ◽  
Catalyst Systems ◽  
Molecular Size Distribution

Abstract Polybutadienes made with six different catalyst systems were examined: (1) butyllithium, (2) “nickel-based”, (3) alfin, (4) “titanium-based”, (5) “cobalt-based”, and (6) free radical emulsion. The microstructure and macrostructure of the polybutadienes have been determined and are compared to the results published in the literature. These polybutadienes may be considered to be random terpolymers of cis, trans, and vinyl addition of butadiene. The glass transition temperature is specified by the vinyl content. The crystalline melting points of the high trans and also the high cis polybutadienes obey to a high measure Flory's equation for melting point depression of a random terpolymer. The molecular weights of the polybutadienes have been determined by light scattering and osmometry and the degree of long chain branching has been determined by the branching index, 〈g′〉. The macro-structural features of the linear polymers are confirmed by fractionation. However, the polydispersities calculated from fractionation data do not agree with those determined from light scattering and osmometry for the branched samples. The discrepancy is attributed to the method of characterization of the fractions. A distinction is made between molecular weight distribution and molecular size distribution.

scholarly journals Melliferous flora and pollen characterization of honey samples of Apis mellifera L., 1758 in apiaries in the counties of Ubiratã and Nova Aurora, PR

2013 ◽  
Vol 85 (1) ◽  
pp. 307-326 ◽  
Author(s):  
ELIZABETE S. SEKINE ◽  
VAGNER A.A. TOLEDO ◽  
MARCELO G. CAXAMBU ◽  
SUZANE CHMURA ◽  
ELIZA H. TAKASHIBA ◽  
...  
Keyword(s):  
Glycine Max ◽  
Apis Mellifera ◽  
Native Species ◽  
Ricinus Communis ◽  
Cultivated Plants ◽  
Melia Azedarach ◽  
Native Vegetation ◽  
Pollen Types ◽  
Pollen Analyses

The aim of this study was to carry out a survey of the flora with potential for beekeeping in the counties of Ubiratã and Nova Aurora-PR through the collection of plants and pollen analyses in honey samples collected monthly. 208 species of plants were recorded, distributed in 66 families. The families that showed the major richness of pollen types were: Asteraceae, Myrtaceae and Solanaceae. Approximately 80 pollen types were found in honey samples, most of them were characterized as heterofloral. Cultivated plants, such as Glycine max (soybean) and Eucalyptus spp., were representative in some months of the year. Exotic species, such as Ricinus communis and Melia azedarach, were also frequent. However, over than 50% of the pollen types belong to native species of the region, such as Schinus terebinthifolius, Baccharis spp. Alchornea triplinervia, Parapiptadenia rigida, Hexaclamys edulis, Zanthoxylum sp. and Serjania spp., indicating the importance of the native vegetation for the survival of the colonies.

scholarly journals Sequencing of the Canine Cytochrome P450 CYP2C41 Gene and Genotyping of Its Polymorphic Occurrence in 36 Dog Breeds

2021 ◽  
Vol 8 ◽  
Author(s):  
Emre Karakus ◽  
Clarissa Prinzinger ◽  
Silke Leiting ◽  
Joachim Geyer
Keyword(s):  
Cytochrome P450 ◽  
Drug Metabolism ◽  
Gene Deletion ◽  
Homologous Sequence ◽  
Pharmacokinetic Studies ◽  
Metabolizing Enzymes ◽  
Genomic Level ◽  
Very High

Cytochrome P450 (CYP) drug metabolizing enzymes play an important role in efficient drug metabolism and elimination. Many CYPs are polymorphic and, thereby, drug metabolism can vary between individuals. In the case of canine CYP2C41, gene polymorphism was identified. However, as the first available canine genome sequences all were CYP2C41 negative, this polymorphism could not be clarified at the genomic level. The present study provides an exact characterization of the CYP2C41 gene deletion polymorphism at the genomic level and presents a PCR-based genotyping method that was used for CYP2C41 genotyping of 1,089 individual subjects from 36 different dog breeds. None of the Bearded Collie, Bernese Mountain, Boxer, Briard, French Bulldog or Irish Wolfhound subjects had the CYP2C41 gene in their genomes. In contrast, in the Chinese Char-Pei, Siberian Husky, Schapendoes and Kangal breeds, the CYP2C41 allele frequency was very high, with values of 67, 57, 43, and 34%, respectively. Interestingly, the site of gene deletion was identical for all CYP2C41 negative dogs, and all CYP2C41 positive dogs showed highly homologous sequence domains upstream and downstream from the CYP2C41 gene. CYP2C41 genotyping can now be routinely used in future pharmacokinetic studies in canines, in order to identify genetically-based poor or extensive drug metabolizers. This, together with more extensive in vitro drug screening for CYP2C41 substrates will help to determine the clinical relevance of CYP2C41, and to optimize drug treatment. Although the relative abundance of the CYP2C41 protein in the canine liver seems to not be very high, this CYP could substantially contribute to hepatic drug metabolism in dogs expressing CYP2C41 from both alleles and, when CYP2C41 shows higher catalytic activity to a given drug than other hepatic metabolic enzymes.

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