scholarly journals Origins of the difference in Ca2+ requirement for activation of μ- and m-calpain

2002 ◽  
Vol 367 (1) ◽  
pp. 263-269 ◽  
Author(s):  
Previn DUTT ◽  
Cherie N. SPRIGGS ◽  
Peter L. DAVIES ◽  
Zongchao JIA ◽  
John S. ELCE
Keyword(s):  
Large Subunit ◽  
Cysteine Proteases ◽  
Intramolecular Interactions ◽  
Global Response ◽  
X Ray ◽  
Kd Values ◽  
Ef Hand ◽  
The Difference ◽  
Type Sequence

The μ- and m-calpains are closely related Ca2+-dependent cysteine proteases having different in vitro Ca2+ requirements (Kd), of approx. 25 and 325μM respectively. The two isoforms are heterodimers of slightly different large (80kDa) subunits and an identical small (28kDa) subunit, so that the difference in Kd values must reside in the large subunits. As assayed here, these Kd values relate to the Ca2+ required for the first phase of calpain activation and do not reflect the lower Ca2+ then required by fully activated calpain. On the basis of sequence comparison and the X-ray structure of m-calpain, many m-type residues in the C-terminal EF-hand-containing domain IV were converted into the corresponding μ-type residues, but these mutations did not produce the expected decrease in Kd. In a series of hybrid (μ/m) large-subunit calpains, the Kd values decreased progressively towards that of μ-calpain as the proportion of μ-type sequence increased from 0 to 90%. Kd values cannot therefore be ascribed to one or a few specific intramolecular interactions, but reflect the global response of the whole molecule to Ca2+ binding. Nonetheless, 25% of the difference in Kd values between μ- and m-calpain can be ascribed to the N-terminal peptide of the large subunit, whereas the C-terminal EF-hand-containing domain IV accounts for 65% of the difference.

Is intervertebral disc degeneration related to segmental instability? An evaluation with two different grading systems based on clinical imaging

2017 ◽  
Vol 59 (3) ◽  
pp. 327-335 ◽  
Author(s):  
David Volkheimer ◽  
Fabio Galbusera ◽  
Christian Liebsch ◽  
Sabine Schlegel ◽  
Friederike Rohlmann ◽  
...  
Keyword(s):  
Intervertebral Disc Degeneration ◽  
Statistical Significance ◽  
Axial Rotation ◽  
Spinal Motion ◽  
X Ray ◽  
Grading Systems ◽  
Flexion Extension ◽  
The Difference ◽  
Magnetic Resonance Imaging Mri

Background Several in vitro studies investigated how degeneration affects spinal motion. However, no consensus has emerged from these studies. Purpose To investigate how degeneration grading systems influence the kinematic output of spinal specimens. Material and Methods Flexibility testing was performed with ten human T12-S1 specimens. Degeneration was graded using two different classifications, one based on X-ray and the other one on magnetic resonance imaging (MRI). Intersegmental rotation (expressed by range of motion [ROM] and neutral zone [NZ]) was determined in all principal motion directions. Further, shear translation was measured during flexion/extension motion. Results The X-ray grading system yielded systematically lesser degeneration. In flexion/extension, only small differences in ROM and NZ were found between moderately degenerated motion segments, with only NZ for the MRI grading reaching statistical significance. In axial rotation, a significant increase in NZ for moderately degenerated segments was found for both grading systems, whereas the difference in ROM was significant only for the MRI scheme. Generally, the relative increases were more pronounced for the MRI classification compared to the X-ray grading scheme. In lateral bending, only relatively small differences between the degeneration groups were found. When evaluating shear translations, a non-significant increase was found for moderately degenerated segments. Motion segment segments tended to regain stability as degeneration progressed without reaching the level of statistical significance. Conclusion We found a fair agreement between the grading schemes which, nonetheless, yielded similar degeneration-related effects on intersegmental kinematics. However, as the trends were more pronounced using the Pfirrmann classification, this grading scheme appears superior for degeneration assessment.

Two new zinc(II) and mercury(II) complexes based on N,N′-(cyclohexane-1,2-diylidene)bis(4-fluorobenzohydrazide): synthesis, crystal structures and antibacterial activities

2020 ◽  
Vol 76 (5) ◽  
pp. 476-482
Author(s):  
Al-Ameen Bariz OmarAli ◽  
Ahmed Jasim M. Al-Karawi ◽  
Adil A. Awad ◽  
Necmi Dege ◽  
Sevgi Kansız ◽  
...  
Keyword(s):  
Organic Ligand ◽  
Antibacterial Activities ◽  
Structural Features ◽  
Bacterial Strains ◽  
Tetrahedral Geometry ◽  
X Ray ◽  
X Ray Crystallography ◽  
Growth Inhibitory ◽  
The Difference

Reaction of N,N′-(cyclohexane-1,2-diylidene)bis(4-fluorobenzohydrazide), C20H18F2N4O2, (LF ), with zinc chloride and mercury(II) chloride produced different types and shapes of neutral coordination complexes, namely, dichlorido[N,N′-(cyclohexane-1,2-diylidene)bis(4-fluorobenzohydrazide)-κ2 N,O]zinc(II), [ZnCl2(C20H18F2N4O2)], (1), and dichlorido[N,N′-(cyclohexane-1,2-diylidene)bis(4-fluorobenzohydrazide)-κ4 O,N,N′,O′]mercury(II), [HgCl2(C20H18F2N4O2)], (2). The organic ligand and its metal complexes are characterized using various techniques: IR, UV–Vis and nuclear magnetic resonance (NMR) spectroscopies, in addition to powder X-ray diffraction (PXRD), single-crystal X-ray crystallography and microelemental analysis. Depending upon the data from these analyses and measurements, a typical tetrahedral geometry was confirmed for zinc complex (1), in which the ZnII atom is located outside the bis(benzhydrazone) core. The HgII atom in (2) is found within the core and has a common octahedral structure. The in vitro antibacterial activities of the prepared compounds were evaluated against two different bacterial strains, i.e. gram positive Bacillus subtilis and gram negative Pseudomonas aeruginosa bacteria. The prepared compounds exhibited differentiated growth-inhibitory activities against these two bacterial strains based on the difference in their lipophilic nature and structural features.

scholarly journals Multiple interactions of the ‘transducer’ govern its function in calpain activation by Ca2+

2005 ◽  
Vol 388 (3) ◽  
pp. 741-744 ◽  
Author(s):  
Zoltán BOZÓKY ◽  
Anita ALEXA ◽  
Peter TOMPA ◽  
Peter FRIEDRICH
Keyword(s):  
Structural Changes ◽  
Catalytic Domain ◽  
Intramolecular Interactions ◽  
Site Directed Mutagenesis ◽  
Structural Element ◽  
X Ray ◽  
Calpain Activation ◽  
Ef Hand ◽  
Domain Iii ◽  
Multiple Interactions

Typical calpains in mammals become activated on binding of 8–12 Ca2+ ions per enzyme molecule, giving an example of integrated, manifold regulation by calcium. Besides two identified Ca2+ sites in catalytic domain II and several EF-hand motifs in domains IV and VI, an acidic loop in the centrally positioned domain III seems to harbour Ca2+. The mediator of distant Ca2+-induced structural transitions is an elongated structural element, the ‘transducer’. By site-directed mutagenesis along the transducer, we have generated various forms of rat m-calpain in which critical intramolecular interactions, as judged from the X-ray structure, would be abolished or modified. The kinetic parameters of these mutant enzymes support a model featuring shrinkage of transducer as a contributor to structural changes involved in calpain activation.

scholarly journals Structural analysis of new ligands against Schistosoma mansoni

2014 ◽  
Vol 70 (a1) ◽  
pp. C972-C972
Author(s):  
Ana Mafud ◽  
Yvonne Mascarenhas
Keyword(s):  
Structural Analysis ◽  
Dihedral Angle ◽  
Density Functional ◽  
Augmented Lagrangian Method ◽  
Anthelmintic Activity ◽  
Density Functional Theory Calculations ◽  
Intramolecular Interactions ◽  
X Ray

Natural compounds have been an alternative to treat several diseases. In this sense, epiisopiloturine (EPI) [1] is an alkaloid found in Pilocarpus microphyllus (Rutaceae) leaves, with anthelmintic activity in vitro [2] and in vivo (To be publish). Unfortunately, EPI is slightly soluble in water. To solve this question, complexes and new derivatives were synthetized. Here, we present a x-ray structural analysis of the compounds: EPICu (CCDC 947608), C64H42N8O23Cl2Cu, P21, and EPIZn (CCDC 959718) C32H36N4O6Cl2Zn, P212121, both complexes with anthelmintic activity; EpiiHCl (CCDC 945616), C16H18N2O3Cl, P21, a derivative with reduce anthelmintic activity even high solubility; and epiisopilosine, EPIss C16H18N2O3, (CCDC 957103), P212121, a new active compound found in the same leaves but seasonally. The ligands conformational analysis were determined using Density Functional Theory calculations with the operator correlation of Lee–Yang–Parr (B3LYP), 6-31G++ base set, implementing two functions of polarization (d,p), to obtain the molecular electrostatic potential map. X-ray powder diffraction and thermogravimetry analysis were also performed to EPIss. The dihedral angle between the benzene and imidazole rings is feature common to such compounds, which range from 3 to 73 degrees. It is interesting to observe that the dihedral angle may be associated with the activity of these compounds, as well as other factors, since the smaller the dihedral angle, the activity is increased, even taking into consideration the EPI to this study. To compare the structures of the ligands, they were aligned and the overlay optimizer was the augmented Lagrangian method (local, no-derivative), shown in Figure 1, EPICl in green, EPI in blue and EPIss in yellow. Results showed that hydrophobic regions are conservative except for EPIss, due to its larger volume. And it may be understood through the occurrence of the CH/pi intramolecular interactions in the crystal stacking, which guarantee the EPIss larger globularity. Experimental parameters for TGA curve indicates that EPIss degradation occurs in two steps, associated with an endothermic DTA signal. XRD were performed in order to exclude the occurrence of isoforms in the crystalline powder.

Intermolecular interaction energies and molecular conformations in N-substituted 4-aryl-2-methylimidazoles with promising in vitro antifungal activity

2019 ◽  
Vol 75 (6) ◽  
pp. 1197-1207 ◽  
Author(s):  
Nerith-Rocio Elejalde ◽  
Estefanía Butassi ◽  
Susana Zacchino ◽  
Mario A. Macías ◽  
Jaime Portilla
Keyword(s):  
Antifungal Activity ◽  
High Resolution Mass Spectrometry ◽  
X Ray Diffraction ◽  
One Pot ◽  
Molecular Conformations ◽  
X Ray ◽  
The Difference ◽  
Molecule Interactions ◽  
In Vitro Antifungal Activity

A convenient one-pot synthesis of 4-aryl-2-methyl-N-phenacylimidazoles (4) through a microwave-assisted pseudo-tricomponent reaction of α-bromoacetophenones (1) with acetamidine hydrochloride (2) is reported. Ketones (4) were successfully used as substrates for the preparation of the respective N-(2-hydroxyethyl)imidazoles (5) with yields up to 87%. The synthesized compounds were characterized by NMR and high-resolution mass spectrometry analyses, and several structures were confirmed and studied by single-crystal X-ray diffraction. The analysis of the whole-of-molecule interactions shows that, despite the difference in the atom–atom contacts forming the crystals, dispersion energies make the largest contribution to the formation of the solids, giving an isotropic tendency in the topology of the energy framework diagrams for pairs of molecules. In addition, the in vitro antifungal activity of both families of compounds [ketones (4) and alcohols (5)] against Candida albicans and Cryptococcus neoformans was evaluated, where the 2,4-dichlorophenyl-substituted alcohol (5f), an isomer of the drug miconazole, showed the highest activity (IC50 = 7.8 µg ml−1 against C. neoformans).

scholarly journals Calpain-mediated protein targets in cardiac mitochondria following ischemia–reperfusion

2022 ◽  
Vol 12 (1) ◽  
Author(s):  
Ling Li ◽  
Jeremy Thompson ◽  
Ying Hu ◽  
Edward J. Lesnefsky ◽  
Belinda Willard ◽  
...  
Keyword(s):  
Mitochondrial Function ◽  
Mitochondrial Permeability Transition ◽  
Ischemia Reperfusion ◽  
Large Subunit ◽  
Cardiac Injury ◽  
Cysteine Proteases ◽  
Complex Iii ◽  
Regulatory Subunit ◽  
Protein Targets

AbstractCalpain 1 and 2 (CPN1/2) are calcium-dependent cysteine proteases that exist in cytosol and mitochondria. Pharmacologic inhibition of CPN1/2 decreases cardiac injury during ischemia (ISC)–reperfusion (REP) by improving mitochondrial function. However, the protein targets of CPN1/2 activation during ISC–REP are unclear. CPN1/2 include a large subunit and a small regulatory subunit 1 (CPNS1). Genetic deletion of CPNS1 eliminates the activities of both CPN1 and CPN2. Conditional cardiomyocyte specific CPNS1 deletion mice were used in the present study to clarify the role of CPN1/2 activation in mitochondrial damage during ISC–REP with an emphasis on identifying the potential protein targets of CPN1/2. Isolated hearts from wild type (WT) or CPNS1 deletion mice underwent 25 min in vitro global ISC and 30 min REP. Deletion of CPNS1 led to decreased cytosolic and mitochondrial calpain 1 activation compared to WT. Cardiac injury was decreased in CPNS1 deletion mice following ISC–REP as shown by the decreased infarct size compared to WT. Compared to WT, mitochondrial function was improved in CPNS1 deletion mice following ischemia–reperfusion as shown by the improved oxidative phosphorylation and decreased susceptibility to mitochondrial permeability transition pore opening. H2O2 generation was also decreased in mitochondria from deletion mice following ISC–REP compared to WT. Deletion of CPNS1 also resulted in less cytochrome c and truncated apoptosis inducing factor (tAIF) release from mitochondria. Proteomic analysis of the isolated mitochondria showed that deletion of CPNS1 increased the content of proteins functioning in regulation of mitochondrial calcium homeostasis (paraplegin and sarcalumenin) and complex III activity. These results suggest that activation of CPN1 increases cardiac injury during ischemia–reperfusion by impairing mitochondrial function and triggering cytochrome c and tAIF release from mitochondria into cytosol.

scholarly journals Roles of individual EF-hands in the activation of m-calpain by calcium

2000 ◽  
Vol 348 (1) ◽  
pp. 37-43 ◽  
Author(s):  
Previn DUTT ◽  
J. C. Simon ARTHUR ◽  
Pawel GROCHULSKI ◽  
Miroslaw CYGLER ◽  
John S. ELCE
Keyword(s):  
Driving Force ◽  
Specific Activity ◽  
Small Subunit ◽  
Wild Type ◽  
X Ray ◽  
X Ray Crystallography ◽  
Kd Values ◽  
Ef Hand ◽  
Ef Hands ◽  
Made In

m-Calpain is a heterodimeric, cytosolic, thiol protease, which is activated by Ca2+-binding to EF-hands in the C-terminal domains of both subunits. There are four potential Ca2+-binding EF-hands in each subunit, but their relative affinities for Ca2+ are not known. In the present study mutations were made in both subunits to reduce the Ca2+-binding affinity at one or more EF-hands in one or both subunits. X-ray crystallography of some of the mutated small subunits showed that Ca2+ did not bind to the mutated EF-hands, but that its binding at other sites was not affected. The structures of the mutant small subunits in the presence of Ca2+ were otherwise identical to that of the Ca2+-bound wild-type small subunit. In the whole enzyme the wild-type macroscopic Ca2+ requirement (Kd) was approx. 350 μM. The mutations did not affect the maximum specific activity of the enzyme, but caused increases in Kd, which were characteristic of each site. All the EF-hands could be mutated in various combinations without loss of activity, but preservation of at least one wild-type EF-hand 3 sequence was required to maintain Kd values lower than 1 mM. The results suggest that all the EF-hands can contribute co-operatively to calpain activation, but that EF-hand 3, in both subunits, has the highest intrinsic affinity for Ca2+ and provides the major driving force for conformational change.

scholarly journals Precision of aiming with a portable X-ray device (Nomad Pro 2) compared to a wall-mounted device in intraoral radiography

2019 ◽  
Vol 48 (5) ◽  
pp. 20180221 ◽  
Author(s):  
Reinier C. Hoogeveen ◽  
Bram R. Meertens ◽  
W. Erwin R. Berkhout
Keyword(s):  
Mixed Model ◽  
Rank Test ◽  
Limiting Factor ◽  
Intraoral Radiography ◽  
X Ray ◽  
Safety Issues ◽  
Quality Aspects ◽  
Signed Rank Test ◽  
The Difference

Background: Hand-held (HH) X-ray devices are currently introduced for routine dentistry in some global areas. Safety issues are resolved but imaging quality aspects remain to be explored. Aim: To compare the aiming accuracy of two intraoral radiography devices: the portable Nomad Pro2 and the wall-mounted (WM) Planmeca Intra. Setting and design: In vitro experimentation involving radiologic mannequins and unbiased student operators. Methods and materials: 20 operators obtained intraoral radiographs of four regions (bitewing, upper molar, lower molar and upper anterior) in five mannequins, using HH and WM devices. Beam-aiming devices were fitted with metal cross-wires to project on image sensors. Deviation from ideal perpendicular incidence of beam was calculated, based on positions of cross-wires relative to gold-standard positions (i.e. average of 10-fold precise aiming by authors via WM system). Analytic models relied on Wilcoxon signed-rank test and mixed model analyses. Results: Mean deviations from perfect aim were 2.88˚ (± 1.80˚) for WM and 3.06˚ (± 1.90˚) for HH methods. The difference among all operators (HH vs WM) was 0.17˚ (± 2.48˚), which was not significant. Seven operators showed better aim by HH device (13 by WM system); and in one instance, this difference was significant. Conclusions: Aiming precision proved similar for HH and WM methods of intraoral radiography, although individual operators may perform better using one of these modalities. Aim is not an expected limiting factor for image quality in HH (vs WM) diagnostics.

A novel human small subunit of calpains

2002 ◽  
Vol 362 (2) ◽  
pp. 383-388 ◽  
Author(s):  
Éva SCHÁD ◽  
Attila FARKAS ◽  
Gáspár JÉKELY ◽  
Peter TOMPA ◽  
Peter FRIEDRICH
Keyword(s):  
Expressed Sequence Tag ◽  
Large Subunit ◽  
Cysteine Proteases ◽  
Small Subunit ◽  
Regulatory Subunit ◽  
Intronless Gene ◽  
Functional Equivalent

Typical calpains are heterodimeric cysteine proteases which have distinct large catalytic subunits (80kDa) but share a common small regulatory subunit (30kDa; css1). Here we report the identification, cloning and characterization of a novel human small subunit (css2) encoded by an intronless gene, capns2, located on chromosome 16. This new protein displays 73% sequence identity within the Ca2+-binding region but lacks two oligo-Gly stretches characteristic of the N-terminal domain of the conventional small subunit. css2 appears to be the functional equivalent of the conventional small subunit in vitro in that it helps the large subunit fold into the active conformation of similar Ca2+ sensitivity when the two proteins are co-expressed in Escherichia coli. The purification of various chimaeric rat 80kDa—human css2 constructs, on the other hand, shows that css2 binds the large subunit much more weakly than css1. Further, it does not undergo the autolytic conversion typical of the classical small subunit. The expression of this protein in vivo, as assessed from its appearance in expressed sequence tag clones, is rather limited, making it an example of a tissue-specific, rather than ubiquitous, small subunit.

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