Enzyme Promiscuous Activity: How to Define it and its Evolutionary Aspects

2020 ◽  
Vol 27 (5) ◽  
pp. 400-410
Author(s):  
Valentina De Luca ◽  
Luigi Mandrich
Keyword(s):  
Gene Evolution ◽  
Sequence Divergence ◽  
High Specificity ◽  
Industrial Applications ◽  
Point Of View ◽  
Enzyme Promiscuity ◽  
Primary Activity ◽  
Starting Point ◽  
Main Activity

: Enzymes are among the most studied biological molecules because better understanding enzymes structure and activity will shed more light on their biological processes and regulation; from a biotechnological point of view there are many examples of enzymes used with the aim to obtain new products and/or to make industrial processes less invasive towards the environment. Enzymes are known for their high specificity in the recognition of a substrate but considering the particular features of an increasing number of enzymes this is not completely true, in fact, many enzymes are active on different substrates: this ability is called enzyme promiscuity. Usually, promiscuous activities have significantly lower kinetic parameters than to that of primary activity, but they have a crucial role in gene evolution. It is accepted that gene duplication followed by sequence divergence is considered a key evolutionary mechanism to generate new enzyme functions. In this way, promiscuous activities are the starting point to increase a secondary activity in the main activity and then get a new enzyme. The primary activity can be lost or reduced to a promiscuous activity. In this review we describe the differences between substrate and enzyme promiscuity, and its rule in gene evolution. From a practical point of view the knowledge of promiscuity can facilitate the in vitro progress of proteins engineering, both for biomedical and industrial applications. In particular, we report cases regarding esterases, phosphotriesterases and cytochrome P450.

scholarly journals Development of High Affinity and High Specificity Inhibitors of Matrix Metalloproteinase 14 through Computational Design and Directed Evolution

2017 ◽  
Vol 292 (8) ◽  
pp. 3481-3495 ◽  
Author(s):  
Valeria Arkadash ◽  
Gal Yosef ◽  
Jason Shirian ◽  
Itay Cohen ◽  
Yuval Horev ◽  
...  
Keyword(s):  
Directed Evolution ◽  
Surface Display ◽  
High Specificity ◽  
Computational Design ◽  
Computational Method ◽  
Yeast Surface Display ◽  
Mmp Inhibitor ◽  
Starting Point ◽  
Yeast Surface

Degradation of the extracellular matrices in the human body is controlled by matrix metalloproteinases (MMPs), a family of more than 20 homologous enzymes. Imbalance in MMP activity can result in many diseases, such as arthritis, cardiovascular diseases, neurological disorders, fibrosis, and cancers. Thus, MMPs present attractive targets for drug design and have been a focus for inhibitor design for as long as 3 decades. Yet, to date, all MMP inhibitors have failed in clinical trials because of their broad activity against numerous MMP family members and the serious side effects of the proposed treatment. In this study, we integrated a computational method and a yeast surface display technique to obtain highly specific inhibitors of MMP-14 by modifying the natural non-specific broad MMP inhibitor protein N-TIMP2 to interact optimally with MMP-14. We identified an N-TIMP2 mutant, with five mutations in its interface, that has an MMP-14 inhibition constant (Ki) of 0.9 pm, the strongest MMP-14 inhibitor reported so far. Compared with wild-type N-TIMP2, this variant displays ∼900-fold improved affinity toward MMP-14 and up to 16,000-fold greater specificity toward MMP-14 relative to other MMPs. In an in vitro and cell-based model of MMP-dependent breast cancer cellular invasiveness, this N-TIMP2 mutant acted as a functional inhibitor. Thus, our study demonstrates the enormous potential of a combined computational/directed evolution approach to protein engineering. Furthermore, it offers fundamental clues into the molecular basis of MMP regulation by N-TIMP2 and identifies a promising MMP-14 inhibitor as a starting point for the development of protein-based anticancer therapeutics.

scholarly journals The Role of the Cell Volume-Area Ratio in Thermodynamic Analysis of the Cancer Growth Control for In Vitro Experiments

2018 ◽  
Author(s):  
Umberto Lucia ◽  
Giulia Grisolia
Keyword(s):  
Growth Control ◽  
Area Ratio ◽  
Point Of View ◽  
Cancer Growth ◽  
Cyclic Process ◽  
Cell Life ◽  
Starting Point ◽  
Cell Changes

From a thermodynamic point of view, living cell life is no more than a cyclic process. It starts with the newly separated daughter cells and restarts when the next generations grow as free entities. In this cycle the cell changes its entropy. In cancer the growth control is damaged. In this paper we analyze the role of the volume-area ratio in cell in relation to the heat exchange between cell and its environment in order to point out the effect on the cancer growth. The result holds to a possible control of the cancer growth based on the heat exchanged by the cancer towards its environment, and the membrane potential variation, with the consequence of controlling the ions fluxes and the related biochemical reactions. This second law approach could represent a starting point for a possible future support for the anticancer therapies, in order to improve their effectiveness for the untreatable cancers.

scholarly journals ANCESTRAL RECONSTRUCTION OF A β-LACTAMASE AND COMPARISON WITH ITS EXTANT PROTEINS

2021 ◽  
pp. 155-170
Author(s):  
Gonzalo Fernández Balaguer ◽  
Carmen del Águila ◽  
Carolina Hurtado Marcos ◽  
Rubén Agudo Torres
Keyword(s):  
High Capacity ◽  
Hydrolytic Activity ◽  
Point Of View ◽  
New Drugs ◽  
Ancestral Reconstruction ◽  
Ancestral Sequences ◽  
Starting Point ◽  
Evolution Of Proteins ◽  
Ancestral Protein

The β-lactamases are proteins of bacterial origin that are characterized by hydrolyzing antibiotics β-lactams, conferring microbial resistance against them. They are a heterogeneous family of proteins very relevant from a health point of view due to the ease they present to acquire resistance to new drugs due to their high capacity for evolution. The in vitro evolution of these proteins has served not only to develop their characterization and improve their knowledge, but as a new line of research that allows to predictively identify residues involved in the acquisition of antibiotic resistance. At the same time, the method of ancestral protein reconstruction has been revealed as a novel and useful tool to understand the evolution of β-lactamases and understand some of their characteristics such as their promiscuity. In this work, a study of ancestral β-lactamases reconstructed from the phylogeny of existing class A β-lactamases has been carried out. Of the four ancestral proteins studied, one has been obtained that is functional and has compared its hydrolytic activity with that of four of its current counterparts against eight β-lactam drugs. This ancestral protein has been shown to have a more generalistic antibiotic activity than any of the current proteins studied. In addition, the active ancestral protein showed more resistance to one of the drugs used than the rest of β-lactamases existing. Finally these results have been discussed and from them it is argued why reconstructed ancestral sequences can be a very attractive starting point when it comes to direct evolution of proteins for obtaining proteins of biotechnological interest.

Melanoma Cell Lines Role in Obtaining New Drug Candidates for Combating the Malignant Pathology of the Cutaneous Organ

2019 ◽  
Vol 70 (3) ◽  
pp. 943-945
Author(s):  
Zsolt Gyori ◽  
Monica Susan ◽  
Razvan Susan ◽  
Andrada Iftode ◽  
Cristina Trandafirescu ◽  
...  
Keyword(s):  
Cell Lines ◽  
Point Of View ◽  
In Vitro Studies ◽  
Drug Candidates ◽  
Starting Point ◽  
In Vivo Animal Models ◽  
Culture Growth ◽  
Cell Culture Growth

As prophylactic and therapeutic approaches for melanoma, of great interest and importance are the in vitro studies using cell lines to elucidate several tumoral phenomena. Therefore, the similarities and differences between the different tumor cells must be known and understood in order to obtain a more accurate correlation with processes that occur in vivo. In this study, six cell lines of melanoma, both of mouse and human origin were analyzed from the point of view of cell culture growth, morphology and use in the research of new therapies. In brief, the current paper exhibits a comparison of melanoma cells which can be utilized as a starting point for further in vitro studies and in vivo animal models.

scholarly journals Analysis of Gastrointestinal Acoustic Activity Using Deep Neural Networks

Sensors ◽  
2021 ◽  
Vol 21 (22) ◽  
pp. 7602
Author(s):  
Jakub Ficek ◽  
Kacper Radzikowski ◽  
Jan Krzysztof Nowak ◽  
Osamu Yoshie ◽  
Jaroslaw Walkowiak ◽  
...  
Keyword(s):  
Neural Networks ◽  
Data Exchange ◽  
High Specificity ◽  
Medical Professional ◽  
Point Of View ◽  
Bowel Sound ◽  
Night Time ◽  
Open Platform ◽  
Starting Point ◽  
Analytical Approaches

Automated bowel sound (BS) analysis methods were already well developed by the early 2000s. Accuracy of ~90% had been achieved by several teams using various analytical approaches. Clinical research on BS had revealed their high potential in the non-invasive investigation of irritable bowel syndrome to study gastrointestinal motility and in a surgical setting. This article proposes a novel methodology for the analysis of BS using hybrid convolutional and recursive neural networks. It is one of the first methods of using deep learning to be widely explored. We have developed an experimental pipeline and evaluated our results with a new dataset collected using a device with a dedicated contact microphone. Data have been collected at night-time, which is the most interesting period from a neurogastroenterological point of view. Previous works had ignored this period and instead kept brief records only during the day. Our algorithm can detect bowel sounds with an accuracy >93%. Moreover, we have achieved a very high specificity (>97%), crucial in diagnosis. The results have been checked with a medical professional, and they successfully support clinical diagnosis. We have developed a client-server system allowing medical practitioners to upload the recordings from their patients and have them analyzed online. This system is available online. Although BS research is technologically mature, it still lacks a uniform methodology, an international forum for discussion, and an open platform for data exchange, and therefore it is not commonly used. Our server could provide a starting point for establishing a common framework in BS research.

scholarly journals The Role of the Cell Volume-Area Ratio in Thermodynamic Analysis of the Cancer Growth Control for In Vitro Experiments

2018 ◽  
Author(s):  
Umberto Lucia ◽  
Giulia Grisolia
Keyword(s):  
Growth Control ◽  
Area Ratio ◽  
Point Of View ◽  
Cancer Growth ◽  
Cyclic Process ◽  
Cell Life ◽  
Starting Point ◽  
Cell Changes

From a thermodynamic point of view, living cell life is no more than a cyclic process. It starts with the newly separated daughter cells and restarts when the next generations grow as free entities. In this cycle the cell changes its entropy. In cancer the growth control is damaged. In this paper we analyze the role of the volume-area ratio in cell in relation to the heat exchange between cell and its environment in order to point out the effect on the cancer growth. The result holds to a possible control of the cancer growth based on the heat exchanged by the cancer towards its environment, and the membrane potential variation, with the consequence of controlling the ions fluxes and the related biochemical reactions. This second law approach could represent a starting point for a possible future support for the anticancer therapies, in order to improve their effectiveness for the untreatable cancers.

A Physiologic Regulator of Collagen-Induced Platelet Aggregation: Inhibition by Clq

1981 ◽  
Vol 45 (02) ◽  
pp. 110-115 ◽  
Author(s):  
György Csákó ◽  
Eva A Suba
Keyword(s):  
Platelet Aggregation ◽  
Human Platelet ◽  
Platelet Reactivity ◽  
High Specificity ◽  
Turbidimetric Method ◽  
Specific Manner ◽  
Aggregation Inhibition ◽  
Different Tissues

SummaryPlatelet aggregations were studied by a turbidimetric method in citrated human platelet-rich plasmas (PRP) in vitro. Human Clq inhibited the aggregations caused by collagens derived from different tissues and species. Clq was needed by weight in comparable quantities to collagen for neutralizing the aggregating effect. The dependence of the inhibitory reaction on the preincubation of platelets with Clq and the differences in the occurrence of aggregating substances in supernatants of PRP triggered with collagen in the presence or absence of Clq, confirmed that Clq exerts its effect by preventing fixation of collagen to platelets. In addition, the high specificity of the inhibitory action of Clq for collagen-induced platelet aggregation was demonstrated by results obtained for testing a variety of aggregating agents in combination with Clq and/or collagen.Since normal concentrations of Clq in the blood are in the range of inhibitory doses of Clq for collagen-induced platelet aggregations in vitro and upon activation of complement Clq is known to dissociate from Cl, it is proposed that Clq may participate in a highly specific manner in regulating platelet reactivity to collagen in vivo.

LES AXES DE LA CRÉATION ET LA NAISSANCE DE LʹIMAGINAIRE COMME LANGAGE MYTHIQUE

Trictrac ◽  
2016 ◽  
Vol 9 ◽  
Author(s):  
Liliana Danciu ◽  
Petru Adrian Danciu
Keyword(s):  
Sacred Space ◽  
Physical World ◽  
Spoken Word ◽  
Point Of View ◽  
Philosophical Approach ◽  
Starting Point ◽  
Written Form ◽  
The Creation ◽  
Religious Systems ◽  
Rich Spectrum

The axes of the creation and birth of the imaginary as a mythical language. Our research follows the relationships of the concepts that are taking into account creation on the double axis of verticality and horizontality. We highlight those symbolic elements which would later constitute the mythical language about the sacred space-temporality. Inside this space-temporality a rich spectrum of mythical images develops; images capable of explaining the relationships of the creation plans. Without a religious perception of the temporality, the conceptualization of the axis would remain a philosophical approach. Through our point of view, the two are born simultaneously. Thanks to them, creation can be imagined. The first “frozen” formula of the mystical human spirit can be thought, brought to a palpable reality, expressed in an oral and then a written form. Studied together, temporality (sacred or not) and space are permanently imagined together. For example, a loss of mundane temporality in the secret ecstasy that offers to the soul an ascending direction does not mean getting out of universal temporality, but of its mundane section. In the sacred space the soul relates to time. Even the gods are submitted by the sacred, Aeon sometimes being synonymous to destiny. The universal creator seems to evade every touch, but not consistently, only when he avoids the descent into its created worlds. In sacredness, time and space seem or become confused, both expressing the same reality, by the immediate swing from thinking to deed. The mythical imagery conceives the displacement in the primary space-temporality by the spoken word. So, for something to appear and live, the spoken word is required. Even the divine dream appears as a pre-word of a creator’s thought. The thought follows the spoken word, the spoken word follows the gestures which finally indicate the meanings of the creative act, controlling the rhythm of the creation days. These three will later be adapted through imitation in rite. We are now situated at the limit of the physical world, a real challenge for the mythical imagery. The general feature of the mythical expression on the creation of the material world is the state of the divinity’s exhaustion, most often conceptualized by sacrifice or divine fatigue. The world geography identifies with the anatomy of a self-gutted god. Practically, material creation is most likely the complete revelation of God’s body autopsy. As each body decomposes, everything in it is an illusion. An axial approach of the phenomenon exists in all religious systems. The created element’s origin is exterior, with or without a pre-existing matter, by a god’s sacrifice or only because it has to be that way. This is the starting point of the discussion on the symbolism of axiality as a reason for the constitution of the language of creation, capable of retelling the imaginary construction of myth in an oral and then written form.

L’absence chez Michel Butor. L’Emploi du temps et Degrés

2012 ◽  
pp. 66-80
Author(s):  
Michał Mrozowicki
Keyword(s):  
Human Memory ◽  
Point Of View ◽  
Narrative Discourse ◽  
The Novel ◽  
Michel Butor ◽  
Starting Point ◽  
One Year ◽  
The Moment ◽  
Narrative Mode ◽  
The City

Michel Butor, born in 1926, one of the leaders of the French New Novel movement, has written only four novels between 1954 and 1960. The most famous of them is La Modification (Second thoughts), published in 1957. The author of the paper analyzes two other Butor’s novels: L’Emploi du temps (Passing time) – 1956, and Degrés (Degrees) – 1960. The theme of absence is crucial in both of them. In the former, the novel, presented as the diary of Jacques Revel, a young Frenchman spending a year in Bleston (a fictitious English city vaguely similar to Manchester), describes the narrator’s struggle to survive in a double – spatial and temporal – labyrinth. The first of them, formed by Bleston’s streets, squares and parks, is symbolized by the City plan. During his one year sojourn in the city, using its plan, Revel learns patiently how to move in its different districts, and in its strange labyrinth – strange because devoid any centre – that at the end stops annoying him. The other, the temporal one, symbolized by the diary itself, the labyrinth of the human memory, discovered by the narrator rather lately, somewhere in the middle of the year passed in Bleston, becomes, by contrast, more and more dense and complex, which is reflected by an increasinly complex narration used to describe the past. However, at the moment Revel is leaving the city, he is still unable to recall and to describe the events of the 29th of February 1952. This gap, this absence, symbolizes his defeat as the narrator, and, in the same time, the human memory’s limits. In Degrees temporal and spatial structures are also very important. This time round, however, the problems of the narration itself, become predominant. Considered from this point of view, the novel announces Gerard Genette’s work Narrative Discourse and his theoretical discussion of two narratological categories: narrative voice and narrative mode. Having transgressed his narrative competences, Pierre Vernier, the narrator of the first and the second parts of the novel, who, taking as a starting point, a complete account of one hour at school, tries to describe the whole world and various aspects of the human civilization for the benefit of his nephew, Pierre Eller, must fail and disappear, as the narrator, from the third part, which is narrated by another narrator, less audacious and more credible.

OWNERSHIP IN THE REPUBLIC OF MACEDONIA AND THE STATE AS THE HOLDER OF THE PROPERTY RIGHT UNDER THE CONSTITUTION IN 1991

2018 ◽  
Vol 28 (6) ◽  
pp. 1993-2005
Author(s):  
Shemsije Demiri ◽  
Rudina Kaja
Keyword(s):  
Legal Aspect ◽  
State Ownership ◽  
The State ◽  
Point Of View ◽  
State Function ◽  
Dominant Form ◽  
Republic Of Macedonia ◽  
General Terms ◽  
Starting Point ◽  
The Republic

This paper deals with the right to property in general terms from its source in Roman law, which is the starting point for all subsequent legal systems. As a result of this, the acquisition of property rights is handled from the historical point of view, with the inclusion of various local and international literature and studies, as well as the legal aspect devoted to the respective civil codes of the states cited in the paper.Due to such socio-economic developments, state ownership and its ownership function have changed. The state function as owner of property also changed in Macedonia's property law.The new constitutional sequence of the Republic of Macedonia since 1991 became privately owned as a dominant form of ownership, however, state ownership also exists.This process of transforming social property into state or private (dissolves), in Macedonia starts from Yugoslavia through privatization, return and denationalization measures, on which basis laws on privatization have been adopted. Because of this, there will be particularly intensive negotiations regaring the remaining state assets.

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