Úvod: V důsledku metabolických dějů dochází v živých strukturách k endogenní produkci chemiluminiscence, kterou také označujeme jako biologickou autochemiluminiscenci (BAL). Generování BAL je úzce spojeno s oxidačními procesy, tvorbou volných radikálů a obecně oxidačně-redukční homeostázou zkoumaného biologického materiálu. BAL byla již dříve studována v savčích buněčných modelech a tkáních. Doposud ovšem nebyl tento jev popsán v případě struktur zubní tkáně. Kromě endogenně generované BAL lze BAL indukovat i exogenně, a to jak fyzikálními (UV záření, mechanické poškození, teplo), tak i chemickými (oxidační činidla, např. H2O2) a biotickými (patogeny) faktory. Metodika: V předložené práci byla zkoumána endogenně produkovaná i exogenně indukovaná BAL v povrchových a vnitřních strukturách semiretinovaných a retinovaných třetích molárů, které byly indikovány k extrakci zubním lékařem pro jejich nevhodné uložení v čelisti u dvou pacientů (žena, 21 let, muž, 22 let). Detekce BAL byla provedena po mechanickém odstranění zubního plaku rotačním kartáčkem. Pomocí piezoelektrické pily byly připraveny podélné řezy vedené tak, aby došlo k odhalení všech vnitřních částí zubu. Takto připravené vzorky – celého vnitřního řezu a vnější části celého zubu – byly podrobeny detekci BAL ve světlotěsné komoře za použití fotonásobičového modulu. Následně byly vzorky ošetřeny roztokem oxidačního činidla 3% H2O2 a redukčního činidla 10 mM TCEP (tris(karboxyethyl)fosfin). Výsledky: U obou vzorků zubu bylo prokázáno, že produkují BAL. Produkce endogenní chemiluminiscence byla pozorována ve vnitřních strukturách zubu (18 600 pulzů/600 s), která byla přibližně 2,7krát vyšší než BAL detekovaná na povrchových strukturách zubu (6 900 pulzů/600 s). Po ošetření H2O2 došlo k významnému (až 14násobnému) nárůstu BAL pro vnitřní struktury zubu ve srovnání s bazální intenzitou endogenně produkované BAL. Aplikace TCEP (negativní kontrola) vedla k mírnému potlačení produkce BAL. Závěr: Výsledky této pilotní studie ukazují, že BAL může být produkována nejenom měkkými tkáněmi, ale i tvrdou zubní tkání. Získané výsledky by mohly být využity k výzkumu metabolické aktivity a reaktivity vnitřních i vnějších částí zubu, a to především v kontextu výzkumu oxidačněredukční homeostázy. Detekce BAL by také mohla být aplikována pro vývoj nových zobrazovacích technik.
Introduction: As a result of metabolic processes, the endogenous production of chemiluminescence occurs in living biological structures, which we also refer to as biological autochemiluminescence (BAL). The generation of BAL is closely connected with oxidation processes, the formation of free radicals, and in general the redox homeostasis of the investigated biological material. BAL has previously been studied in mammalian cells and tissues. So far, however, this phenomenon has not been described in dental tissue structures. In addition to endogenously generated BAL, BAL can be exogenously induced by physical (UV radiation, mechanical damage, heat), chemical (oxidizing agents, e.g. H2O2) or biotic (pathogens) factors. Methods: Endogenously and exogenously induced BAL were investigated on the surface and internal structures of semi-impacted and impacted third molars, which were indicated for extraction by a dentist due to their inappropriate placement in the jaw in two patients (a 21-year-old woman and a 22-year-old man). BAL detection was performed with samples after dental plaque was mechanically removed with a rotating brush. Using a piezosurgery unit with a saw headpiece, longitudinal sections were made to reveal all internal parts of the tooth. The samples prepared in this way – the entire internal section and the external part of the entire tooth – were subjected to BAL detection in a dark chamber using H7360-01 PMT photomultiplier. Subsequently, the samples were treated with a solution of the oxidizing agent 3% H2O2 or the reducing agent 10 mM TCEP (tris(carboxyethyl)phosphine). Results: Both tooth samples were shown to produce BAL. Endogenous chemiluminescence production was observed in the internal structures of the tooth (18,600 counts/600 s), which was 2.7-fold higher than the BAL detected on the tooth outer surfaces (6,900 counts/600 s). After H2O2 treatment, there was a significant (up to 14-fold) increase in BAL for internal tooth structures compared to the basal intensity of endogenously produced BAL. The application of TCEP (negative control) resulted in a residual suppression of BAL production. Conclusion: The results of this pilot study show that BAL can be produced not only by soft tissues but also by hard dental tissue. The obtained results could be used for further research of the metabolic activity and reactivity of the inner and outer parts of the tooth, especially in the context of redox biology research. BAL detection could also be applied in the development of new imaging techniques.
Remodeling of nanoscopic structures is not just crucial for cell biology, but it is also at the core of bioinspired materials. While the microtubule cytoskeleton in cells undergoes fast adaptation, adaptive materials still face this remodeling challenge. Moreover, the guided reorganization of the microtubule network and the correction of its abnormalities is still a major aim. This work reports new findings for externally triggered microtubule network remodeling by nanosecond electropulses (nsEPs). At first, a wide range of nsEP parameters, applied in a low conductivity buffer, is explored to find out the minimal nsEP dosage needed to disturb microtubules in various cell types. The time course of apoptosis and microtubule recovery in the culture medium is thereafter assessed. Application of nsEPs to cells in culture media result in modulation of microtubule binding properties to end-binding (EB1) protein, quantified by newly developed image processing techniques. The microtubules in nsEP-treated cells in the culture medium have longer EB1 comets but their density is lower than that of the control. The nsEP treatment represents a strategy for microtubule remodeling-based nano-biotechnological applications, such as engineering of self-healing materials, and as a manipulation tool for the evaluation of microtubule remodeling mechanisms during various biological processes in health and disease.
- MeSH
- elektřina * MeSH
- lidé MeSH
- mikrotubuly metabolismus MeSH
- nádorové buněčné linie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Intense pulsed electric fields are known to act at the cell membrane level and are already being exploited in biomedical and biotechnological applications. However, it is not clear if electric pulses within biomedically-attainable parameters could directly influence intra-cellular components such as cytoskeletal proteins. If so, a molecular mechanism of action could be uncovered for therapeutic applications of such electric fields. To help clarify this question, we first identified that a tubulin heterodimer is a natural biological target for intense electric fields due to its exceptional electric properties and crucial roles played in cell division. Using molecular dynamics simulations, we then demonstrated that an intense - yet experimentally attainable - electric field of nanosecond duration can affect the bβ-tubulin's C-terminus conformations and also influence local electrostatic properties at the GTPase as well as the binding sites of major tubulin drugs site. Our results suggest that intense nanosecond electric pulses could be used for physical modulation of microtubule dynamics. Since a nanosecond pulsed electric field can penetrate the tissues and cellular membranes due to its broadband spectrum, our results are also potentially significant for the development of new therapeutic protocols.
- MeSH
- elektrická stimulace * metody MeSH
- lidé MeSH
- simulace molekulární dynamiky * MeSH
- statická elektřina MeSH
- tubulin fyziologie MeSH
- vazebná místa MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The mechanical properties of microtubules are of great importance for understanding their biological function and for applications in artificial devices. Although microtubule mechanics has been extensively studied both theoretically and experimentally, the relation to its molecular structure is understood only partially. Here, we report on the structural analysis of microtubule vibration modes calculated by an atomistic approach. Molecular dynamics was applied to refine the atomic structure of a microtubule and a C α elastic network model was analyzed for its normal modes. We mapped fluctuations and local deformations up to the level of individual aminoacid residues. The deformation is mode-shape dependent and principally different in α-tubulins and β-tubulins. Parts of the tubulin dimer sequence responding specifically to longitudinal and radial stress are identified. We show that substantial strain within a microtubule is located both in the regions of contact between adjacent dimers and in the body of tubulins. Our results provide supportive evidence for the generally accepted assumption that the mechanics of microtubules, including its anisotropy, is determined by the bonds between tubulins.
- MeSH
- aminokyseliny chemie metabolismus MeSH
- anizotropie MeSH
- konformace proteinů * MeSH
- mechanický stres MeSH
- mikrotubuly chemie metabolismus MeSH
- multimerizace proteinu MeSH
- sekvence aminokyselin MeSH
- simulace molekulární dynamiky * MeSH
- tubulin chemie metabolismus MeSH
- vibrace MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The regulation of chromosome separation during mitosis is not fully understood yet. Microtubules forming mitotic spindles are targets of treatment strategies which are aimed at (i) the triggering of the apoptosis or (ii) the interruption of uncontrolled cell division. Despite these facts, only few physical models relating to the dynamics of mitotic spindles exist up to now. In this paper, we present the first electromechanical model which enables calculation of the electromagnetic field coupled to acoustic vibrations of the mitotic spindle. This electromagnetic field originates from the electrical polarity of microtubules which form the mitotic spindle. The model is based on the approximation of resonantly vibrating microtubules by a network of oscillating electric dipoles. Our computational results predict the existence of a rapidly changing electric field which is generated by either driven or endogenous vibrations of the mitotic spindle. For certain values of parameters, the intensity of the electric field and its gradient reach values which may exert a not-inconsiderable force on chromosomes which are aligned in the spindle midzone. Our model may describe possible mechanisms of the effects of ultra-short electrical and mechanical pulses on dividing cells--a strategy used in novel methods for cancer treatment.
Physical processes in living cells were not taken into consideration among the essentials of biological activity, regardless of the fact that they establish a state far from thermodynamic equilibrium. In biological system chemical energy is transformed into the work of physical forces for various biological functions. The energy transformation pathway is very likely connected with generation of the endogenous electrodynamic field as suggested by experimentally proved electrodynamic activity of biological systems connected with mitochondrial and microtubule functions. Besides production of ATP and GTP (adenosine and guanosine triphosphate) mitochondria form a proton space charge layer,
- MeSH
- adenosintrifosfát metabolismus MeSH
- apoptóza fyziologie genetika imunologie MeSH
- biomedicínský výzkum metody trendy MeSH
- elektromagnetická pole škodlivé účinky MeSH
- financování organizované MeSH
- fyziologie buňky fyziologie genetika imunologie MeSH
- glykolýza fyziologie genetika imunologie MeSH
- guanosintrifosfát metabolismus MeSH
- kyselina dichloroctová aplikace a dávkování škodlivé účinky terapeutické užití MeSH
- lidé MeSH
- mikrotubuly fyziologie metabolismus patologie MeSH
- mitochondrie fyziologie metabolismus patologie MeSH
- nádorová transformace buněk genetika imunologie účinky léků MeSH
- nádory etiologie metabolismus terapie MeSH
- Check Tag
- lidé MeSH
Spontaneous mechanical oscillations were predicted and experimentally proven on almost every level of cellular structure. Besides morphogenetic potential of oscillatory mechanical force, oscillations may drive vibrations of electrically polar structures or these structures themselves may oscillate on their own natural frequencies. Vibrations of electric charge will generate oscillating electric field, role of which in morphogenesis is discussed in this paper. This idea is demonstrated in silico on the conformation of two growing microtubules.
