Temperature Dependence of Tryptophan Fluorescence Lifetime as an Indicator of Its Microenvironment Dynamics.

The spectral-kinetic characteristics of the fluorescence of the tryptophan molecule in an aqueous solution and in the composition of a protein (albumin) were studied in the temperature range from -170 to 25°C. To explain the observed changes in the spectra and the tryptophan fluorescence lifetime with temperature, a model of transitions between the excited and ground states involving a charge-transfer state was used, which takes into account the nonlinear nature of the dynamics of these transitions. In these processes, an important role is played by the interaction of tryptophan molecules with its microenvironment, as well as rearrangements in the system of hydrogen bonds of the water-protein matrix surrounding the tryptophan molecule.

Intramolecular Mobility Affects the Energy Migration from Quantum Dots to Reaction Centers of Photosynthesizing Bacterium Rb. sphaeroides.

The temperature dependence of the efficiency of energy migration from the CdSe/CdS/ZnS quantum dots (QDs) with a fluorescence maximum at 580 nm to the reaction centers (RCs) of the bacteria Rb. sphaeroides is practically constant over the temperature range from 100 to ~230-240 K but then decreases 2.5-3 times as temperature further increases to 310 K. The analysis on this dependence on the basis of Förster's theory showed that the major changes in the energy transfer efficiency are associated with the temperature change in the quantum yield of QD fluorescence, which is due to the activation of intramolecular mobility in the RC structure.

Temperature Dependence of Tryptophan Fluorescence Lifetime in Aqueous Glycerol and Trehalose Solutions.

The temperature dependences of tryptophan fluorescence decay kinetics in aqueous glycerol and 1 M trehalose solutions were examined. The fluorescence decay kinetics were recorded in the spectral region of 292.5-417.5 nm with nanosecond time resolution. The kinetics curves were approximated by the sum of three exponential terms, and the spectral distribution (DAS) of these components was determined. An antisymbatic course of fluorescence decay times of two (fast and medium) components in the temperature range from -60 to +10°C was observed. The third (slow) component showed only slight temperature dependence. The antisymbatic behavior of fluorescence lifetimes of the fast and medium components was explained on the assumption that some of the excited tryptophan molecules are transferred from a short-wavelength B-form with short fluorescence lifetime to a long-wavelength R-form with an intermediate fluorescence lifetime. This transfer occurred in the indicated temperature range.

Generation of radical form of dipyridamole at illumination of photosynthetic reaction centers of Rb. sphaeroides.

The study of the effect of vasodilator, antiplatelet agent, and inhibitor P-glycoprotein dipyridamole (DIP) on the functioning of the transmembrane protein of the reaction center (RC) of Rb. sphaeroides showed that the activation of RC by constant light generates the DIP radical cation, which significantly affects the kinetics of recombination of charges divided between photoactive bacteriochlorophyll and quinone acceptors. Thus, the antioxidant properties of DIP may affect the functional activity of membrane proteins, and this apparently should be taken into account in the studies of the mechanisms of therapeutic action of this drug.

Influence of trehalose on high-temperature stability of the photosynthetic reaction centers.

The effect of heating at 65°C for 20 min on the absorption spectra and kinetics of the dark recombination of charges separated between photoactive bacteriochlorophyll and quinone acceptors was studied in dry films of bacterial photosynthetic reaction centers (RCs), RC films in polyvinyl alcohol, and trehalose. A pronounced protective effect of trehalose against pheophytinizaiton of molecules bacteriochlorophylls in RC structure and in maintaining their higher photochemical activity was found.

Investigation of Stability of Photosynthetic Reaction Center and Quantum Dot Hybrid Films.

The efficiency of interaction (efficiency of energy transfer) between various quantum dots (QDs) and photosynthetic reaction centers (RCs) from the purple bacterium Rhodobacter sphaeroides and conditions of long-term stability of functioning of such hybrid complexes in film preparations were investigated. It was found that dry films containing RCs and QDs and maintained at atmospheric humidity are capable to keep their functional activity for at least some months as judging by results of measurement of their spectral characteristics, efficiency of energy transfer from QDs to RCs, and RC electron-transport activity. Addition of trehalose to the films giving them still greater stability is especially expressed for films maintained at low humidity. These stable hybrid film structures are promising for further biotechnological studies for developing new phototransformation devices.

Temperature dependence of protein fluorescence in Rb. sphaeroides reaction centers frozen to 80 K in the dark or on the actinic light as the indicator of protein conformational dynamics.

The differences in the average fluorescence lifetime (τav) of tryptophanyls in photosynthetic reaction center (RC) of the purple bacteria Rb. sphaeroides frozen to 80 K in the dark or on the actinic light was found. This difference disappeared during subsequent heating at the temperatures above 250 K. The computer-based calculation of vibration spectra of the tryptophan molecule was performed. As a result, the normal vibrational modes associated with deformational vibrations of the aromatic ring of the tryptophan molecule were found. These deformational vibrations may be active during the nonradiative transition of the molecule from the excited to the ground state. We assume that the differences in τav may be associated with the change in the activity of these vibration modes due to local variations in the microenvironment of tryptophanyls during the light activation.

Properties of hybrid complexes composed of photosynthetic reaction centers from the purple bacterium Rhodobacter sphaeroides and quantum dots in lecithin liposomes.

Quantum dots (QDs) can absorb ultraviolet and long-wavelength light energy much more efficiently than natural light-harvesting proteins and transfer the excitation energy to photosynthetic reaction centers (RCs). Inclusion into liposomes of RC membrane pigment-protein complexes combined with QDs as antennae opens new opportunities for using such hybrid systems as a basis for artificial energy-transforming devices that potentially can operate with greater efficiency and stability than devices based only on biological components. RCs from Rhodobacter sphaeroides and QDs with fluorescence maximum at 530 nm (CdSe/ZnS with hydrophilic covering) were embedded in lecithin liposomes by extrusion of a solution of multilayer lipid vesicles through a polycarbonate membrane or by dialysis of lipids and proteins dispersed with excess detergent. The dimensions of the resulting hybrid systems were evaluated using dynamic light scattering and by transmission cryoelectron microscopy. The efficiency of RC and QD interaction within the liposomes was estimated using fluorescence excitation spectra of the photoactive bacteriochlorophyll of the RCs and by measuring the fluorescence decay kinetics of the QDs. The functional activity of the RCs in hybrid complexes was fully maintained, and their stability was even increased.

[On the mechanism for stabilizing a long-living charge separated state of photosynthetic reaction centers frozen under intensive illumination].

It is shown that freezing of the photosynthetic reaction centers from purple bacteria Rhodobacter sphaeroides under intensive illumination leads to the appearance of long-living charge separated states of reaction centers (P(+)QA-). This implies that the recombination reactions is blocked or charge separated state is stabilized. Experimental data are presented. It is also shown that this stabilization effect is caused by the structural relaxation of reaction centers to a new equilibrium state, and the free energy difference decreases as a result of this relaxation. The possible mechanism of such relaxation is determined by the effect of the polar water molecules orientation in the semiquinone local electrostatic field. The detailed analysis of the stabilization effect has been carried out, and its result supports a hypothesis of non equilibrium state of many electron transfer reactions in biological systems.

Online KidClot education for patients and families initiating warfarin therapy: The eKITE study.

Anticoagulation with Vitamin K antagonists (VKA) has always posed challenges in terms of monitoring requirements. These challenges were further exacerbated in the setting of the COVID-19 pandemic, with limited access to and/or avoidance of laboratory testing. The importance of utilizing point of care (POC) health technology for individualized patient management is salient. The foundation of effective home INR monitoring is establishing patient knowledge about their therapy and INR testing proficiency. The eKITE series was developed to support patients in establishing foundational knowledge required for VKA (warfarin) management and INR monitoring. The primary objectives were to evaluate eKITE, a patient-oriented innovative online education program for warfarin therapy, participant learning stress, and patient preference for online learning. This multi-center prospective study provided patients access to warfarin online education. Participants were required to complete written quizzes assessing warfarin knowledge of key concepts proficiency and identifying knowledge deficits. Patient preference, evaluating calm (lack of anxiety) while learning, and an INR on a home meter was completed. Participants performed INR tests at home and reported INRs by telephone. The analysis included 144 children and caregivers enrolled at five US and CDN sites. Most indications for anticoagulation were cardiac (congenital or acquired heart disease) with varied INR target ranges. Mean knowledge scores for warfarin and INR self-testing modules were 97%, with low anxiety with TTR of 84%. Patient preferred online learning. eKITE is an effective teaching modality for warfarin/home INR monitoring with safe INR testing and warfarin management that is appropriate for pediatrics and adults alike. PROLOGUE: The whir in the hallways is deafening. Lights bright, alarms are ringing in a chorus of unsynchronized beeps and screeches. It has been more than a week since I have slept. Snuggled beside me is my precious child, whining and equally irritated with the asynchronous symphony, further compounded by anxiety, procedures, and pain. The sun has broken. The staff smiles are welcoming and incessant, as one after one, they approach hurried, urgent, assiduous, their need to coach me for our upcoming departure to the warmth of home. Each provides essential information that I will require to keep my child, my treasure, safe and healthy. Yet, my eyes are heavy, blurred, and my brain foggy, trapped in a dark heavy cloud. How am I to follow? Comprehend? and retain anything? As they instruct, my precious child yearns for loving arms, compassion and love, whining, crying in disquiet. Overwhelmed does not adequately describe my ineffable exhaustion. Amidst this, how am I to learn about warfarin? Such a challenging medication, with so much to know. Concentrate, I tell myself, focus; now is my only opportunity to learn. I must be alert. It seems to be nonsensical.

Study of effect of molecular mobility in chromatophore membranes of the bacterium E. shaposhnikovii on processes of photoinduced electron transport using the NMR-spin-echo method with isotope substitution and dehydration.

The effect of dehydration and (2)H2O/H2O isotope substitution on electron transport reactions and relaxation of proton-containing groups was studied in chromatophore membranes of Ectothiorhodospira shaposhnikovii. During dehydration (including isotope substitution of hydrate water) of preliminarily dehydrated isolated photosynthetic membranes there was a partial correlation between hydration intervals within which activation of electron transport from high-potential cytochrome c to photoactive bacteriochlorophyll dimer P890 of photosynthetic reaction center and variation of spin-lattice and spin-spin proton relaxation time was observed. Partial correlation between hydration intervals can be considered as evidence of correlation between mobility of non-water proton-containing groups with proton relaxation frequency approximately 10(8) sec(-1) with efficiency of electron transfer at the donor side of the chain.

Role of fibronectin in the migration of fibroblasts into plasma clots.

The adhesion and migration of human diploid fibroblasts on plasma clots were measured. The role of plasma fibronectin was examined by depleting plasma of fibronectin before clotting. Fibronectin was not essential for cell adhesion and spreading, although rates were slightly slower on depleted clots. Rates of migration on the surface of clots were unaffected by fibronectin depletion. In contrast, fibronectin was an absolute requirement for migration of cells into plasma clots. Cells migrated rapidly into control clots but completely failed to penetrate the surface of fibronectin-depleted clots. The effect of depletion could only be reversed by adding fibronectin to depleted plasma before clotting. Adsorption of fibronectin after clotting failed to reverse the effect of depletion, suggesting that fibronectin had to be cross-linked by transglutaminase during the clotting process.

Beef production options and requirements for fossil fuel.

A large percentage of the feed resources used in beef production cannot be used by man or most other animals. These noncompetitive feeds could be used in different ways to increase beef production, but fossil fuel consumption by the beef industry would not be greatly reduced.

Use of a system of differential equations to analyze the functioning of a catalytic bio macromolecule under non equilibrium conditions.

The aim of the work is to analyze the response of a biomolecule to an external influence based on the study of its hidden states by identifying differential equations with constant coefficients. The relevance of the work lies in the fact that often the main reaction of an object to an external action can be represented as a sum of various exponential functions with a common starting point and a material balance equation. In this case, the response of an object to an external action corresponds to a system of differential equations with constant coefficients. This character of the main reaction may be due to the influence of the hidden properties of the object, which play the role of regulatory parameters. The problem is that the hidden factors and the system of differential equations are not identified. As an object, isolated reaction centers (RC) of the bacteria Rhodobacter sphaeroides , which possess the above properties, has used. Their structure is well studied. As result of studying of photo excitation processes of the reaction center has shown that electron transfer kinetics (the main reaction) can be approximated by three normalized exponential functions. Program was developed to identify for four differential equations of electron transfer and the balance equation, the behavior of hidden states of the reaction center. It was concluded that time the dependence the probability density of finding an electron in different conformational states of the reaction center characterizes the space-time changes in the structure of the reaction center.

The influence of hydrogen bonds on electron transfer rate in photosynthetic RCs.

Hydrogen bonds formed between photosynthetic reaction centers (RCs) and their cofactors were shown to affect the efficacy of electron transfer. The mechanism of such influence is determined by sensitivity of hydrogen bonds to electron density rearrangements, which alter hydrogen bonds potential energy surface. Quantum chemistry calculations were carried out on a system consisting of a primary quinone Q(A), non-heme Fe(2+) ion and neighboring residues(.) The primary quinone forms two hydrogen bonds with its environment, one of which was shown to be highly sensitive to the Q(A) state. In the case of the reduced primary quinone two stable hydrogen bond proton positions were shown to exist on [Q(A)-His(M219)] hydrogen bond line, while there is only one stable proton position in the case of the oxidized primary quinone. Taking into account this fact and also the ability of proton to transfer between potential energy wells along a hydrogen bond, theoretical study of temperature dependence of hydrogen bond polarization was carried out. Current theory was successfully applied to interpret dark P(+)/Q(A)(-) recombination rate temperature dependence.

Relaxation processes accompanying electron stabilization in the quinone acceptor part of Rb. sphaeroides reaction centers.

The temperature dependence of the dark recombination rate in photooxidized bacteriochlorophyll (P) and photoreduced quinone acceptors (ubiquinones) QA and QB of photosynthetic reaction centers of purple bacteria Rhodobacter sphaeroides (Rb. sphaeroides) was studied. Photoinduced changes in the absorption were detected in the Qx absorption band of photooxidized bacteriochlorophyll at 600 nm and in the bands corresponding to the redox changes of ubiquinones at 335 and 420-450 nm. Kinetic analysis was used to evaluate the activation energy and the characteristic time of the transient process of relaxation accompanying electron stabilization at the final quinone acceptor. A comparative study of the kinetics of oxidation-reduction reactions of photoactive bacteriochlorophyll RC purple bacteria and quinone acceptors in their individual absorption bands is an informative approach to studying the mechanisms of this stabilization. The analysis of the revealed kinetic differences makes it possible to estimate the activation energy and the characteristic times of the transition relaxation processes associated with the stabilization of the electron in the quinone acceptor part of RC. Purple bacterial reaction centers have fundamental similarities with PSII reaction centers. Such a similarity represents evolutional closeness between the two types of RC. So it is possible that the photoinduced charge separation in PSII RC, as well as in purple bacteria RC, is also accompanied by definite conformational changes. The possible role of hydrogen bonds of surrounding protein in the relaxation processes accompanying the electron transfer to quinone acceptors is discussed.

The effect of acetaminophen (four grams a day for three consecutive days) on hepatic tests in alcoholic patients--a multicenter randomized study.

BACKGROUND: Hepatic failure has been associated with reported therapeutic use of acetaminophen by alcoholic patients. The highest risk period for alcoholic patients is immediately after discontinuation of alcohol intake. This period exhibits the largest increase in CYP2E1 induction and lowest glutathione levels. Our hypothesis was that common liver tests would be unaffected by administration of the maximum recommended daily dosage of acetaminophen for 3 consecutive days to newly-abstinent alcoholic subjects. METHODS: Adult alcoholic subjects entering two alcohol detoxification centers were enrolled in a prospective double-blind, randomized, placebo-controlled trial. Subjects were randomized to acetaminophen, 4 g/day, or placebo for 3 consecutive days. The study had 95% probability of detecting a 15 IU/L difference in serum ALT. RESULTS: A total of 443 subjects were enrolled: 308 (258 completed) received acetaminophen and 135 subjects (114 completed) received placebo. Study groups did not differ in demographics, alcohol consumption, nutritional status or baseline laboratory assessments. The peak mean ALT activity was 57 +/- 45 IU/L and 55 +/- 48 IU/L in the acetaminophen and placebo groups, respectively. Subgroup analyses for subjects presenting with an elevated ALT, subjects fulfilling a diagnosis of alcoholic hepatitis and subjects attaining a peak ALT greater than 200 IU/L showed no statistical difference between the acetaminophen and control groups. The one participant developing an increased international normalized ratio was in the placebo group. CONCLUSION: Alcoholic patients treated with the maximum recommended daily dose of acetaminophen for 3 consecutive days did not develop increases in serum transaminase or other measures of liver injury. Treatment of pain or fever for 3 days with acetaminophen appears safe in newly-abstinent alcoholic patients, such as those presenting for acute medical care.

CD40 induces apoptosis in carcinoma cells through activation of cytotoxic ligands of the tumor necrosis factor superfamily.

CD40, a tumor necrosis factor (TNF) receptor (TNFR) family member, conveys signals regulating diverse cellular responses, ranging from proliferation and differentiation to growth suppression and cell death. The ability of CD40 to mediate apoptosis in carcinoma cells is intriguing given the fact that the CD40 cytoplasmic C terminus lacks a death domain homology with the cytotoxic members of the TNFR superfamily, such as Fas, TNFR1, and TNF-related apoptosis-inducing ligand (TRAIL) receptors. In this study, we have probed the mechanism by which CD40 transduces death signals. Using a trimeric recombinant soluble CD40 ligand to activate CD40, we have found that this phenomenon critically depends on the membrane proximal domain (amino acids 216 to 239) but not the TNFR-associated factor-interacting PXQXT motif in the CD40 cytoplasmic tail. CD40-mediated cytotoxicity is blocked by caspase inhibitors, such as zVAD-fmk and crmA, and involves activation of caspase 8 and caspase 3. Interestingly, CD40 ligation was found to induce functional Fas ligand, TRAIL (Apo-2L) and TNF in apoptosis-susceptible carcinoma cells and to up-regulate expression of Fas. These findings identify a novel proapoptotic mechanism which is induced by CD40 in carcinoma cells and depends on the endogenous production of cytotoxic cytokines and autocrine or paracrine induction of cell death.