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1.
Enthalpy and conformational volume changes in photolyses of oxy-hemoglobin (HbO(2)) of human, bovine, pig, horse and rabbit are investigated by photoacoustic calorimetry. In the experiment, a pulsed Nd:YAG laser is used as an exciting source, and a PVDF film transducer and a PZT transducer are used to detect the photoacoustic signals. Based on the time scales of the excitation and detection systems as well as the photolysis processes of HbO(2), it can be indicated that the measured enthalpy and conformational volume changes are related to slow geminate recombination and tertiary relaxation in photolyses of HbO(2), which are with the time scale of 30-40 ns and 100-150 ns, respectively. The results show that the enthalpy and conformational volume changes are different for both photolysis processes of HbO(2) and also for various mammals. The different results among the five mammals are analyzed and discussed briefly. 相似文献
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A photoacoustic calorimetry study of horse carboxymyoglobin on the 10-nanosecond time scale. 总被引:3,自引:3,他引:0 下载免费PDF全文
The development of a photoacoustic calorimeter with a time resolution of 10 ns is presented, and the dynamics of the enthalpy and volume changes found in the photodissociation of CO from horse carboxymyoglobin are examined. With this enhanced time resolution a new transient species, the lifetime of which is 29 ns at 20 degrees C, is observed in the ligand dissociation process. 相似文献
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The structures and functions of the cellular acidic compartments are strongly dependent on the pH gradients across vesicular membranes. Measurement and imaging of the vesicular pH require fluorophores with appropriate pK(a) values. In this report, we characterized the pH-dependent lifetime responses of a family of acidotropic probes, LysoSensors, to evaluate their usefulness to low-pH lifetime imaging. LysoSensors are cell-permeable weak bases that selectively accumulate in acidic vesicles after being protonated. They have higher quantum yields at lower pH ranges to allow visualization of the lysosomes. For LysoSensors DND-167, DND-189, and DND-153, raising the buffer pH increased the quenching effects of their basic side chains and substantially reduced their steady-state fluorescence and lifetimes. The apparent pK(a) values determined from their lifetime responses were shifted to near neutral values because of the dominant intensity contribution from their protonated species. One unique property of LysoSensor DND-189 is its nonmonotonic lifetime responses of the maxima occurring between pH 4 and 5. LysoSensor DND-192 did not show significant lifetime changes over a wide pH range. LysoSensor DND-160, which was the only excitation and emission ratiometric probe, showed significant pH-dependent lifetime changes as well as its spectral shifts. Its apparent pK(a) values determined from the lifetime responses were comparable to the lysosomal pH because of its bright basic form. Because of the pH-dependent absorption spectra, the apparent pK(a) values could be manipulated between 3 and 5 by changing the excitation and/or emission wavelengths. These results indicate that LysoSensor DND-160 is a promising probe for lifetime imaging to determine lysosomal pH. 相似文献
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Feig AL 《Biopolymers》2007,87(5-6):293-301
Isothermal titration calorimetry (ITC) has been applied to the study of proteins for many years. Its use in the biophysical analysis of RNAs has lagged significantly behind its use in protein biochemistry, however, in part because of the relatively large samples required. As the instrumentation has become more sensitive, the ability to obtain high quality data on RNA folding and RNA ligand interactions has improved dramatically. This review provides an overview of the ITC experiment and describes recent work on RNA systems that have taken advantage of its versatility for the study of small molecule binding, protein binding, and the analysis of RNA folding. 相似文献
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Role of the arginine-45 salt bridge in ligand dissociation from sperm whale carboxymyoglobin as probed by photoacoustic calorimetry 总被引:3,自引:0,他引:3
The dynamics of the enthalpy and volume changes found in the photodissociation of CO from sperm whale carboxymyoglobin and two site-directed mutants in which arginine-45 is replaced by glycine and asparagine are examined by photoacoustic calorimetry. An intermediate is observed whose lifetime at 20 degrees C is 700 ns. The enthalpy of the intermediate increases by approximately 7 kcal/mol upon replacing arginine-45 with either asparagine or glycine. These observations support recent proposals that an arginine-45 salt bridge is broken upon ligand dissociation. 相似文献
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Groundworks for an evolutionary biochemistry: the iron-sulphur world. 总被引:10,自引:0,他引:10
G W?chtersh?user 《Progress in biophysics and molecular biology》1992,58(2):85-201
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《生物化学与分子生物教育》2001,29(2):60-65
Although most biochemistry curricula include some treatment of light absorption and spectrophotometry, discussion of fluorescence spectroscopy is generally omitted. This omission is unfortunate given the increasing use of fluorescence in many fields of biochemical research. In this paper we briefly review the principles and applications of fluorescence in biochemical systems, from the viewpoint of teaching fluorescence in undergraduate curricula. Simple practical demonstrations are presented, which clearly demonstrate important concepts yet require minimal specialized equipment. 相似文献
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To expand the multiplicity of the in situ hybridization (ISH) procedure, which is presently limited by the number of fluorochromes spectrally separable in the microscope, a digital fluorescence ratio method is proposed. For this purpose, chromosome-specific repetitive probes were double-labeled with two haptens and hybridized to interphase nuclei of human peripheral blood lymphocytes. The haptens were immunocytochemically detected with specific antibodies conjugated with the fluorochromes FITC or TRITC. The FITC and TRITC fluorescence intensities of spots obtained with different double-haptenized probes were measured, and the fluorescence ratio was calculated for each ISH spot. Combinations of different haptens, such as biotin, digoxigenin, fluorescein, sulfonate, acetyl amino fluorene (AAF), and mercury (Hg) were used. The fluorescence intensity ratio (FITC/TRITC) of the ISH spots was fairly constant for all combinations used, with coefficients of variation between 10 and 30%. To study the feasibility of a probe identification procedure on the basis of probe hapten ratios, one probe was double-labeled with different ratios, by varying the relative concentrations of the modified nucleotides (biotin-11-dUTP and digoxigenin-11-dUTP) in the nick-translation reaction. Measurement of the FITC and TRITC intensities of the ISH spots showed that the concentration of modified nucleotides used in the labeling procedures was reflected in the mean fluorescence intensity of the ISH spots. Furthermore, the ratio distributions showed little overlap due to the relatively small coefficients of variation. The results indicate that a multiple ISH procedure based on fluorescence ratio imaging of double-labeled probes is feasible. 相似文献
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Summary Differential scanning calorimetry of multilamellar liposomes, interacting with the optical probe Merocyanine 540, yields quantitative information about perturbances of the bilayer structure induced by this dye. At low dye: lipid ratios, the dye perturbs primarily its own microenvironment, which is laterally separated from the unmodified lipid domain and exhibits modified thermotropic properties. A further increase in the dye concentration results in a perturbance of the whole lipid bilayer. The degree of perturbance is sensitive to structural modifications in the head-group region of the lipids. It is concluded that Merocyanine 540 reports in every case, even at infinite dilution, on localized events originating from a perturbed microenvironment.An abstract of this paper was presented at the XI Congress of Biochemistry, Toronto, in July 1979. 相似文献
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Fluorescence in situ hybridisation of multiple probes on a single microscope slide. 总被引:2,自引:1,他引:2 下载免费PDF全文
Z Larin M D Fricker E Maher Y Ishikawa-Brush E M Southern 《Nucleic acids research》1994,22(18):3689-3692
We report a method to analyse multiple samples by fluorescence in situ hybridisation on a single glass microscope slide. Wells were formed in which independent hybridisation reactions could proceed by sealing a silicon rubber gasket to the slide. In the largest format tested, different probes were hybridised simultaneously by applying them directly from a 96-well microtitre dish which was inverted on a glass plate. This technique will increase the rate of analysis of multiple probes against a standard set of chromosomes and could also be used to analyse different karyotypes using a panel of probes such as single chromosome paints during a single operation. It should be useful for both chromosomal mapping projects and screening for chromosome abnormalities in clinical diagnostic laboratories. 相似文献
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We previously demonstrated that the relationship between respiratory gas exchange and metabolic heat production is unexpectedly variable and that conventional approaches to estimating energy expenditure by indirect calorimetry can incorporate large errors. Prior studies, however, comparing direct and indirect calorimetry of animals focused only on endothermic organisms. Given that endothermy and ectothermy represent a fundamental dichotomy of animal energetics, in this analysis we explore how these contrasting physiologies correlate with the relationship between heat production and respiratory gas exchange. Simultaneous indirect and direct calorimetry in an ectotherm, the ball python (Python regius Shaw), revealed that the relationships between gas exchange and heat production were within 1% of those expected when analyses using indirect calorimetry were based on the assumption that the fasting animal catabolized only protein. This accuracy of indirect calorimetry contrasts sharply with our previous conclusions for three species of birds and mammals. 相似文献
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Diubko TS Egorov MI Linnik TP Belonozhko AP Ermolenko IG Semenova ON Patsenker LD Pivovarenko VG Povrozin EA 《Tsitologiia》2007,49(6):521-526
By fluorescent spectroscopy and microscopy methods the possibility of fluorescent probes DSM, E-176, 3-DAB and FME application for study of cryoprotective agents' influence on the dog spermatozoa are investigated. It is established that FME and 3-DAD dyes are suitable for the posed problem solving, and the DSM and E-176 probes have restrictions owing to enough strong fluorescence from cryoprotectant solutions. It is shown that the fluorescent probes investigated influence the cells motility to different degree. The perspectives of investigated dyes application for study of cryoprotective agent' finfluence on spermatozoa are considered. 相似文献
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The transverse location normal to the bilayer surface of a series of n-(9-anthroyloxy) fatty acid probes, where n = 2, 3, 6, 7, 9, 12, and 16, was determined by fluorescence quenching measurements with a flow cytometer. We show that the anthroyloxy moieties of the probes locate at a graded series of depths in the outer leaflet of the plasma membrane of living HeLa cells, in a manner similar to that previously observed for model membrane systems, and mitochondria. For different n, the efficiency of quenching with an aqueous phase quencher, Cu+2, was 2 greater than or equal to 3 greater than 6 greater than or equal to 7 greater than 9 greater than 12 greater than 16. Therefore, flow cytometry permits use of these probes for measurements of dynamic parameters related to membrane fluidity at different depths in the plasma membranes of living cells. 相似文献
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Jeff Davis 《Evolution and human behavior》2012,33(6):647-656
This study presents a test of general life history theory by estimating the association between perceived environmental threat (PET) and reproductive outcomes among American youth. Data for the study came from the National Longitudinal Survey of Youth 1997–2009, which consists of a nationally representative sample of youth. Analyses were based on a subsample of N= 4748 respondents who were aged 12–14 years in 1997; reported having no biological children in 1997; and, for females, were not pregnant at the time of the 1997 interview. PET was measured using 11 questionnaire items that asked respondents about experiences with violent assaults from peers, witnessing violent assaults, and nonviolent crime victimization. First childbirth occurred earlier among respondents who reported higher PET in 1997. Average fertility during the study period was also higher among respondents with higher PET. Male and female respondents' reproductive behaviors were similarly associated with PET. However, only among female respondents was the association between PET and fertility moderated by the quality of the household environment. 相似文献
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Proteins for therapeutic use may contain small amounts of partially misfolded monomeric precursors to postproduction aggregation. To detect these misfolded proteins in the presence of an excess of properly folded protein, fluorescent probes such as 8-anilino-1-naphthalene sulfonate (ANS) are commonly used. We investigated the possibility of using isothermal titration calorimetry (ITC) to improve the detection of this type of conformational change using hydrophobic probes. As a case study, conformational changes in human polyclonal immunoglobulin G (IgG) were monitored by measuring the enthalpies of binding of ANS using ITC. Results were compared with those using fluorescence spectroscopy. IgG heated at 63 °C was used as a model system for “damaged” IgG. Heat-treated IgG can be detected already at levels below 5% with both ITC and fluorescence. However, ITC allows a much wider molar probe-to-protein ratio to be sampled. In particular, using reverse titration experiments (allowing high probe-to-protein ratios not available to fluorescence spectroscopy), an increase in the number of binding sites with a Kd > 10 mM was observed for heat-treated IgG, reflecting subtle changes in structure. Both ITC and fluorescence spectroscopy showed low background signals for native IgG. The nature of the background signals was not clear from the fluorescence measurements. However, further analysis of the ITC background signals shows that a fraction (8%) binds ANS with a dissociation constant of approximately 0.2 mM. Measurements were also carried out at pH 4.5. Precipitation of IgG was induced by ANS at concentrations above 0.5 mM, interfering with the ITC measurements. Instead, with the nonfluorescent probes 4-amino-1-naphthalene sulfonate and 1-naphthalene sulfonate, no precipitation is observed. These probes yield differences in the enthalpies of binding to heated and nonheated IgG similar to ANS. The data illustrate that ITC with low-molecular-weight probes is a versatile tool to monitor conformational changes in proteins with a wider application potential than fluorescence measurements. 相似文献
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The old saying that ‘love heals'' has some truth to it. The intricate dance between two neuropeptides both regulates our ability to love and influences our health and well-being.Love is deeply biological. It pervades every aspect of our lives and has inspired countless works of art. Love also has a profound effect on our mental and physical state. A ‘broken heart'' or a failed relationship can have disastrous effects; bereavement disrupts human physiology and might even precipitate death. Without loving relationships, humans fail to flourish, even if all of their other basic needs are met.As such, love is clearly not ‘just'' an emotion; it is a biological process that is both dynamic and bidirectional in several dimensions. Social interactions between individuals, for example, trigger cognitive and physiological processes that influence emotional and mental states. In turn, these changes influence future social interactions. Similarly, the maintenance of loving relationships requires constant feedback through sensory and cognitive systems; the body seeks love and responds constantly to interaction with loved ones or to the absence of such interaction.Without loving relationships, humans fail to flourish, even if all of their other basic needs are metAlthough evidence exists for the healing power of love, it is only recently that science has turned its attention to providing a physiological explanation. The study of love, in this context, offers insight into many important topics including the biological basis of interpersonal relationships and why and how disruptions in social bonds have such pervasive consequences for behaviour and physiology. Some of the answers will be found in our growing knowledge of the neurobiological and endocrinological mechanisms of social behaviour and interpersonal engagement.Nothing in biology makes sense except in the light of evolution. Theodosius Dobzhansky''s famous dictum also holds true for explaining the evolution of love. Life on Earth is fundamentally social: the ability to interact dynamically with other living organisms to support mutual homeostasis, growth and reproduction evolved early. Social interactions are present in primitive invertebrates and even among prokaryotes: bacteria recognize and approach members of their own species. Bacteria also reproduce more successfully in the presence of their own kind and are able to form communities with physical and chemical characteristics that go far beyond the capabilities of the individual cell [1].As another example, insect species have evolved particularly complex social systems, known as ‘eusociality''. Characterized by a division of labour, eusociality seems to have evolved independently at least 11 times. Research in honey-bees indicates that a complex set of genes and their interactions regulate eusociality, and that these resulted from an “accelerated form of evolution” [2]. In other words, molecular mechanisms favouring high levels of sociality seem to be on an evolutionary fast track.The evolutionary pathways that led from reptiles to mammals allowed the emergence of the unique anatomical systems and biochemical mechanisms that enable social engagement and selectively reciprocal sociality. Reptiles show minimal parental investment in offspring and form non-selective relationships between individuals. Pet owners might become emotionally attached to their turtle or snake, but this relationship is not reciprocal. By contrast, many mammals show intense parental investment in offspring and form lasting bonds with the offspring. Several mammalian species—including humans, wolves and prairie voles—also develop long-lasting, reciprocal and selective relationships between adults, with several features of what humans experience as ‘love''. In turn, these reciprocal interactions trigger dynamic feedback mechanisms that foster growth and health.Of course, human love is more complex than simple feedback mechanisms. Love might create its own reality. The biology of love originates in the primitive parts of the brain—the emotional core of the human nervous system—that evolved long before the cerebral cortex. The brain of a human ‘in love'' is flooded with sensations, often transmitted by the vagus nerve, creating much of what we experience as emotion. The modern cortex struggles to interpret the primal messages of love, and weaves a narrative around incoming visceral experiences, potentially reacting to that narrative rather than reality.