RESUMEN
In the associated main paper (JPBA 2019), substance P was shown to be a valuable neuropeptide reporter substance to monitor the protease activity of serum. The assay was developed based on the predecessor assay using bradykinin (JPBA 2017). Both neuropeptides are of interest in inflammation and pain research and were thus explored for use with capillary blood and blood cards. Here, we present the protocols and set them in perspective to above neuropeptide assays for serum.â¢Neuropeptide reporter substance protease activity assay for use with fresh and dried blood.â¢Dabsylated Substance P and bradykinin are substrates of angiotensin-converting enzyme and other proteases.â¢Neuropeptides of interest in inflammation and pain.
RESUMEN
Recent progress in the domain of time and frequency (T/F) standards requires important improvements of existing time distribution links. Among these, the accuracy of time transfer is actually an important part of the concerns in order to establish and maintain time & space references from ground and/or space facilities. Several time transfers by laser link projects have been carried out over the past 10 years with numerous scientific and metrological objectives. Satellite Laser ranging (SLR) has proven to be a fundamental tool, offering a straightforward, conceptually simple, highly accurate and unambiguous observable. Depending on the mission, LR is used to transmit time over two-way or one-way distances from 500 to several millions of km. The following missions and their objectives employed this technique: European Laser Timing (ELT) at 450 km, Time Transfer by Laser Link (T2L2) at 1,336 km, Laser Time Transfer (LTT) at 36,000 km, Lunar Reconnaissance Orbiter (LRO) at 350,000 km, and MErcury Surface, Space ENvironment, GEochemistry, and Ranging (MESSENGER) at tens of million km. This article describes the synergy between SLR and T/F technologies developed on the ground and in space and as well as the state of the art of their exploitation. The performance and sources of limitation of such space missions are analyzed. It shows that current and future challenges lie in the improvement of the time accuracy and stability of the time for ground geodetic observatories. The role of the next generation of SLR systems is emphasized both in space and at ground level, from the point of view of GGOS and valuable exploitation of the synergy between time synchronization, ranging and data transfer.
RESUMEN
Phycobilin fluorescence of Anacystis nidulans grown at 28 degrees C increases substantially upon cooling below 10 degrees C. A maximal increase is found around -5 degrees C and amounts to 300%, with almost complete reversibility upon re-warming. Illumination with actinic light leads to considerable stimulation of the cold-induced phycobilin fluorescence increase. Analysis of the light stimulation phenomenon reveals: (1) Actinic illumination shifts the fluorescence-temperature characteristic by about 3 degrees C upwards on the T-axis. At temperatures below 5 degrees C the light stimulating effect becomes smaller again and fluorescence-temperature characteristics measured at high and low light intensity converge around -5 degrees C. (2) In the 13-8 degrees C region a large (up to 100%) light-induced phycobilin fluorescence increase is observed, while only negligible changes occur in the dark. (3) 3-(3,4-Dichlorophenyl)-1,1-dimethyl urea (DCMU) as well as uncouplers inhibit the light stimulation, which hence depends on coupled electron transport. In agreement with previous work (Schreiber, U. (1979) FEBS Lett. 107, 4-9) it is concluded that illumination enhances cold-induced phycobilisome detachment by increasing the net negative charge at the outer surface of the thylakoid membrane. The possible role of a fluid leads to ordered transition of membrane lipids (Murata, N. and Fork, D.C. (1975) Plant Physiol. 56, 791-796) is discussed.
Asunto(s)
Clorofila/metabolismo , Frío , Cianobacterias/metabolismo , Pigmentos Biológicos/metabolismo , Transferencia de Energía/efectos de la radiación , Luz , Ficobilisomas , Espectrometría de Fluorescencia , Desacopladores/efectos de la radiaciónRESUMEN
1. The recently described reaction of ATP-induced luminescence is analyzed for its relation to other ATP-induced reactions such as ATP-driven transmembrane proton gradient formation and ATP-driven reverse electron flow. 2. In the absence of phenazine methosulfate ATP-induced luminescence is optimal while the main phase of ATP-driven reverse electron flow is eliminated. 3. DCMU which by itself causes a much smaller luminescence, inhibits the ATP-induced luminescence. 4. Nigericin plus valinomycin, but not each by itself, fully inhibit the ATP-induced luminescence. 5. The observations are interpreted as indicating that ATP stimulates luminescence by a 2-fold mechanism: (a) increasing the amount of the reducing primary electron acceptor of Photosystem II, Q, and (b) creating a transmembrane electrochemical potential which serves to decrease the activation energy required for the charge recombination reaction which leads to luminescence.
Asunto(s)
Adenosina Trifosfato/farmacología , Cloroplastos/metabolismo , Adenosina Trifosfatasas/metabolismo , Clorofila/metabolismo , Cloroplastos/efectos de los fármacos , Ditionita/farmacología , Diurona/farmacología , Transporte de Electrón/efectos de los fármacos , Cinética , Mediciones Luminiscentes , Metosulfato de Metilfenazonio/farmacología , Nigericina/farmacología , Plantas , Valinomicina/farmacologíaRESUMEN
The I-D transient in the chlorophyll fluorescence induction curve (kautsky effect) is investigated in the view of recently discovered rapid changes in energy distribution between the two photosystems (Schreiber, U. and Vidaver, W., FEBS Lett., in the press). Fluorescence induction curves differ appreciably depending on whether measured at lambda less than 690 nm, originating in pigment system II, or at lambda greater than 715 nm, which is in part from pigment system I. The differences occur as well in the rapid part of the induction curve (O-I-D-P) as in the slower P-S decay. Most significant changes in energy distribution are indicated in the region of the I-D dip, being induced by appropriate preillumination. The effect is studied by (a) comparing the individual fluorescence time courses at lambda less than 690 nm and lambda greather than 715, (b) plotting F less than 690 vs. F greater than 715 and (c) recording time courses of F less than 690/F greater than 715 ratios. In (a) the I and D characteristics are delayed at F greater than 715 relative to F less than 690, which is accompanied by periods close to I and D, where the two emissions follow inverse courses. In (b) the I-D dip corresponds to a loop. And in (c) it is shown that a rapid ratio decay, reflecting increasing excitation of System I pigments, is initiated before the I-D dip. These data indicate that the I-D transient is caused by a rapid switch of energy distribution in favor of System I and resulting stimulation of Q reoxidation via the electron transport chain. It is suggested that as with the slow fluorescence transients the rapid also can be understood as a composite of two different changes, (1) direct changes resulting from a switch in energy distribution, which are inverse for F less than 690 and F greater than 715, and (2) indirect changes due to stimulated Q reduction or Q oxidation, which are parallel for both emissions. The rapid ratio decay, correlated to I-D, persists and is even stimulated in the presence of electron transport inhibitors. This and the speed of the phenomenon make it improbable that the rapid energy distribution changes are affected by an ion flux-induced mechanism. It is proposed that the electrical field across the thylakoid membrane is involved in the energy switch mechanism.
Asunto(s)
Fotosíntesis , Plantas/metabolismo , Oscuridad , Transferencia de Energía , Cinética , Luz , Espectrometría de Fluorescencia , Factores de TiempoRESUMEN
The light minus dark difference spectrum and the kinetics of the indicator pigment C-550 have been measured at room temperature in isolate, envelope-free chloroplasts in the presence of 3-(3' ,4'-dichlorophenyl)-1,1-dimethylurea (DCMU). The C-550 spectrum indicates a band shift with peaks at 540 and 550 nm and has an isobestic point at 545 nm. On the assumption of 400 chlorophyll molecules per electron transfer chain the differentaial extinction coefficient delta epsilon (540-550) is calculated to be approximately 5 mM-1 . CM-1. The kinetics of the C-550 absorbance change, occurring upin the onset of continuous illumination, are shown to be biphasic and strictly correlated with the kinetics of the complementary area measured from the fluorescence induction curve under identical cinditions and with those of the absorbance increase at 320 nm due to photoreduction of Q. The lighted-induced change in these three parameters can be described as a function of the variable fluorescence yield change occurring under the same conditions. Such functions are non-linear and reveal a heterogeneous dependence of the variable fluorescence yield on the fraction of closed System II reaction centers. It is concluded that for every molecule of the primary electron acceptor Q of Photosystem II that is photochemically reduced there corresponds an equivalent change in the absorbance of the indicator pigment C-550 and in the size of the complementary area. Ths, C-550 and area are two valid parameters for monitoring the primary photochemical activity of System II at the room temperature.
Asunto(s)
Cloroplastos/metabolismo , Pigmentos Biológicos/metabolismo , Oscuridad , Diurona , Cinética , Luz , Oxidación-Reducción , Fotosíntesis , Plantas , Espectrometría de Fluorescencia , EspectrofotometríaRESUMEN
1. The reverse reactions induced by coupled ATP hydrolysis were studied in spinach chloroplasts by measurements of the ATP-induced increase in chlorophyll fluorescence reflecting reverse electron flow, and of the ATP-induced decrease in 9-aminoacridine fluorescence, representing formation of the transthylakoidal proton gradient (deltapH). ATP-induced reverse electron flow was kinetically analysed into three phases, of which only the second and third one were paralleled by corresponding phases in deltapH formation. The rapid first phase and formation of a deltapH occur also in the absence of the electron transfer mediator phenazine methosulfate. 2. The rate and extent of the reverse reactions were measured at temperatures in the range from 0 to 30 degrees C. The rate of formation of delta pH and of reverse electron flow were faster at high temperatures, but the maximal extent of delta pH and chlorophyll fluorescence increase were observed at the lowest temperature. Considering rate and extent of the ATP-stimulated reactions, a temperature optimum around 15 degrees C was found. Light activation of the ATPase occurred throughout the range studied. At 0 degrees C and in the presence of inorganic phosphate the activated state for ATPase was maintained for more than 10 min. 3. The ATP-induced rise in chlorophyll fluorescence yield was found to be of similar magnitude as the rise induced by 3-(3,4-dichlorophenyl)-1,1-dimethyl-urea (DCMU), when both were measured with an extremely weak measuring beam. It is concluded, that both effects, although derived via distinctly different pathways, are limited by the same electron donating or electron accepting pool.
Asunto(s)
Adenosina Trifosfato/metabolismo , Cloroplastos/metabolismo , Clorofila/metabolismo , Cloroplastos/efectos de los fármacos , Diurona/farmacología , Transporte de Electrón , Concentración de Iones de Hidrógeno , Cinética , Luz , Metosulfato de Metilfenazonio/farmacología , Plantas , Espectrometría de FluorescenciaRESUMEN
A newly observed general chlorophyll fluorescence induction effect in plants is described. Fluorescence yield can rise through as many as four different phases (alpha, beta, gamma, ) in the dark, when intact cells or leaves are rapidly heated (within approx. 2.5 s) from 20 to 40-50 degrees C. An analysis of this temperature-jump fluorescence induction in Scenedesmus obliquus leads to the following: 1. Phase alpha is due to removal of S-quenching and appears to be related to heat deactivation of the water-splitting enzyme system. With prolonged heating, irreversibility of alpha upon recooling reflects irreversible damage to the water-splitting enzyme system. 2. beta is independent of the S-states and of the redox state of primary System II acceptor Q. It is suggested that beta parallels functional separation of Q from the System II trapping centre. This effect is highly reversible. 3. gamma and beta reflect reduction of primary System II acceptor Q by a heat-induced endogenous reductant, which is probably identical to hydrogenase. Critical temperatures for pronounced alpha and beta phases differ markedly in different plants. Possible correlations between temperature-jump fluorescence inductio, thylakoid membrane lipid composition, lipid phase transition and lipid-protein interactions are discussed.
Asunto(s)
Clorofila/metabolismo , Plantas/metabolismo , Chlorophyta/metabolismo , Cianuros/farmacología , Oscuridad , Diurona/farmacología , Cinética , Plantas/efectos de los fármacos , Especificidad de la Especie , Espectrometría de Fluorescencia , TemperaturaRESUMEN
The heat-induced changes of chlorophyll fluorescence excitation and emission properties were studied in isolated chloroplasts of Larrea divaricata Cav. An analysis of the temperature dependency of fluorescence, under Fo and Fmax conditions, of temperature-jump fluorescence induction kinetics, and of 77 degrees K emission spectra of preheated chloroplasts revealed two major components in the heat-induced fluorescence changes: (1) a fluorescence rise, reflecting the block of Photosystem II reaction centers; and (2) a fluorescence decrease, caused by the functional separation of light-harvesting pigment protein complex from the rest of the pigment system. Preferential excitation of chlorophyll a around 420 nm, produced a predominant fluorescence rise. Preferential excitation of chlorophyll b, at 480 nm, gives a predominant fluorescence decrease. It is proposed that the overlapping of the fluorescence decrease on the somewhat faster fluorescence rise, results in the biphasic fluorescence rise kinetics observed in isolated chloroplasts. Both the rise component and the decay component are affected by the thermal stability of the chloroplasts, acquired during growth of the plants in different thermal environments. Mg2+ enhances the stability against heat-damage expressed in the decrease component, but has no effect on the rise component. Heat pretreatment leads to a decrease of the variable fluorescence in the light-induced 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) rise curve, but no change in half-rise time is observed. It is concluded that the block of Photosystem II reaction centers precedes the loss of the light-harvesting pigment protein complex. However, the approximately antiparallel heat-induced Fmax decrease and Fo increase suggest a common cause for the two events. A heat-induced perturbation of the thylakoid membrane is discussed.
Asunto(s)
Clorofila/metabolismo , Cloroplastos/metabolismo , Fotosíntesis , Células Vegetales , Cloroplastos/efectos de la radiación , Diurona/farmacología , Fluorescencia , Calor , Cinética , Luz , Oxígeno/metabolismoRESUMEN
Mounting concerns regarding the environmental impact of herbicides has meant a growing requirement for accurate, timely information regarding herbicide residue contamination of, in particular, aquatic systems. Conventional methods of detection remain limited in terms of practicality due to high costs of operation and the specialised information that analysis provides. A new phytotoxicity bioassay was trialled for the detection of herbicide residues in filter-purified (Milli-Q) as well as natural waters. The performance of the system, which combines solid-phase extraction (SPE) with the ToxY-PAM dual-channel yield analyser (Heinz Walz GmbH), was tested alongside the traditional method of liquid chromatography-mass spectrometry (LC-MS). The assay methodology was found to be highly sensitive (LOD 0.1 ng L(-1) diuron) with good reproducibility. The study showed that the assay protocol is time effective and can be employed for the aquatic screening of herbicide residues in purified as well as natural waters.
Asunto(s)
Técnicas Biosensibles , Pruebas de Toxicidad , Agua/análisisRESUMEN
A phytotoxicity assay based on the ToxY-PAM dual-channel yield analyser has been developed and successfully incorporated into field assessments for the detection of phytotoxicants in water. As a means of further exploring the scope of the assay application and of selecting a model biomaterial to complement the instrument design, nine algal species were exposed to four chemical substances deemed of priority for water quality monitoring purposes (chlorpyrifos, copper, diuron and nonylphenol ethoxylate). Inter-species differences in sensitivity to the four toxicants varied by a factor of 1.9-100. Measurements of photosystem-II quantum yield using these nine single-celled microalgae as biomaterial corroborated previous studies which have shown that the ToxY-PAM dual-channel yield analyser is a highly sensitive method for the detection of PS-II impacting herbicides. Besides Phaeodactylum tricornutum, the previously applied biomaterial, three other species consistently performed well (Nitzschia closterium, Chlorella vulgaris and Dunaliella tertiolecta) and will be used in further test optimisation experiments. In addition to sensitivity, response time was evaluated and revealed a high degree of variation between species and toxicants. While most species displayed relatively weak and slow responses to copper, C. vulgaris demonstrated an IC10 of 51 microgL-1, with maximum response measured within 25 minutes and inhibition being accompanied by a large decrease in fluorescence yield. The potential for this C. vulgaris-based bioassay to be used for the detection of copper is discussed. There was no evidence that the standard ToxY-PAM protocol, using these unicellular algae species, could be used for the detection of chlorpyrifos or nonylphenol ethoxylate at environmentally relevant levels.
Asunto(s)
Bioensayo/métodos , Chlorophyta/efectos de los fármacos , Diatomeas/efectos de los fármacos , Monitoreo del Ambiente/métodos , Fotosíntesis/efectos de los fármacos , Pruebas de Toxicidad/métodos , Contaminantes Químicos del Agua/toxicidad , Cloropirifos/toxicidad , Cobre/toxicidad , Diurona/toxicidad , Glicoles de Etileno/toxicidad , Fluorometría/métodos , Complejo de Proteína del Fotosistema II/metabolismo , Sensibilidad y Especificidad , Especificidad de la Especie , Contaminantes Químicos del Agua/análisisRESUMEN
Valinomycin/K+ is shown to selectively interact at sub-micromolar concentrations with the cytochrome bf complex in thylakoid membranes, inducing a red shift of the ferrohaem b absorbance alpha-band, a slow down of post-illumination b-reoxidation and a corresponding increase of b-reduction level in continuous light. These effects of valinomycin/K+ are not related to its field dissipating action, as they are not affected by nonactin. Presence of K+ is required. Phenomenologically the valinomycin/K+ effects are similar to those caused by 10 times higher MOA stilbene nonactin. Presence of K+ is required. Phenomenologically the valinomycin/K+ effects are similar to those caused by 10 times higher MOA stilbene concentrations. However, synergism is observed between the two inhibitors, suggesting different modes of action. When both inhibitors are combined more than one haem b can be reduced by illumination.
Asunto(s)
Cloroplastos/metabolismo , Grupo Citocromo b/metabolismo , Citocromos/metabolismo , Potasio/farmacología , Estilbenos/farmacología , Valinomicina/farmacología , Verduras/metabolismo , Citocromos f , Sinergismo Farmacológico , Cinética , Luz , Oxidación-Reducción , EspectrofotometríaRESUMEN
Upon onset of saturating continuous light only the first part of the observed polyphasic fluorescence rise follows Q(A) reduction (photochemical phase), whereas the remaining part (thermal phases) is kinetically limited by relatively slow reactions with light saturated half-times in the order of 10-50 ms. A simple hypothesis is presented for the interpretation of these fundamentally different types of variable fluorescence. The hypothesis, which is based on the reversible radical pair model of PSII, assumes stimulation of both prompt and recombination fluorescence upon Q(A) reduction, with only recombination fluorescence being in competition with nonradiative energy loss processes at the reaction centers. It is proposed that changes in the rate constants of these processes modulate the yield of recombination fluorescence in closed centers, thus causing large variations in the maximal fluorescence yield and also giving rise to the 'thermal phases'. This hypothesis can reconcile numerous experimental findings which so far have seemed difficult to interpret.
Asunto(s)
Clorofila/metabolismo , Fotosíntesis , Proteínas del Complejo del Centro de Reacción Fotosintética/metabolismo , Cloroplastos/metabolismo , Fluorescencia , Cinética , Luz , Complejos de Proteína Captadores de Luz , Oxidación-ReducciónRESUMEN
Under normal physiological conditions the state of the cyt bf complex is characterized by rapid reoxidation kinetics of cyt b-563 following flash-illumination. It is known that these kinetics are dramatically slowed down under oxidizing conditions. Here we show that this slow-down of cyt b-563 oxidation is the consequence of a relatively slow (half-time of several minutes) transformation of the cyt bf complex into a distinctly different state (termed state-s). Reversal to the normal state requires strong reductive treatment or light-induced electron transport. The results are in line with a recent model of functional cyt bf dimers [Cramer et al., Annu. Rev. Plant Physiol. Plant Mol. Biol. 47 (1996), 477-5081, if it is assumed that state-s reflects the monomeric state of the bf complex.
Asunto(s)
Cloroplastos/química , Grupo Citocromo b/fisiología , Citocromos/fisiología , Fotosíntesis , Citocromos f , Sustancias Macromoleculares , Oxidación-Reducción , Spinacia oleraceaRESUMEN
In agreement with previous work [Shuvalov, Heber and Schreiber (1988) FEBS Lett. 258, 27-31] two hemes (low potential (LP) and extra low potential (XLP)) per two pheophytins were found in isolated D1D2Cyt b-559 complexes. Reductive and oxidative redox titrations demonstrate that the Em of the LP form is at about +150 mV. It is independent of pH between pH 7.2 and 9.4. The XLP heme is autoxidizable at pH 7.2 and displays, at this pH, an Em of -45 mV. Both the LP and XLP hemes show absorption peaks at 559 nm. They are proposed to have bis-histidine ligation of the heme iron. At pH 9.4, the XLP heme splits into two forms. One of them has an Em of +40 mV, and absorption peaks at 559 nm showing the bis-histidine ligation. The other displays an Em of -220 mV and the peak is shifted to 562 nm. This last form is proposed to be due to the incorporation of OH- which occupies the 6th coordination position of the heme Fe(III) at high pH. The pK value for the conversion of the XLP heme is close to 7.7. In a structure simulation of the alpha-helices of alpha- and beta-polypeptide, the beta-polypeptide, but not the alpha-polypeptide, reveals a distance between the histidine N and the heme Fe which permits stable N-Fe coordination. In the alpha-polypeptide, OH- can be incorporated between N and Fe. The functional role of the two hemes of cyt b-559 is briefly discussed with respect to water oxidation and cyclic electron transfer.
Asunto(s)
Grupo Citocromo b/química , Hemo/química , Complejo de Proteína del Fotosistema II , Plantas/química , Clorofila/análisis , Grupo Citocromo b/metabolismo , Transporte de Electrón , Concentración de Iones de Hidrógeno , Oxidación-Reducción , Feofitinas/análisis , Espectrofotometría , TermodinámicaRESUMEN
EPR properties of Cyt b-559 have been investigated in intact chloroplasts that are functionally competent in O2 evolution and in CO2 fixation. After chemical oxidation of Cyt b-559 by 10 mM 2,3-dicyano, 4,5-dichloro-p-benzoquinone (DDQ) the major part of Cyt b-559 is found to be present in the high spin Fe(III) form. Only a small fraction of low spin heme Fe(III) (less than 5%) was formed by chemical or light-induced oxidation. This fraction increased during aging of intact chloroplasts. A comparison with the EPR signal of Fe(III) in myoglobin (Mb) reveals that the structure of the high spin signal in intact chloroplasts is indicative for the presence of an axial OH- ligand at the heme Fe(III). This type of ligation comprised a considerable part (approximately 40%) of the total Cyt b-559 content. Removal of the Mn-cluster caused a change of the EPR parameters of OH- ligation. When in intact chloroplasts the heme Fe is chemically oxidized to Fe(III) ligated by OH-, this OH- ligation disappeared after a subsequent illumination at 80K by red light. Upon illumination at 140K this disappearance was accompanied by the formation of a high spin Fe(III) that is not ligated by OH-. These results are discussed in terms of removal of OH- from Fe(III) caused by structural changes or photooxidation at a complex of Cyt b-559 that could possibly also comprise the Mn-cluster. This photooxidation is assumed to be accompanied by the formation of a bound OH. radical. The possibility is discussed that this process is related to photosynthetic water oxidation.
Asunto(s)
Cloroplastos/química , Grupo Citocromo b/química , Hemo/química , Fotosíntesis , Complejo de Proteína del Fotosistema II , Benzoquinonas/farmacología , Cloroplastos/metabolismo , Cloroplastos/efectos de la radiación , Frío , Grupo Citocromo b/efectos de los fármacos , Grupo Citocromo b/metabolismo , Espectroscopía de Resonancia por Spin del Electrón , Compuestos Férricos/química , Hemo/metabolismo , Hidróxidos/química , Hidroxilamina , Hidroxilaminas/farmacología , Luz , Oxidación-Reducción , Spinacia oleraceaRESUMEN
Leaves of Vicia faba were collected from the field and the greenhouse and transmittance of epidermal peels from adaxial and abaxial sides was determined in the wavelength range from 250 to 800 nm using a spectrophotometer equipped for the measurement of turbid samples. From the same leaves, epidermal transmittance was estimated by a recently developed fluorometric method. Both methods gave highly correlated results with a slope of the regression line between both methods close to 1 and an intercept close to 0. Transmittances at around 310 nm as low as 3% were detected in the adaxial epidermis of field-grown leaves, while transmittance could be as high as 70% in the abaxial epidermis of greenhouse-grown leaves. There was a strong correlation between UV-A (ca. 366 nm) and UV-B (ca. 310 nm) transmittance detected by both methods which could be explained by the pigment composition in methanolic extracts where flavonols accounted for 90% of the absorption at 310 nm in the extract, while hydroxycinnamic acid derivatives which absorb only at the shorter wavelength constituted about 5%. It is concluded that the fluorescence method which allows rapid measurements on intact leaves can provide a quantitative estimate of epidermal transmittance for UV-B (280-320 nm) and UV-A (320-400 nm) radiation.
RESUMEN
Heat tolerance limits for a variety of vascular plant leaves were determined both with the conventional post-culture necrosis method and by measurements of the heat-induced increase in chlorophyll fluorescence (F-T curves). The reliability of the fluorescence test was improved with the addition of far-red background light which counteracts dark reduction of the Photosystem II acceptor pool by heat-stimulated endogenous electron donors. This was of particular importance in the case of xeromorphic leaves in which the diffusion barrier for oxygen is high. A satisfactory correlation was found between T L50, the temperature at which a 30 min exposure results in 50% necrotic leaf area following post culture, and the critical temperature, T c ,the temperature at which the dark fluorescence level begins to increase during slow heating of a leaf sample at a rate of 0.7 K min-1, in the fluorescence test. The correlation can be described by a linear function, T L50=1.12 T c -5.37,with a correlation coefficient, r=0.87. Maximal deviation of the regression line from the line T L50=T c was 1.2 K, with 22 determinations for leaves with widely varying heat tolerance limits. This shows that heat-induced fluorescence changes within the thylakoid membrane may be connected with the irreversible leaf tissue damage which occurs following prolonged exposure to high temperature. On the basis of the heat dosage equation of Lepeschkin, a more general expression can be obtained which allows calculation of the accumulated heat dosage under the experimental conditions of the standard fluorescence test (slow heating, 0.7 K min-1). Such calculations reveal that for a given species the 'fraction of critical dosage' begins to increase, i.e. accumulating heat reaches an injurious level, at a temperature which approximately coincides both with T L50, obtained with the necrosis method, and with T c ,the critical temperature derived from the fluorescence test. Hence, the increase in fraction of critical dosage and the rise in chlorophyll fluorescence seem to concur. It is concluded that the fluorescence assay provides a rapid and reliable means of determining the heat tolerance limit of leaf tissue.
RESUMEN
Although topical drugs are usually applied at a convenient site, the target for the drug interaction may be systemic. Phonophoresis is the use of ultrasound to enhance the delivery of topical applied drugs. The purposes of our study were to investigate the in vitro penetration and the in vivo transport of flufenamic acid in dependence of ultrasound. Percutaneous absorption studies are performed in various in vitro models to determine the rate of drug absorption via the skin. We designed a phonophoretic drug delivery system to investigate the influence of ultrasound on transmembrane transport of different drugs. We investigated the absorption of flufenamic acid in a buffer medium in dependence of ultrasound energy and application time. For evaluating membrane penetration of flufenamic acid, the concentration range of buffer solution was measured. Ultrasound energy was supplied for between 5 and 30 min at a range of intensities up to 1.5 W/cm2, energy levels commonly used for therapeutic purpose. The pronounced effect of ultrasound on the transmembrane absorption of the drug was observed at all ultrasound energy levels studied. The time of application was found to play an important role in delivery and transport of drug. Dependent on time, we observed a rise of temperature up to 4.5 degrees C. It appears that there was no difference between an intensity of 0.3 and 1.5 W/cm2 and the measured drug concentrations in solution. The highest penetration was observed at an intensity of 1.0 W/cm2 after 30 min. These results were not significantly different from concentration measurements after 30 min and 0.5 and 1.5 W/cm2. It seems that the arise of drug concentration is caused by effects of temperature and by variation of membrane delivery in dependence of temperature. Using this in vitro model we note it is possible to compare the transdermal penetration and absorption of commercial flufenamic ointment in volunteers.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacocinética , Ácido Flufenámico/farmacocinética , Fonoforesis , Absorción Cutánea , Administración Tópica , Adulto , Transporte Biológico , Tampones (Química) , Sistemas de Liberación de Medicamentos , Femenino , Ácido Flufenámico/administración & dosificación , Ácido Flufenámico/sangre , Humanos , Técnicas In Vitro , Rodilla , Región Lumbosacra , Masculino , Temperatura , Factores de TiempoRESUMEN
High frequency ultrasound can resolve the structure of the wall of the gastro-intestinal tract into three highly echogenic lines with poorly echogenic zones between them. Sequential removal of layers of the walls of fresh specimens of the gastro-intestinal tract showed the following correlation between the sonographic and histological findings: the innermost and outermost highly echogenic lines correspond to incident and exit echoes. The middle line corresponds with the sub-mucosa and the junctional zones of the mucosa/muscularis mucosa and sub-mucosa/muscularis propria. The intervening low echo lines correspond with the mucosa and muscularis propria.