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Influence of phospholipid long chain polyunsaturated fatty acid composition on neonatal rat cardiomyocyte function in physiological conditions and during glucose-free hypoxia-reoxygenation.

There is evidence that dietary polyunsaturated fatty acids (PUFA) may protect against cardiovascular diseases, but the involvement of the cardiac muscle cell in this beneficial action remain largely unknown. The present study compared the respective influence of n-3 and n-6 PUFA on the function of cultured neonatal rat cardiomyocytes (CM). Cells were grown for 4 days in media enriched either n-3 (eicosapentaenoic acid, EPA and docosahexaenoic acid, DHA) or n-6 (arachidonic acid, AA) PUFA. The PUFA n-6/n-3 ratio in the phospholipids was close to 1 and 20 in the n-3 and n-6 cells, respectively. The transmembrane potentials were recorded using microelectrodes and the contractions were monitored with a photoelectric device. In physiological conditions, the increase of n-6 PUFA level in the phospholipids resulted in a significant decrease in the maximal rate of initial depolarization (-16%). In opposition, the action potential amplitude and duration were not altered, and the cell contraction outline was not affected. Ischemia was simulated in vitro using a substrate-free, hypoxia-reoxygenation procedure in a specially designed gas-flow chamber. The progressive loss of electrical activity induced by the substrate-free, hypoxic treatment was affected by the n-6/n-3 ratio, since the n-6 rich CM displayed a slower depression of the AP amplitude and duration parameters. Conversely, the recovery of the resting potential (MDP) during reoxygenation was faster in n-3 CM, whereas the recovery of the contraction parameters was unaffected by the fatty acid composition of the cells. These results suggested that, in physiological conditions, the modification of long chain PUFA balance in the phospholipids of cardiac muscle cells may modulate the initial AP upstroke, which is governed by sodium channels. Moreover, the presence of n-3 PUFA appeared to accelerate the electrical depression during substrate-free hypoxia but in turn to allow a faster recovery upon reoxygenation.

Action Potentials↗

Comparison of semiautomated method with official optical somatic cell counting method III for determining somatic cells in milk.

The new method specifying the Fossomatic-90 differs from the official method, 46.105-46.109, in that the modified instrument includes a halogen lamp; a semiconductor photoelectric detector; a less expensive, bench-top cabinet; manual injection of a larger sample, and a reduced capacity. The new instrument was compared with 2 optical somatic cell counters in routine use. On each of 3 days, 12 subsamples were prepared for each of 5 cell count levels from AM milk with half kept fresh and half preserved with 0.05% potassium dichromate. Subsamples were refrigerated and read 30+ h post-collection. Duplicate sets were read in random order on each machine daily (CV 0.77%). Two sets of slides read by 2 technicians each (strip reticle on 2 smears/slide) gave geometric mean direct microscopic somatic cell count (DMSCC) levels of 296, 526, 772, 930, and 1438 th/mL. Within-technician CV values (from day-level means) ranged from 1.68 to 2.28%. Geometric mean cells in th/mL on the new machine were significantly higher than those on the other two (674 vs 621) and were closer to the DMSCC (694). On the new machine, cell counts were 8.5% greater than on the original machines, were only 2.9% lower than the DMSCC, and showed no significant evidence of bias. Preserved samples averaged slightly greater than fresh (5.3%) but only on the original machines. Carryover by covariance analysis was insignificant. Except for cell levels, high machine precision (error CV value of 1.18%) gave differences with statistical but not practical significance, even for regulatory laboratories.

Animals↗

A simple method for screening photoelectric dyes towards their use for retinal prostheses.

Photoelectric dyes absorb light and convert photon energy to electric potentials. To test whether these dyes could be used for retinal prostheses, a simple in vitro screening system was developed. Retinal neurons were cultured from the eyes of chick embryos at the 10-day embryonic stage, at which time no retinal photoreceptor cells have yet developed. Intracellular calcium elevation was observed with Fluo-4 in cultured retinal neurons before and after photoelectric dye was applied at varying concentrations to the culture medium. Five of 7 photoelectric dyes tested in this in vitro system induced intracellular calcium elevation in cultured chick retinal neurons. The intracellular calcium elevation generated by the 5 photoelectric dyes was blocked by extracellular calcium depletion in the case of all 5 dyes, and, except for one dye, by the presence of voltage-gated calcium channel blockers. The photoelectric dyes absorbed light under an inverted microscope and stimulated retinal neurons. This simple in vitro system allows the screening of photoelectric dyes which can be used for retinal prostheses.

Animals↗

[Inhibitory effect on sulfur dioxide on ciliary motility in rabbit tracheal epithelium and its prevention by intracellular cyclic AMP].

To elucidate the role of endogeneous cyclic AMP in the protection against airway mucosal dysfunction induced by air pollutants, we studied the effect of sulfur dioxide (SO2) on ciliary motility in rabbit cultured tracheal epithelium in vitro. Exposure of cells to perfusate bubbled with SO2 rapidly decreased ciliary beat frequency (CBF), as assessed by a photoelectric method, from 971 +/- 12 to 718 +/- 28 beats/min by 3 ppm SO2 and from 963 +/- 22 to 635 +/- 34 beats/min by 10 ppm SO2 (p < 0.001, in each case). This effect was reversed by washing out the SO2-containing medium and was accompanied by a corresponding decrease in intracellular levels of cyclic AMP. Preincubation of the epithelial cells with salbutamol, vasoactive intestinal peptide, prostaglandin E2 or 3-isobutyl-1-methylxanthine increased cyclic AMP levels and inhibited the decreases in both CBF and cyclic AMP in response to the subsequent application of SO2 at 3 ppm, whereas dexamethasone had no effect. These results suggest that SO2 decreases airway ciliary motility through the reduction of intracellular cyclic AMP concentration, and that drugs that increase endogeneous cyclic AMP may prevent the SO2-induced impairment of mucociliary transport in the respiratory tract.

Animals↗

Photoelectric dye-coupled polyethylene film as a prototype of retinal prostheses.

Photoelectric dyes, which absorb light and convert photon energy to electric potentials, have been previously shown to stimulate retinal neurons in culture. In this study, a photoelectric dye was coupled to a polyethylene film surface and tested in vitro using retinal tissues from chick embryos at the 12-day embryonic stage, at which time outer segments of retinal photoreceptor cells have not yet developed. Carboxyl moieties were introduced to a polyethylene film surface by fuming nitric acid, and then a photoelectric dye, 2-[2-[4-(dibutylamino)phenyl]ethenyl]-3-carboxymethylbenzothiazolium bromide, was coupled to the film through amide linkage. Intracellular calcium elevation was observed with Fluo-4 in retinal tissues placed on the dye-coupled polyethylene film, in contrast to retinal tissues which had no contact with the film. The response was inhibited by calcicludine, a voltage-gated calcium channel blocker, and also by extracellular calcium depletion. The photoelectric dye, coupled to the polyethylene film surface, absorbed light under a dissecting microscope and stimulated neurons in retinal tissues, showing that the dye-coupled film could be used as a prototype of retinal prostheses.

Aniline Compounds↗

Transmission X-ray microscopy of intact hydrated PtK2 cells during the cell cycle.

Transmission X-ray microscopy makes it possible to investigate biological specimens, i.e. cells and organelles, in their natural wet environment. The main processes determining the contrast in X-ray microscopy are photoelectric absorption and phase shift. X-ray microscopic experiments can therefore be carried out in both amplitude and phase contrast. The Göttingen X-ray microscope at the BESSY storage ring in Berlin is described. PtK2 cells were examined during different stages of the cell cycle. All major constituents of the mitotic apparatus, e.g. chromosomes, centromeres, microtubules and centrosomes, could be visualized, as well as the main structural compartments and organelles of the interphase cell, e.g. nuclear membrane, interphase chromatin, nucleolus and cytoplasmic mitochondria, as well as parts of the cytoskeletal apparatus. In this way new information can be obtained with regard to the ultrastructure of the constituents of intact and unstained cells at a resolution which bridges the gap between light microscopy and electron microscopy. The prospects for the future application of transmission X-ray microscopy in biomedical research are discussed.

Animals↗

Observations on a new non-invasive monitor of skin blood flow.

1. A 'tissue perfusion monitor' (TPM) to non-invasively provide an index of skin blood flow (SkBF) has been developed; it employs photoelectric plethysmographic principles to measure changes in the nett flux of red blood cells in superficial microvasculature. 2. The 'tissue perfusion index' (TPI) varies in proportion to SkBF, provided local haemoglobin concentration does not change significantly. TPI of humans and experimental animals has been shown to indicate reliably, well established phenomena such as decreased SkBF in response to mechanical restriction, cold or Valsalva's manoeuvre, or increased SkBF in response to heat, acetylcholine, sodium nitrite or local nerve blockade. 3. SkBF in sheep was varied between 1 and 156 mL/100g per min as measured with radioactive microspheres. Simultaneous measurements were made using the TPM and four laser-Doppler instruments. The TPI yielded a correlation coefficient of 0.938, and when data were expressed as percentage change, the regression line did not differ significantly from the line of identity and the root-mean-square-error was 6.2%. Data for the laser-Doppler indices of SkBF were, respectively, 0.549-0.786, highly significant deviations in slopes, and 13.6-16.7%. 4. Thus, the TPI is a reliable index of changes in SkBF. Compared with some other available instruments, the TPM is more precise; it is also less sensitive to movement artefact, can be completely portable by battery operation, probes can be multiplexed to a single meter and it is likely to be much less expensive than current lasers. 5. Applications include, for example, experimental investigations of SkBF in man and animals, clinical uses such as evaluation of the efficacy of regional nerve blockade or of circulatory restitution after reconstructive surgery, and clinical tests of neurovascular function.

Adult↗

Modification of the n-6/n-3 fatty acid ratio in the phospholipids of rat ventricular myocytes in culture by the use of synthetic media: functional and biochemical consequences in normoxic and hypoxic conditions.

The respective roles of exogenous polyunsaturated fatty acids on the lipid composition, physiological properties and enzyme release was investigated on isolated cardiac muscle cells in normoxia and hypoxia. Rat neonatal ventricular myocytes were grown for 5 days in conventional serum-supplemented medium. Cells were then incubated for 24 h in fully chemically-defined media featuring a balanced fatty acid composition containing either linoleic acid (18:2 n-6) or linolenic acid (18:3 n-3) as sole polyunsaturated fatty acid source. Transmembrane potentials were monitored with microelectrodes and contractions with a photoelectric device. The radio of n-6 to n-3 phospholipid fatty acids increased from 6.3 in control cells to 20.2 in cells exposed to n-6 fatty acids (SM6) and decreased to 1.4 in those exposed to n-3 fatty acids (SM3). These modifications had no influence on the electrical and mechanical activities and on automaticity in normoxic conditions. The action potential depression under hypoxia was less severe in SM6 cells, whereas there was a better electrophysiological recovery upon reoxygenation in SM3 cells. However, the loss of lactate dehydrogenase during sustained hypoxic treatment was not affected by changes in phospholipid fatty acid pattern. These results suggest that the effect of the polyunsaturated fatty acid balance depends on the cellular function under study and on the environmental conditions.

Animals↗

Novel carboxylated oligothiophenes as sensitizers in photoelectric conversion systems.

Novel carboxylated oligothiophenes with different thiophene units were designed and synthesized as photosensitizers in dye-sensitized solar cells (DSSCs) for efficient opto-electric materials. The introduction of -COOH into thiophene molecules can lead to a red shift of UV-visible absorption, increase light-harvesting efficiency, and enhance photoinduced charge transport by forming efficient covalent bonds to the substrate surface. A red shift of the absorption spectrum of oligothiophene is also achieved by the increase in the number of thiophene units. The DSSCs based on the oligomers have excellent photovoltaic performances. Under 100 mW cm(-2) irradiation a short-circuit current of 10.57 mA cm(-2) and an overall energy conversion efficiency of 3.36 % is achieved when pentathiophene dicarboxylated acid was used as a sensitizer. The incident photo-to-current conversion efficiency (IPCE) has a maximum as high as 80 %. In addition, photovoltage and photocurrent transients show that slow charge recombination in DSSCs is important for efficient charge separation and excellent photoelectric conversion properties of the oligomers. These initial and promising results suggest that carboxylated oligothiophenes are efficient photosensitizers.

Journal Article↗

Ciliary activity of the middle ear lining in guinea pigs.

Since the middle ear lining is an extension and a modification of the respiratory epithelium, it is conceivable that it has a mucociliary system and plays an important role in clearing the middle ear cavity. It has already been noted in morphological investigations that the middle ear mucosa has ciliated cells. To our knowledge, however, ciliary activity has never been observed directly. In our research, we used the photoelectric method to study ciliary activity of the middle ear mucosa directly and quantitatively. We made special reference to the frequency of ciliary beating at various sites within the middle ear cavity. Ciliary activity was found to exist in the eustachian tube and the middle ear, the same as in other respiratory organs, and this activity was stronger in cells distal to the eustachian tube.

Animals↗

Translational and Brownian motion in laser-Doppler flowmetry of large tissue volumes.

This study reports the derivation of a precise mathematical relationship existing between the different p-moments of the power spectrum of the photoelectric current, obtained from a laser-Doppler flowmeter (LDF), and the red blood cell speed. The main purpose is that both the Brownian (defining the 'biological zero') and the translational movements are taken into account, clarifying in this way what the exact contribution of each parameter is to the LDF derived signals. The derivation of the equations is based on the quasi-elastic scattering theory and holds for multiple scattering (i.e. measurements in large tissue volumes and/or very high red blood cell concentration). The paper also discusses why experimentally there exists a range in which the relationship between the first moment of the power spectrum and the average red blood cells speed may be considered as 'linear' and what are the physiological determinants that can result in nonlinearity. A correct way to subtract the biological zero from the LDF data is also proposed. The findings should help in the design of improved LDF instruments and in the interpretation of experimental data.

Animals↗

[Effect of roxithromycin on ciliary motility of rabbit tracheal epithelium in culture].

Mucociliary transport plays an important role in the lung defense mechanism by clearing inhaled particles and bacteria from the respiratory tract. In the present study, we tried to determine whether roxithromycin (RXM), a new semisynthetic macrolide, can affect airway mucociliary functions and, if so, what the mechanism of its action is. To do so we measured ciliary beat frequency (CBF) of rabbit cultured tracheal epithelium by a photoelectric method. Addition of RXM (10(-4) M) to a Rose chamber containing epithelial cells caused a rapid increase in CBF from 12.1 +/- 0.6 to 16.2 +/- 0.8 Hz (mean +/- SE, p less than 0.001) within 5 min. This effect was dose-dependent, the maximal increase from the baseline CBF and EC50 being 34.6 +/- 5.0% and 3 x 10(-6)M, respectively. The increase in CBF produced by RXM (3 x 10(-6)M) was not influenced by pretreatment of cells with the beta-adrenergic blocker propranolol, the lipoxygenase inhibitor AA-861, or the Ca(2+)-channel blocker verapamil, but it was partially reduced by the cyclooxygenase inhibitor indomethacin (p less than 0.05). Intracellular cyclic AMP levels were increased by 10(-4)M RXM from 42.3 +/- 8.7 to 72.7 +/- 7.0 pmoles/mg protein (p less than 0.05). These results indicate that RXM stimulates ciliary motility; an effect that may be attributed at least in part to the synthesis of cyclooxygenase products and cyclic AMP, and suggest that this macrolide could be useful in treating patients with impaired mucociliary transport in the airway.

Animals↗

Intracellular calcium response and adhesiveness of chick embryonic retinal neurons to photoelectric dye-coupled polyethylene films as prototypes of retinal prostheses.

Photoelectric dyes, which absorb light and convert photon energy to electric potentials, were shown to stimulate retinal neurons in culture. One of these dyes coupled with polyethylene film surface, as a prototype of retinal prostheses, could induce intracellular calcium elevation in chick embryonic retinal tissues. In this study, we used retinal cells from chick embryos in which no photoreceptor outer segments yet developed, and assessed cell adhesiveness and response to the original and modified types of our retinal prostheses. Extruded-blown film of high density polyethylene was used as the original polyethylene film. Polyethylene film surface was modified either by coupling with type 1 collagen or by crystallization from the melt of the original polyethylene film. A photoelectric dye, 2-[2-[4-(dibutylamino)phenyl]ethenyl]-3-carboxymethylbenzothiazolium bromide, was coupled to these different types of polyethylene films through amide linkage. Retinal cells from chick embryos at the 12-day embryonic stage were cultured on the surface of dye-coupled films. The response of retinal cells to the dye-coupled films was observed by calcium imaging. Cell types, either neurons or glial cells, were detected by immunocytochemical staining and also observed by scanning electron microscopy (SEM). Intracellular calcium elevation was observed in cells, including both neurons and glial cells, on the surface of the original dye-coupled film, collagen-coated dye-coupled film, and dye-coupled recrystallized film, in contrast with no response of cells cultured on the dye-uncoupled plain film. Retinal neurons grew upon colonies of glial cells settling on the surface of all different types of the films. The number of retinal neurons on the collagen-coated dye-coupled film and dye-coupled recrystallized film was significantly larger than the number of neurons on the dye-uncoupled plain film (P = 0.0004, analysis of variance; P < 0.05, Tukey-Kramer test). With SEM, Muller cell-like large cells covered the original dye-coupled film and collagen-coated dye-coupled film, while neuron-like small cells settled directly on the dye-coupled recrystallized film and also formed large colonies on Muller-like cells. Retinal neurons and glial cells were cultured directly on the different types of dye-coupled polyethylene films. Photoelectric dye-coupled polyethylene films could be used as prototypes of retinal prostheses.

Aniline Compounds↗

In vivo studies on the effect of UV-radiation on the eye lens in animals.

Ultraviolet light is a non-ionizing radiation that induces photochemical reactions in the tissue. Its spectral A and B ranges are partially absorbed by the cornea and/or lens thus causing damage on the cellular, cell physiological and molecular level. UV-A does not seem to damage the cornea permanently and its effects in the lens have a very prolonged latency period. Typical reactions of the cornea are oedema, punctuate keratitis (photoelectric keratitis) and neovascularization. In the lens all reactions that could be evidenced, were located in the epithelium and in the outer cortical fiber cells. In vivo UV-A induces swelling and slight vacuolation of the anterior suture system, but apart from these transient effects, only very limited permanent damage could be demonstrated. UV-B induces the formation of an anterior subcapsular cataract, starting also with vacuolation of the suture system. These morphological characteristics can be visualized at the slitlamp microscope. Histologically, sutural irregularities (UV-A) and epithelial hyperplasia with capsular multiplication (UV-B) as well as disintegration of the anterior suture system could be observed. Patho-physiologically, a reduction of lens fresh weight (UV-B) as well as changes of the equilibrium of reduced and oxidized glutathione (GSH/GSSG) could be demonstrated. On the protein-biochemical level, changes in the ratio of water-soluble versus water-insoluble protein could be evidenced, as well as effects on specific crystallin fractions, namely alpha-crystallin. In addition, the appearance of a newly synthetized 31 kDa protein could be demonstrated in UV-B irradiated mice.

Animals↗

Photon activation of iododeoxyuridine: biological efficacy of Auger electrons.

Photon activation therapy is a binary system being investigated as a potential therapeutic modality to improve the treatment of malignancies, particularly the highly lethal and malignant brain tumor, glioblastoma multiforme. Its success relies upon the incorporation of a target atom in the immediate vicinity of a tumor cell's critical site, followed by the activation of this atom with photons of energies suitable for the induction of the photoelectric effect and its concomitant Auger cascades. The collective action of the Auger electrons imparts high-LET type damage at the critical site. Photon activation therapy uses iodine from stable iododeoxyuridine (IdUrd) as the target atom, and monochromatic photons above the K absorption edge of iodine (33.2 keV) as the activating agent. Although IdUrd is a cell-sensitizing agent, work described was designed to separate the biological efficacy due to sensitization from that of the Auger effect. Chinese hamster V79 cells with and without IdUrd in cellular DNA were irradiated at the X17B1 beam line in the National Synchroton Light Source of Brookhaven National Laboratory. Monochromatic photons above (33.4 keV) and below (32.9 keV) the K absorption edge were used to determine if any additional biological damage would accrue from the Auger cascades. The 33.4-keV photons were found to be a factor of 1.4 times more effective than 32.9-keV photons in damaging iodinated cells. The sensitizing effect, evaluated separately, was found to be a factor of 2.2 at 10% survival, regardless of photon energy. Thus the total therapeutic gain was 1.4 x 2.2 = 3.1. Irradiations of noniodinated control cells showed no difference in their response to energies above and below the iodine K edge.

Animals↗

Rapid inhibition of the glutamate-induced increase of intracellular free calcium by magnesium in rat hippocampal neurons.

By using Fura-2/AM, the effects of magnesium (Mg2+) on the glutamate-induced increase of intracellular free calcium ([Ca2+]i) in the cultured hippocampal neurons and the features were investigated by integrated photoelectric detecting system. The experiments were designed to three groups (The drug was spit to the cells for 20 s): Group A receiving 1 x 10(-5) mol/L glutamate; Group B receiving 1 x 10(-5) mol/L glutamate and 1 x 10(-5) mol/L Mg2+ simultaneously; Group C receiving 1 x 10(-5) mol/L glutamate again after [Ca2+]i in group B back to the baseline. The results showed that in group A, [Ca2+]i was obviously increased. In group B, the changes in [Ca+] i and the peak value were significantly decreased. Moreover, the elevation of Phase 1 was slowed down and Phase 2 was shortened to some extent, and the plateau phase between them was relatively prolonged. In group C, calcium oscillation similar to that in group A occurred, but both the Phase 1 and Phase 2 were shortened and the delta[Ca2+]i was slightly decreased. It was suggested that Mg2+ could quickly inhibit the rise of [Ca2+]i induced by glutamate in the cultured hippocampal neurons in rats.

Animals↗

Slowing of proton transport processes in the structure of bacterial reaction centers and bacteriorhodopsin in the presence of dipyridamole.

Dipyridamole, 2,6-bis(diethanolamino)-4,8-dipiperidinopyrimido(5, 4-d)pyrimidine, is employed in clinical practice as a vasodilator. It can also inhibit a specific membrane protein (glycoprotein P) which pumps anticancer drugs out of tumor cells. Dipyridamole (10-4 M) markedly slows down the kinetics of the electrogenic phase of the photoelectric response in Rhodobacter sphaeroides chromatophores. This phase is due to proton transfer from the external medium to the secondary quinone acceptor in the reaction center. In purple membranes of bacterium Halobacterium salinarium containing bacteriorhodopsin dipyridamole (in its charged state) significantly slowed the kinetics of proton transfer from the primary donor, Asp-96 (in membranes from bacteria of wild type), or from the external medium (in D96N mutant) to the Schiff base. It is suggested that dipyridamole can influence the structural-dynamic state of membrane proteins including modification of the structure of their hydrogen bonds involved in proton-transport processes.

Bacteriorhodopsins↗

[Stimulation of ciliary motility by beta 3-adrenoceptor agonist rabbit tracheal epithelium].

To determine whether atypical beta-adrenoceptor (beta 3-adrenoceptor) agonist, BRL 37344, stimulates airway ciliary motility and, if so, what its mechanism of action is, we studied rabbit tracheal epithelium in vitro. Epithelial cells cultured for 7 days were mounted in the Rose chamber and ciliary beat frequency (CBF) was measured by a photoelectric method. Addition of BRL 37344 rapidly increased CBF from 15.5 +/- 0.6 to 20.3 +/- 1.4 Hz (mean +/- SE, p < 0.01), which remained elevated for at least 20 min. This effect was concentration-dependent, the maximal increase from the baseline value and the EC50 being 27.2 +/- 0.4% (mean +/- SE, p < 0.01) and 1.75 x 10(-7) M, respectively. Other beta-adrenoceptor agonists likewise increased CBF, where the rank order of potency was salbutamol (1) > BRL 37344 (0.57) >> norepinephrine (1.3 x 10(-3)). Preincubation of cells with propranolol inhibited the increase in CBF produced by salbutamol, and BRL 37344 significantly raised intracellular cyclic AMP contents. These results suggest that beta 3-adrenoceptor probably exists in rabbit tracheal epithelium and that stimulation of this type of receptor increases ciliary motility through a cyclic AMP-dependent mechanism, an effect that might be of value in enhancing mucociliary transport function in the airway.

Adrenergic beta-Agonists↗