Cerebrospinal fluid-contacting neurons in the paraventricular organ and in the spinal cord of the quail embryo: a fluorescence-histochemical study

Although the cerebrospinal fluid-contacting neurons of the avian paraventricular organ exhibit considerable amounts of catecholamines, they show no tyrosine hydroxylase immunoreactivity. In the quail embryo, the development of these neurons has been studied using the paraformaldeyde-glutaraldeyde method for the fluorescence-histochemical localization of catecholamines. The timing of the appearance of catecholamine fluorescence in cerebrospinal fluid-contacting neurons and that in catecholamine-containing neurons of the brainstem have been compared. The first neurons displaying catecholamine fluorescence are found within the locus coeruleus and the nucleus subcoeruleus ventralis on the 5.5th day of incubation. Catecholaminergic neuronal groups of the medulla and mesencephalon can be identified by embryonic day 7, and fluorescent cerebrospinal fluid-contacting neurons of the hypothalamic paraventricular organ can be first recognized at the 8th day of incubation. If the catecholamine content of cerebrospinal fluid-contacting neurons that lack tyrosine hydroxylase depends upon an uptake mechanism, it may be significant that, in fluorescence-histochemical preparations, these neurons can be identified 1–3 days later than those in which catecholamines are synthesized and from which catecholamines are released at an earlier developmental stage. Moreover, cerebrospinal fluid-contacting neurons that have previously been shown to be tyrosine-hydroxylase immunoreactive, and that lie at the spinal-medullary junction display a different developmental pattern. By fluorescence histochemistry, they can be detected only by embryonic day 10.5. The chemical, developmental and topographical differences suggest that the catecholamine-containing cerebrospinal fluid-contacting elements of the paraventricular organ and those of the spinal cord represent two different subsets of cerebrospinal fluid-contacting neurons whose respective functional roles remain to be investigated.     

Rapid Determination of Conidial Viability for Entomopathogenic Hyphomycetes Using Fluorescence Microscopy Techniques

The viability of conidia from two species of deuteromycetes fungi pathogenic to insects was determined using two fluorochrome stains, fluorescein diacetate (FDA) and propidium iodide (PI). These stains were used either alone or in combination, and results were compared with standard conidial germination tests. FDA fluoresces bright green in viable conidia and PI fluoresces red in non-viable conidia, when viewed using specific fluorescence microscopic techniques. Conidia from two isolates of Paecilomyces fumosoroseus (Wize) Brown and Smith and two isolates of Beauveria bassiana (Balsamo) Vuillemin were evaluated. Conidia were suspended in deionized water and half of each suspension was treated with microwave radiation to kill all the conidia. Conidia were tested for viability in non-microwaved suspensions in a mixture (ca. 1:1) of viable and non-viable conidial suspensions, and in the microwaved suspensions that contained all non-viable conidia. No significant differences were observed for the four isolates tested between germination tests on water and agar and viability tests conducted with FDA alone or FDA in combination with PI. One isolate of B. bassiana that had been damaged in storage was also tested. Differences were observed between the actual germination and the percentage of viability determined using FDA or FDA plus PI. Damaged conidia maintained a measure of viability and fluoresced green, but did not fully germinate.     

高灵敏度荧光显微镜量化技术及其在光生物学研究中的应用

介绍一种用像增器接收荧光图像的高灵敏度荧光显微镜,相对于普通荧光显微镜的灵敏度提高了４×１０４倍,并用宽量程微光光亮度计对仪器的微弱成像性能进行了实验标定,得到了图像采集数据和图像发光强度的线性数量关系。高灵敏度荧光显微镜在给出细胞荧光图像的同时,可以给出图像上每一像元的发光强度和细胞平均发光强度,仪器对图像细微变化的判断能力远大于人眼直接观察图像。高灵敏度荧光显微镜可应用于研究细胞中荧光物质在细胞生理过程中的分布变化和发光强度变化。使用此仪器已得到了光敏竹红菌甲素（ＨＡ）在Ｈｅｌａ细胞（人体子宫癌细胞）中的分布图像和更为直观的三维显示图形,以及加入ＨＡ后Ｈｅｌａ细胞受到强先照射后的细胞损伤图像。     

Structure of the histone tetramer (H3-H4)2: 2. Position of λmax in the tyrosyl fluorescence spectra and tyrosyl accessibility to quenchers

It was shown that the histone tetramer (H3-H4)₂ fluorescence spectra were shifted by about 2 nm towards the long-wave region and had a larger halfwidth than the free tyrosine fluorescence spectra. Denaturation with 8 m urea resulted in a shift towards the short-wave region and a decrease in the halfwidth of the histone tetramer (H3-H4)₂ tyrosine fluorescence spectra. Fluorescence quenching of the histone tetramer (H3-H4)₂ by iodine ions was analysed by the Stern-Volmer equation. It was estimated that at 0.1 m NaCl and 0.3–0.8 m NaCl, 45% and 60% tyrosyl fluorescence, respectively, was quenched by I^? ions. The results obtained suggests that histone tetramer (H3-H4)₂ may have several structural forms distinguished by the amount of ‘exposed’ and ‘buried’ tyrosyls depending upon the conditions of the medium.     

Fluorescence studies using synchrotron radiation on normal and differentiated cells labeled with parinaric acids

Changes in membrane properties during the differentiation process in K562 cells have been investigated. A decrease of lectin-induced agglutination has been detected. The agglutination assay revealed to be an early and sensitive test to monitor the induced differentiation of the K562 cells. Naturally occurring fluorescent fatty acids (cis- and trans-parinaric acids) and the recently developed multifrequency phase and modulation technique were used to study cell membrane properties. Changes in fluorescence lifetime and polarization are clearly associated with cell differentiation, suggesting the involvement of the cellular plasma membrane in the differentiation process.     

The effect of chloroplast coupling factor ATP synthetase (CF1 · CF0) reconstitution on fluidity properties of isolated thylakoid lipid vesicles

The reconstitution of chloroplast coupling factor ATP synthetase (CF₁ · CF₀) with thylakoid lipids by cholate dialysis produced vesicles that displayed higher steady-state anisotropy (r_s) values for both 1,6-diphenyl-1,3,5-hexatriene (DPH) and trimethylammonium-diphenyl hexatriene fluorescence than the pure lipid alone. This is interpreted as meaning that the insertion of protein into the lipid bilayer brings about an increase in the ordering of acyl chains. This ordering effect became more obvious as the protein-to-lipid ratio was increased. Time-resolved decay analyses of DPH fluorescence anisotropy confirmed the conclusion drawn from the steady-state measurements, but further indicated that the dynamic motion of the probe was also slightly restricted after CF₁ · CF₀ incorporation. The restriction of DPH motion and the change in the half-angle for its cone of rotation was observed at relatively low protein-to-lipid ratios as compared with other reconstituted or biological membranes, suggesting that perhaps lipid-protein interactions occur with the inserted CF₁ · CF₀ complex.     

Identity of the Photosystem II reaction center polypeptide

A Photosystem-II (PS-II)-enriched chloroplast submembrane fraction has been subjected to non-denaturing gel-electrophoresis. Two chlorophyll a (Chl a)-binding proteins associated with the core complex were isolated and spectrally characterized. The Chl protein with apparent apoprotein mass of 47 kDa (CP47) displayed a 695 nm fluorescence emission maximum (77 K) and light-induced absorption characteristics indicating the presence of the reaction center Chl, P-680, and its primary electron acceptor, pheophytin. A Chl protein of apparent apoprotein mass of 43 kDa (CP43) displayed a fluorescence emission maximum at 685 nm. We conclude that CP43 serves as an antenna Chl protein and the PS II reaction center is located in CP47.     

Proton-induced membrane fusion role of phospholipid composition and protein-mediated intermembrane contact

Glycolipid-phospholipid vesicles containing phosphatidate and phosphatidylethanolamine were found to undergo proton-induced fusion upon acidification of the suspending medium from pH 7.4 to pH 6.5 or lower, as determined by an assay for lipid intermixing based on fluorescence resonance energy transfer. Lectinmediated contact between the vesicles was required for fusion. Incorporation of phosphatidylcholine in the vesicles inhibited proton-induced fusion. Vesicles in which phosphatidate was replaced by phosphatidylserine underwent fusion only when pH was reduced below 4.5, while no significant fusion occured (pH ? 3.5) when the anionic phospholipid was phosphatidylinositol. It is suggested that partial protonation of the polar headgroup of phosphatidate and phosphatidylserine, respectively, causes a sufficient reduction in the polarity and hydration of the vesicle surface to trigger fusion at sites of intermembrane contact.     

Chloroplast thylakoid membrane fluidity and its sensitivity to temperature

In order to investigate membrane fluidity, the hydrophobic probe, 1,6-diphenyl-1,3,5-hexatriene (DPH), has been incorporated into intact isolated thylakoids and separated granal and stromal lamellae obtained from the chloroplasts of Pisum sativum. The steady-state polarization of DPH fluorescence was measured as a function of temperature and indicated that at physiological values the thylakoid membrane is a relatively fluid system with the stromal lamellae being less viscous than the lamellae of the grana. According to the DPH technique, neither region of the membrane, however, showed a sharp phase transition of its bulk lipids from the liquid-crystalline to the gel state for the temperature range -20° to 50° C. Comparison of intact thylakoids isolated from plants grown at cold (4°/7°C) and warm (14°/17° C) temperatures indicate that there is an adaptation mechanism operating which seems to maintain an optimal membrane viscosity necessary for growth. Using a modified Perrin equation the optimal average viscosity for the thylakoid membrane of the chill-resistant variety used in the study (Feltham First) is estimated to be about 1.8 poise.Abbreviations DPH 1,6-diphenyl-1,3,5-hexatriene - Hepes N-(2-hydroxyethyl)-1-piperazineethanesulphonic acid     

Effect of ligand binding and conformational changes in proteins on oxygen quenching and fluorescence depolarization of tryptophan residues

The rotational freedom of tryptophan residues in protein-ligand complexes was studied by measuring steady-state fluorescence anisotropies under conditions of oxygen quenching. There was a decrease in the oxygen bimolecular quenching constant upon complexation of trypsin and alpha-chymotrypsin with proteinaceous trypsin inhibitors, of lysozyme with N-acetylglucosamine (NAG) and di(N-acetyl-D-glucosamine) ((NAG)2) and of hexokinase with glucose. Binding of the bisubstrate analogue N-phosphonacetyl-L-aspartate (PALA) to aspartate transcarbamylase (ATCase) and binding of biotin to avidin resulted in increased oxygen quenching constants. The tryptophan of human serum albumin (HSA) in the F state was more accessible to oxygen quenching than that in the N state. With the exception of ATCase, the presence of subnanosecond motions of the tryptophan residues in all the proteins is suggested by the short apparent correlation times for fluorescence depolarization and by the low apparent anisotropies obtained by extrapolation to a lifetime of zero. Complex formation evidently resulted in more rigid structures in the case of trypsin, alpha-chymotrypsin and lysozyme. The effects of glucose binding on hexokinase were not significant. Binding of biotin to avidin resulted in a shorter correlation time for the tryptophan residues. The N --> F transition in HSA resulted in a more rigid environment for the tryptophan residue. Overall, these changes in the dynamics of the protein matrix and motional freedom of tryptophan residues due to complex formation and subsequent conformational changes are in the same direction as those observed by other techniques, especially hydrogen exchange. Significantly, the effects of complex formation on protein dynamics are variable. Among the limited number of cases we examined, the effects of complex formation were to increase, decrease or leave unchanged the apparent dynamics of the protein matrix.