A microcytofluorometric method for quantitative and qualitative evaluation of CFU-E and BFU-E colonies.

A new method has been developed for the precise identification of human bone marrow colony forming unit erythroid (CFU-E) and burst forming unit erythroid (BFU-E) colonies, and for determination of the hemoglobin contents using microcytofluorometry. The method relies on a photochemical reaction in which intracellular hemoglobin is converted into fluorescent porphyrin under violet light (lambda = 405 nm) in the presence of an SH-donor (mercaptoethylamine hydrochloride). The CFU-E and BFU-E colonies showed red fluorescence with two spectrum peaks at 600 and 650 nm when illuminated by violet light. These two peaks are consistent with those of porphyrin fluorescence. The porphyrin fluorescence was not inducible in colony forming unit granulocyte-macrophage (CFU-GM) colonies, while 20% of the CFU-GM colonies were false positive with respect to the conventional benzidine reaction. The photochemically inducible fluorescence began to appear in BFU-E colonies on the 4th day of culture, while the same colonies started to be positive for the benzidine reaction on the 9th day. Therefore, the photochemical reaction was more specific and sensitive than the benzidine reaction for the identification of CFU-E and BFU-E colonies. In addition, this method enabled us to measure the hemoglobin level in the cells forming the colonies because the intensity of the fluorescence was proportional to the amount of hemoglobin when the photochemical reaction was carried out for 50 min.(ABSTRACT TRUNCATED AT 250 WORDS)     

The photodestruction of hemoglobin]

It was shown that hemoglobin photodestruction occurs in the process of light absorption by protein globin, as well as by its hem. Quantum yields of photoreactions decreased with the increase of irradiation wave length. We observed two mechanisms of photodestruction: dependent and independent on the existence of the dissolved oxygen in solution. Hemoglobin photodestruction due to hem absorption did not exist in the absence of dissolved oxygen.     

A microcytofluorometric method for quantitative and qualitative evaluation of CFU-E and BFU-E colonies

Summary A new method has been developed for the precise identification of human bone marrow colony forming unit erythroid (CFU-E) and burst forming unit erythroid (BFU-E) colonies, and for determination of the hemoglobin contents using microcytofluorometry. The method relies on a photochemical reaction in which intracellular hemoglobin is converted into fluorescent porphyrin under violet light (=405 nm) in the presence of an SH-donor (mercaptoethylamine hydrochloride). The CFU-E and BFU-E colonies showed red fluorescence with two spectrum peaks at 600 and 650 nm when illuminated by violet light. These two peaks are consistent with those of porphyrin fluorescence. The porphyrin fluorescence was not inducible in colony forming unit granulocyte-macrophage (CFU-GM) colonies, while 20% of the CFU-GM colonies were false positive with respect to the conventional benzidine reaction. The photochemically inducible fluorescence began to appear in BFU-E colonies on the 4th day of culture, while the same colonies started to be positive for the benzidine reaction on the 9th day. Therefore, the photochemical reaction was more specific and sensitive than the benzidine reaction for the identification of CFU-E and BFU-E colonies. In addition, this method enabled us to measure the hemoglobin level in the cells forming the colonies because the intensity of the fluorescence was proportional to the amount of hemoglobin when the photochemical reaction was carried out for 50 min. As a result of qualitative and quantitative analysis of CFU-E colonies by this method, it was possible to detect the hemoglobin levels in the colonies from 1 of 4 cases of untreated acute nonlymphocytic leukemia and from 2 of 4 cases of myelodysplastic syndrome in which the hemoglobin levels were too low to be detected by the benzidine reaction. These cases, where the CFU-E colonies showed very low levels of hemoglobin, were associated with poor prognosis. Thus, our method is useful for identifying CFU-E colonies, determining their hemoglobin synthesis, and as a cue to predict the clinical course of the patients.     

Photochemical and mutational analysis of the FMN-binding domains of the plant blue light receptor, phototropin

The plant photoreceptor phototropin is an autophosphorylating serine-threonine protein kinase activated by UV-A/blue light. Two domains, LOV1 and LOV2, members of the PAS domain superfamily, mediate light sensing by phototropin. Heterologous expression studies have shown that both domains function as FMN-binding sites. Although three plant blue light photoreceptors, cry1, cry2, and phototropin, have been identified to date, the photochemical reactions underlying photoactivation of these light sensors have not been described so far. Herein, we demonstrate that the LOV domains of Avena sativa phototropin undergo a self-contained photocycle characterized by a loss of blue light absorbance in response to light and a spontaneous recovery of the blue light-absorbing form in the dark. Rate constants and quantum efficiencies for the photoreactions indicate that LOV1 exhibits a lower photosensitivity than LOV2. The spectral properties of the photoproduct produced for both LOV domains are unrelated to those found for photoreduced flavins and flavoproteins, but are consistent with those of a flavin-cysteinyl adduct. Flavin-thiol adducts are generally short-lifetime reaction intermediates formed during the flavoprotein-catalyzed reduction of protein disulfides. By site-directed mutagenesis, we have identified several amino acid residues within the putative chromophore binding site of LOV1 and LOV2 that appear to be important for FMN binding and/or the photochemical reactivity. Among those is Cys39, which plays an important role in the photochemical reaction of the LOV domains. Replacement of Cys39 with Ala abolished the photochemical reactions of both LOV domains. We therefore propose that light sensing by the phototropin LOV domains occurs via the formation of a stable adduct between the FMN chromophore and Cys39.     

Kinetic study on the photostability of riboflavin in the presence of barbituric acid

The photochemical fate of riboflavin (vitamin B2) in the presence of barbituric acid was examined employing polarographic detection of dissolved oxygen and steady-state and time-resolved spectroscopy. Under visible light, riboflavin reacts with barbituric acid--the latter being transparent to this type of photo-irradiation--via radicals and reactive oxygen species, such as singlet molecular oxygen [O2(1delta(g))] and superoxide radical anion, which are generated from the excited triplet state of the vitamin. As a result, both the vitamin and barbituric acid are photodegraded. Kinetic and mechanistic studies on the photoreactions of riboflavin in the presence of barbituric acid indicate the excellent quenching ability of the latter towards O2(1delta(g)).     

Using photolabile protecting groups for the controlled release of bioactive volatiles

To develop their biological activity, bioactive volatile compounds, such as pheromones or fragrances, have to evaporate from surfaces. Because these surfaces are usually exposed to natural daylight, the preparation of non-volatile precursors using photoremovable protecting groups is an ideal tool to control the release of caged volatile molecules from various surfaces by light-induced covalent bond cleavage. Many photoreactions occur under mild environmental conditions and are highly selective. To break covalent bonds under typical application conditions, the photoreaction has to proceed at ambient daylight, to tolerate the presence of oxygen and to run in polar media (e.g. in water). The amount of volatiles generated from photochemical delivery systems depends on the light intensity to which the systems are exposed. Both photoisomerisations and photofragmentations have successfully been investigated for the slow release of caged pheromones and fragrances from their corresponding precursors.     

Single cell observations of gas reactions and shape changes in normal and sickling erythrocytes.

Microspectrophotometry has been applied to single red blood cells to reinvestigate the linked processes of diffusion of gases inside the erythrocyte and their combination with hemoglobin. The experiments took advantage of the photosensitivity of the cabron monoxide derivative of hemoglobin, which allows ligand release from the CO-saturated red cells under strong illumination and recombination when the light is switched off. The photochemical method was also used to study the kinetics of sickling on ligand removal in single erythrocytes of Hb S carriers. The results give new information on the mechanism of the sickling process.     

Features of photochemical properties of hemoglobin under native conditions

The photochemistry of human hemoglobin (Hb) in the blood and blood serum (lambda = 254-578 nm) was studied, using spectrophotometric methods. The Hb photochemistry is a complex set of photoreactions leading to successive photoconversions of Hb forms: from oxy- to met- to deoxy- and, finally, to carboxy-form. The photodestruction of Hb and the photoreactions involving other serum proteins were found to occur simultaneously. In the blood Hb photomodifications are localized directly in erythrocytes. The conditions necessary for the photo--induced rupture of erythrocyte membranes and the subsequent release of Hb into the blood plasma, were determined. Although the general characteristics of Hb photochemistry are the same for model systems and for native conditions, there are some distinctions in the effectiveness of the photoconversion. It seems likely that the observed effects are due to the antioxidant properties of the serum. These properties may be the cause of the inhibition of blood photohemolysis upon irradiation (lambda greater than or equal to 300 nm).     

Photobiophysics of furanocoumarins

Furocoumarins (psoralens) are photosensitizers of plant origin, which increase the sensitivity of biological objects to near ultraviolet (UV-A, 320-400 nm). In combination with UV-A, they are successfully used for treating many dermal and autoimmune diseases (PUVA therapy and photophoresis). Along with therapeutic effects, the furocoumarin photochemotherapy induces a number of side-effects (erythema, edema, hyperpigmentation, and premature aging of skin). All photobiological effects of furocoumarins result from their photochemical reactions. Therefore, in order to advance the therapy, it is necessary to know the photochemical mechanisms of induction of both side- and therapeutic effects. The types of photoreactions of furocoumarins classified with respect to reactive photoproducts interacting with substrate were considered. Primary emphasis was placed on reactions proceeding with the participation of photooxidation products of furocoumarins. Among these photoproducts, at least two types can be distinguished. Some of them possess membranotoxic properties, others produce the immunosuppressory action in vivo. The photochemical mechanisms of the formation of the photoproducts of furocoumarins are different. It was found that, by varying the illumination conditions (intensity of UV-A radiation or the concentration of the photosensitizer), it is possible to obtain the photoproducts of furocoumarins that have either membranotoxic or immunosuppressory properties. It was found that the mechanisms of the immunosuppressive action of the photooxidation products of furocoumarins have some features in common with those underlying the PUVA therapy and photophoresis. It is assumed that the photochemical basis of the therapeutic action of furocoumarins is the reactions with the involvement of the products of their photooxidation.     

Kinetic study on the photostability of riboflavin in the presence of barbituric acid

Abstract

The photochemical fate of riboflavin (vitamin B₂) in the presence of barbituric acid was examined employing polarographic detection of dissolved oxygen and steady-state and time-resolved spectroscopy. Under visible light, riboflavin reacts with barbituric acid – the latter being transparent to this type of photo-irradiation – via radicals and reactive oxygen species, such as singlet molecular oxygen [O₂(¹Δ_g)] and superoxide radical anion, which are generated from the excited triplet state of the vitamin. As a result, both the vitamin and barbituric acid are photodegraded. Kinetic and mechanistic studies on the photoreactions of riboflavin in the presence of barbituric acid indicate the excellent quenching ability of the latter towards O₂(¹Δ_g).     

Effect of carbon dioxide concentration on pigmentation in the blue-green alga Anacystis nidulans

The pigment content in the blue-green alga Anacystis nidulanswas found to be dependent upon CO₂ concentration during growth.In cells grown with 1% CO₂ in air the total pigment constituted20.5% of the dry weight while it was only 11.1% of dry weightof cells grown in air (0.03% CO₂). This decrease in total pigmentwas found to be almost entirely ascribable to decrease in phycocyanin.Since light absorbed by phycocyanin has been shown to providenearly equal rates of photoreactions I and II, the "CO₂ control"of phycocyanin is viewed as an effective means of regulationof the photoreactions without upsetting the balance of operationof the two photoreactions. (Received December 25, 1970; )     

Effects of shading on photosynthetic characteristics and chlorophyll fluorescence parameters in leaves of Hydrangea macrophylla

Aims The objectives were to investigate the effects of different light intensities on photosynthetic characteristics and chlorophyll fluorescence parameters, to clarify the physiological responses and photo-protective mechanisms of Hydrangea macrophylla to changes in light regimes in view of the distribution of energy absorbed and photosynthetic characteristics.Methods Three light regimes including natural and shade (shading rate 50% and 75% of natural light) were applied to plants for 60 days. After the treatment, the gas-exchange, chlorophyll a fluorescence and photosynthesis-light curves were measured by a portable leaf gas exchange system (LI-6400).Important findings The results showed that the weak light intensity treatment reduced dark respiration rate, light compensation point and light saturation point of plant, but increased apparent quantum yield, suggesting that plants had the physiological strategy to utilize the weakening light by reducing respiration. The net photosynthetic rate, intercellular CO₂ concentration, transpiration rate and water use efficiency of plants grown below 50% of natural light showed significant difference compared with natural and shading rate 75% of natural light. There were significant difference between natural and shade treatments in the maximal quantum efficiency of PSII (F_v/F_m), as indicated that it was significantly less at full light than that at 50% of natural light. Initial fluorescence intensity (F_o) of plants was higher at full light than that at 50% of natural light, suggesting that photoinhibition occurred in natural light. The non-photochemical quenching (NQP) decreased with the aggravation of shade stress, indicating that shading decreased the efficiency of photochemical reaction by reducing the fraction of incident light in photochemical energy utilization and decreased thermal dissipation through regulating energy distribution in photosystem II (PSII) in the leaves of Hydrangea macrophylla. In general, the 70% of incident light in photochemical energy utilization was distributed to thermal dissipation, 20% was distributed to non-regulated energy dissipation and 4% was distributed to effective photochemical reaction. In conclusion, responses of plants to increased irradiance are governed by strategy: to utilize a high fraction of incident light in photochemistry and regulate energy dissipation in PSII and weaken the accumulation of excess excitation energy in PSII to protect the photosynthetic apparatus in the leaves of H. macrophylla under saturated radiation.     

In vivo photochemical micronucleus induction due to certain quinolone antimicrobial agents in the skin of hairless mice

The skin micronucleus test combined with irradiation due to a sunlight simulator having a spectrum almost identical to solar irradiation was used as a novel in vivo testing method for detecting or comparing the photochemical chromosome damage of quinolone antibacterial agents (quinolones). Eight-week-old male SKH1 hairless mice were orally administered once lomefloxacin (LFLX), a strong in vitro photochemical clastogen, at 25 or 50 mg/kg, followed by light irradiation at 7.9-9.4J/cm2 of ultraviolet A (UVA). Animals were killed on Days 2, 3, 4, 5 or 8 (the dosing day was designated as Day 1), and the incidence of micronucleus in the epidermis was determined. As results, LFLX at either dose caused significant increases in the micronucleus frequency, which peaked on Day 4. These changes tended to return to the control level on Day 8. Then, the micronucleus induction potential of the quinolone derivatives levofloxacin (LVFX) and clinafloxacin (CLFX) at 10, 20 or 40 mg/kg was assessed on Day 4 under the same experimental conditions as for LFLX. Although LVFX was negative even at 40 mg/kg, CFLX dose-dependently induced significant increases in micronucleus frequency at all doses. The correlation of magnitude among the three quinolones in the skin micronucleus test with light irradiation was similar to that in our previous in vitro photochemical clastogenicity study. No significant increase in micronucleus frequency was observed in any of three quinolones employed without light irradiation. In conclusion, the experimental method presented here would be a useful tool for detecting in vivo photochemical chromosome damage and for research on photochemical carcinogenesis of chemicals.     

硝酸根胁迫对黄瓜幼苗叶片光合速率、PSⅡ光化学效率及光能分配的影响

研究了不同浓度NO₃^-胁迫对黄瓜幼苗叶片光合速率、PSⅡ光化学效率及光能分配的影响.结果表明,当NO₃^-浓度较低时（14～98 mmol·L^-1）,适当增加NO₃^-浓度,可增强黄瓜幼苗叶片对光的捕获能力,促进光合作用.随着NO₃^-浓度的进一步增加（140～182 mmol·L^-1）,PSⅡ光化学效率降低,电子传递受到抑制,净光合速率降低;吸收的光能中,通过天线色素的热耗散增加,用于光化学反应的能量降低,光化学效率下降.140和182 mmol·L^-1 NO₃^-处理黄瓜幼苗叶片6 d后净光合速率(P_n)极显著下降,分别比对照降低了35%和78%;PSⅡ最大光化学效率(F_v/F_m)、天线转化效率(F_v’/F_m’)、实际光化学效率(Φ_PSⅡ)、光化学猝灭系数(q_P)均低于对照,非光化学猝灭(NPQ)高于对照,激发能在两个光系统间的分配不平衡性(β/α-1)增大.高浓度NO₃^-处理的黄瓜幼苗叶片各荧光参数变化幅度比低浓度大.当光照增强时,高浓度NO₃^-胁迫下黄瓜幼苗叶片吸收的光能中应用于光化学反应的份额(P) 显著降低,天线热耗散的份额(D)显著增加. 天线热耗散是耗散过剩能量的主要途径.     

The transducer protein HtrII modulates the lifetimes of sensory rhodopsin II photointermediates.

We studied the photochemical reaction cycle of sensory rhodopsin II (SRII) by flash photolysis of Halobacterium salinarum membranes genetically engineered to contain or to lack its transducer protein HtrII. Flash photolysis data from membranes containing HtrII were fit well in the 10 micros-10 s range by three rate constants and a linear unbranched pathway from the unphotolyzed state with 487 nm absorption maximum to a species with absorption maximum near 350 nm (M) followed by a species with maximum near 520 nm (O), as has been found in previous studies of wild-type membranes. Data from membranes devoid of HtrII exhibited similar M and O intermediates but with altered kinetics, and a third intermediate absorbing maximally near 470 nm (N) was present in an equilibrium mixture with O. The modulation of SRII photoreactions by HtrII indicates that SRII and HtrII are physically associated in a molecular complex. Arrhenius analysis shows that the largest effect of HtrII, the acceleration of O decay, is attributable to a large decrease in activation enthalpy. Based on comparison of SRII photoreactions to those of sensory rhodopsin I and bacteriorhodopsin, we interpret this kinetic effect to indicate that HtrII interacts with SRII so that it alters the reaction process involving deprotonation of Asp73, the proton acceptor from the Schiff base.     

Effect of short-term heat treatment of rice seedlings on sensitivity of thylakoid membranes to photoinhibition

The after-effects of 24 h high temperature (35 or 45 °C) treatment on the photochemical activities and photooxidative lipid peroxidation, subsequent to their irradiation were studied in 7-d-old etiolated rice (Oryza sativa) seedlings. Photosystem (PS) 1 and PS 2 mediated photoreactions of thylakoids isolated from the seedlings exposed to high temperature did not differ significantly from the thylakoids isolated from control seedlings (25 °C). Hence, all kinds of tested thylakoids were equally efficient in capturing and utilizing radiant energy. The high irradiance induced loss in PS 2 activity and lipid peroxidation measured in terms of malondialdehyde production was more rapid in thylakoids isolated from stressed seedlings as compared to that of control seedlings. Thus the thylakoids isolated from the stressed seedlings were more prone to photodamage than those from the control seedlings.     

The Branched Photocycle of the Slow-Cycling Channelrhodopsin-2 Mutant C128T

Channelrhodopsins (ChRs) of green algae such as Chlamydomonas are used as neuroscience tools to specifically depolarize cells with light. A crude model of the ChR2 photocycle has been recently established, but details of the photoreactions are widely unknown. Here, we present the photoreactions of a slow-cycling ChR2 mutant (step function rhodopsin), with C128 replaced by threonine and 200-fold extended lifetime of the conducting-state P520. At a late state of the photocycle, a fraction of the proteins branches off into an inactive species, P380, which accumulates during prolonged illumination. At neutral pH, P380 is converted into P353, a species with a characteristic fine-structured spectrum that is interpreted as retroretinyl chromophore. The described branching reactions should be considered, when ChR is used as a neuroscience tool, especially in the case of fluorescence imaging at high light intensities.     

LIGHT DEPENDENCY OF PHOTOSYNTHETIC RECOVERY DURING WETTING AND THE ACCLIMATION OF PHOTOSYNTHETIC APPARATUS TO LIGHT FLUCTUATION IN A TERRESTRIAL CYANOBACTERIUM NOSTOC COMMUNE1

The PSII photochemical activity in a terrestrial cyanobacterium Nostoc commune Vaucher ex Bornet et Flahault during rewetting was undetectable in the dark but was immediately recognized in the light. The maximum quantum yield of PSII (F_v/F_m) during rewetting in the light rose to 85% of the maximum within ～30 min and slowly reached the maximum within 6 h, while with rewetting in the darkness for 6 h and then exposure to light the recovery of F_v/F_m required only ～3 min. These results suggested that recovery of photochemical activity might depend on two processes, light dependence and light independence, and the activation of photosynthetic recovery in the initial phase was severely light dependent. The inhibitor experiments showed that the recovery of F_v/F_m was not affected by chloramphenicol (CMP), but severely inhibited by 3‐(3,4‐dichlorophenyl)‐1,1‐dimethylurea (DCMU) in the light, suggesting that the light‐dependent recovery of photochemical activity did not require de novo protein synthesis but required activation of PSII associated with electron flow to plastoquinone. Furthermore, the test indicated that the lower light intensity and the red light were of benefit to its activation of photochemical activity. In an outdoor experiment of diurnal changes of photochemical activity, our results showed that PSII photochemical activity was sensitive to light fluctuation, and the nonphotochemical quenching (NPQ) was rapidly enhanced at noon. Furthermore, the test suggested that the repair of PSII by de novo protein synthesis played an important role in the acclimation of photosynthetic apparatus to high light, and the heavily cloudy day was more beneficial for maintaining high photochemical activity.     

Adaptation of photosystem II to high and low light in wild-type and triazine-resistant Canola plants: analysis by a fluorescence induction algorithm

Plants of wild-type and triazine-resistant Canola (Brassica napus L.) were exposed to very high light intensities and after 1?day placed on a laboratory table at low light to recover, to study the kinetics of variable fluorescence after light, and after dark-adaptation. This cycle was repeated several times. The fast OJIP fluorescence rise curve was measured immediately after light exposure and after recovery during 1?day in laboratory room light. A fluorescence induction algorithm has been used for resolution and analysis of these curves. This algorithm includes photochemical and photo-electrochemical quenching release components and a photo-electrical dependent IP-component. The analysis revealed a substantial suppression of the photo-electrochemical component (even complete in the resistant biotype), a partial suppression of the photochemical component and a decrease in the fluorescence parameter F (o) after high light. These effects were recovered after 1?day in the indoor light.