Freezing injury in cold-acclimated and unhardened spinach leaves

Spinach plants (Spinacia oleracea L.) were frost-hardened by cold-acclimation to 1° C or kept in an unhardy state at 20°/14° C in phytotrons. Detached leaves were exposed to temperatures below 0°C. Rates of photosynthetic CO₂ uptake by the leaves, recorded after frost treatment, served as a measure of freezing injury. Thylakoid membranes were isolated from frost-injured leaves and their photosynthetic activities tested. Ice formation occurred at about-4° to-5° C, both in unhardened and cold-acclimated leaves. After thawing, unhardened leaves appeared severely damaged when they had been exposed to-5° to-8° C. Acclimated leaves were damaged by freezing at temperatures between-10° to-14° C. The pattern of freezing damage was complex and appeared to be identical in hardened and unhardened leaves: 1. Inactivation of photosynthesis and respiration of the leaves occurred almost simultaneously. 2. When the leaves were partly damaged, the rates of photosynthetic electron transport and noncyclic photophosphorylation and the extent of light-induced H⁺ uptake by the isolated thylakoids were lowered at about the same degree. The dark decay of the proton gradient was, however, not stimulated, indicating that the permeability of the membrane to-ward protons and metal cations had not increased. 3. As shown by partial reactions of the electron transport system, freezing of leaves predominantly inhibited the oxygen evolution, but photosystem II and photosystem I-dependent electron transport were also impaired. 4. Damage of the chloroplast envelope was indicated by a decline in the percentage of intact chloroplasts found in preparations from injured leaves. The results are discussed in relation to earlier studies on freezing damage of thylakoid membranes occurring in vitro.Abbreviations Chl chlorophyll - DCPIP 2,6-dichlorophenol indophenol - HEPES N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid - MES 2(N-morpholino) ethane sulfonic acid     

Increased phenolic content and antioxidant capacity of the heated leaves of yacon (Smallanthus sonchifolius)

ABSTRACT

We investigated the content of phenolic compounds and antioxidant capacity of two batches of non-heated and heated leaves of the yacon cultivar “Andes no yuki”, grown in Japan. Lyophilized yacon leaves heated at 160°C for 20 min and 100°C for 60 min had a 1.96 to 9.69-times higher total phenolic content than that of the non-heated leaves. Heated leaves exhibited a 1.98 to 4.07-times higher antioxidant capacity than that of the non-heated leaves in three different free radical scavenging assays. Heated leaves were more efficient at attenuating the superoxide anion radical production in human granulocytic cells than the non-heated leaves. High-performance liquid chromatography analysis revealed that, in the heated leaves, the caffeic acid content was 2.13 to 3.64-times higher and the chlorogenic acid content was slightly lower than those in the non-heated leaves. Hence, heat processing may affect the active constituent contents in yacon leaves, potentiating its antioxidant capacity.

Abbreviations: ABTS⁺: 2,2′-azinobis(2-ethylbenzothiazoline-6-sulfonic acid) cation; DPPH: 1,1-diphenyl-2-picrylhydrazyl, HPLC: high-performance liquid chromatography; NBT: nitroblue tetrazolium; O₂^?: superoxide anion; PMA: phorbol 12-myristate 13-acetate; PMS: phenazine methosulfate; TEAC: Trolox equivalent antioxidant capacity     

Photodynamic Activity of Compounds Structurally Related to Cercosporin

The photodynamic activity of three compounds structurally related to cercosporin (iso-cercosporin, phleichrome, iso-phleichrome) and rubellin, a pigment possessing an anthra-quinone-like structure, was studied. As previously reported for cercosporin, iso-cercosporin, phleichrome, iso-phleichrome and rubellin, when irradiated by an incandescent lamp, induced oxygen uptake and malondialdehyde (MDA) formation in pea stem and rat liver mitochondria and microsomes. All these compounds were also capable of inhibiting MDA formation induced by the peroxidizing system ascorbate-FeSO₄. Circumstantial evidence suggests that the compounds, as shown for cercosporin, induce a lipoperoxidative degradation of the polyunsaturated fatty acids of cell membranes when irradiated by light, but at the same time, they are able to lower the lipoperoxidation induced by ferrous ions.     

Photodynamic properties of pyridoxal phosphate bound to cystathionase-gamma-lyase

The cofactor pyridoxal phosphate bound through an aldimine linkage to lysine residues of the enzyme cystathionase (L-Cystathione cysteine-lyase (deaminating), EC 4.4.1.1) is very stable to irradiation with light of 420 nm. The catalytic function of the enzyme remains unaffected indicating that the cofactor is not an efficient photosensitizer of essential amino acid residues. This unusual stability of the cofactor to irradiation can be ascribed to the presence of aldimine linkages as demonstrated by studies conducted on model compounds. The binding of a reversible inhibitor (L-allylglycine) to the catalytic site of the enzyme does not facilitate photooxidation of the cofactor. On the contrary, irradiation of the cofactor in the presence of the inhibitor results in photodestruction of the inhibitor.     

竹红菌素光敏剂与微血管病变光动力治疗

光动力疗法已被用于临床治疗除实体肿瘤以外的一些微血管类疾病,包括鲜红斑痣(portwine stains,PWS)和老年视网膜黄斑变性(age-related macular degeneration,AMD)等。竹红菌素是一种苝醌类光敏剂,因为在光疗窗口(600～900 nm)的吸收较少,它不适用于实体肿瘤的光动力治疗,但对于治疗微血管类疾病却有其独特的优势。本文根据竹红菌素光物理特性提出其主要适应症范围,并根据其临床实用化问题提出应对策略。通过构造竹红菌素水溶性纳米制剂或具有优化脂水双亲性的衍生物,实现脂溶性光敏剂既可以安全给药又最大程度保持生物利用度和光动力活性。生物学实验证明竹红菌素类光敏剂对生物靶体具有超强的光动力活性。由此推测:竹红菌素类光敏剂在光动力治疗微血管疾病(如鲜红斑痣和老年黄斑变性)及其它浅表型疾病方面具有广阔的应用前景。     

Photodynamic protein cross-linking

Exposure of spectrin to visible light in the presence of a photosensitizer results in photo-oxidation of sensitive amino acid residues and covalent cross-linking of the polypeptides. In a previous paper the cross-linking was ascribed to a secondary reaction between photo-oxidized histidine residues and amino groups. The following observations, described in this paper, are in accordance with this supposition. (1) During illumination of spectrin in the presence of a photosensitizer a pronounced photo-oxidation of histidine residues takes place. (2) Simultaneously a decrease of free amino groups is observed. (3) Semicarbazide protects against cross-linking and is bound to a histidine photo-oxidation product in spectrin. (4) The pH profile of histidine photo-oxidation and subsequent reaction with amino groups is similar to the pH profile of spectrin cross-linking. Amidination of NH₂ groups in spectrin does not inhibit cross-linking, as visualized by gel electrophoresis. On the other hand aminidation of denatured myoglobin causes a 50% inhibition of cross-linking. These observations support the notion of NH₂-involvement in cross-linking but also demonstrate, that other photodynamic cross-linking mechanisms exist.     

Photodynamic antimicrobial therapy

Photodynamic antimicrobial therapy (PACT) involves the utilisation of photosensitizers activated by exposure to visible light in order to eradicate microbes (this method has already been applied in photodynamic therapy of tumours). Photodynamic effect of the particular photosensitive substance (PS) is attributed to its ability to penetrate susceptible microorganisms, to absorb the light of certain wavelength, and to generate reactive cytotoxic oxygen products. The target microorganisms for photoinactivation are bacteria, fungi, viruses and protozoa. Photodynamic antimicrobial therapy is proposed as a potentially topical, non-invasive approach suitable for treatment of locally occurring infection. The fact that bacteria are becoming increasingly resistant to antibiotics and antiseptics has lead to an increased interest in the development of new alternative eradication methods, such as PACT. Research and development of photosensitive substances are aimed at finding effective antimicrobial substances, which would have a broad-spectrum potency.     

Nucleic Acid and protein changes in relation to cold acclimation and freezing injury of korean boxwood leaves

Quantitative and qualitative differences in nucleic acids of Korean boxwood (Buxus microphylla var. Koreana) leaves were determined by methylated albumin kieselguhr chromatography at different levels of cold hardiness. During cold acclimation there was an increase in RNA, mainly ribosomal RNA, with little or no change in DNA. The increase in ribosomal RNA was closely paralleled by an increase in water soluble and membrane bound proteins. As cold hardiness increased, ribonuclease activity declined.     

Identification of a tyrosine phosphatase involved in the response to mechanical injury in leaves of Ricinus communis

Plants defend themselves against biotic or abiotic stress by triggering intracellular signaling pathways that regulate gene expression and responses to the offending agent. Phosphorylation and dephosphorylation represent major mechanisms for the regulation of plant defense pathways. Therefore, MAP kinases and phosphatases have been the focus of many studies in this area. This study identified three phosphatase activities, namely RcPPase I, II and III. Wounding increased the activity levels of RcPPase III, while the activities of RcPPase I and II remained constant compared to the control. N-terminal partial amino acid sequence, biochemical characterization with use of specific substrates and inhibitors indicated that the RcPPase III belong to the family of tyrosine phosphatases (PTPs).     

Influence of olive leaves extract on hepatorenal injury in streptozotocin diabetic rats

Medicinal plants have always been an important source of new alternative effective compounds for human therapy. Currently, there are many of scientific evidences indicate that the medicinal plants contain a lot of hypoglycemic chemical compounds. The purpose of the present study was to determine the influence of olive leaves extract on hepatorenal injury in diabetic male rats. Experimental diabetes was induced by streptozotocin (STZ). The levels of serum glucose, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, gamma glutamyl transferase, total bilirubin, creatinine, blood urea nitrogen, uric acid and malondialdehyde were significantly increased, while the levels of serum superoxide dismutase, glutathione and catalase were statistically decreased in untreated diabetic rats. Moreover, the histopathological examination showed several alterations in the structure of liver and kidney in untreated diabetic rats. Treatments with low dose and high dose of olive leaves extract in diabetic rats showed remarkable reducing and protecting influences of physiological and histopathological alterations. Moreover, the highly treatment efficiency was noted in diabetic rats treated with high dose followed by low dose of olive leaves extract. Additionally, the results of this study proved that the antioxidant activities of olive leaves extract played a vital role against the hepatorenal injury induced by diabetes. Finally, this study indicates to the importance of the use of olive leaves extract as promising alternative and complementary therapeutic agent against diabetes and its complications.     

Photodynamic action inSpirostomum ambiguum

Summary The photodynamic action inSpirostomum ambiguum has been studied measuring the time lag between a flash of visible light and the induced contraction of spirostomum. This quantity shows an exponential dependence on the flash strength and on the dye concentration. These measurements indicate that there is in spirostomum a natural content of photodynamic substances and that there is a natural rhythm of contraction of about 900 sec (at the temperature of 20 °C) even in darkness and without added photodynamic substances. Also the temperature dependence of has an exponential behaviour with an activation energy of 0.37 eV.The effect of the partial pressure of oxygen on has also been investigated and a hyperbolic dependence is shown to occur. The action of the oxygen on the photodynamic effect can be described by a relationship which is similar to that employed to describe the biological damage by X-rays.The results are analyzed in terms of a model which accounts for the action of two kinds of molecules activated by light: molecules which are always active (n ₂) and molecules which are active only when reacting with oxygen (n ₁). This model suggests that the photodynamic action in spirostomum is regulated by an autocatalytic process.     

Chilling injury and recovery in detached and attached leaves measured by chlorophyll fluorescence

Smillie, R. M., Nott, R., Hetherington, S. E. and Öyustt, G. 1987. Chilling injury and recovery in detached and attached leaves measured by chlorophyll fluorescence Chilling injury was compared in detached and attached leaves chilled at 0 or 0.5°C by measuring the decrease in induced chlorophyll fluorescence in vivo. The fluorescence parameter measured was F_R, the maximal rate of rise of induced chlorophyll fluorescence emission after irradiating dark-adapted leaves. The plants used were bean, Phaseolus vulgaris L. cv. Pioneer, and maize, Zea mays L. cvs hybrid GH 390 and Northern Belle. Leaves were detached and placed on wet paper and covered with thin polyethylene film to prevent water loss during chilling. Leaves left attached on plants were treated similarly. When chilled in this way at 100% relative humidity, the chilling-induced decrease in F_R was the same in detached and attached leaves. For the attached leaves, the same result was obtained whether just a single leaf was chilled or the whole plant. Expression of chilling injury was greatest in fully turgid leaves and comparisons can be invalid unless the water status of the detached and attached leaves are the same. Problems arising from diurnal fluctuations in water potential of plants grown in a glasshouse were circumvented by placing leaves on the wet filter paper under polyethylene film prior to chilling, which allowed high water potentials to be regained, or mist sprays in the glasshouse were employed. Determinations of the time course for changes in F_R of maize (cv. Northern Belle) during chilling at 0°C showed that F_R decreased exponentially, at the same rate (time to 50% decrease in F_R was 9.3 h) in detached and attached leaves. Chilling injury was largely reversible for the first 20 h of chilling stress as both detached and attached leaves recovered their pre-chilling values of F_R after a further 20 h at 20°C in darkness. Leaves chilled for 48 h showed partial recovery, while those chilled for 72 h did not recover. Recovery was impeded by light. Inability to recover from chilling as indicated by measurements of F_R was paralleled by the incidence of visible symptoms of injury. It is concluded that detached and attached leaves behave similarly during chilling and short-term recovery, provided a similarity in treatments is rigorously maintained.     

Luminescence characteristics of strawberry leaves at early stages of injury by spider mite

In this work, we have investigated how the luminescence characteristics and the total content of antioxidants in leaves of strawberry (Fragaria × ananassa Duch.) change after infestation of plants by spider mites (Tetranychus atlanticus McGregor). At early stages of plant injury, the chlorophyll concentration remained unchanged. However, the total amount of antioxidants decreased and the intensity of high-temperature thermoluminescence band (60–100°C) increased, indicating degradation of membrane lipids caused by oxidative stress. In infested leaves, the light-induced rise of nonphotochemical quenching of chlorophyll fluorescence was slower, while the relaxation of nonphotochemical quenching in the dark was faster than in control leaf samples. These data may indicate that the thylakoid membranes in infested leaves were more permeable to ions than in control leaves.     

Adherence of bacteria to leaves.

Members of seven genra of bacteria, pathogens and nonpathogens of plants, adhered to young leaves when leaves were suspended in cell suspensions for 10 min. With Pseudomonas lachrymans, the adherence rate (cells applied vs. cells adhering) to host (cucumber) and nonhost (chrysanthemum) leaves was a straight-line, log-log function, as was the adherence of Escherichia coli and Serratia marcescens to cucumber leaves. Adhering cells of these three bacteria were washed with water from cucumber leaves at a straight-line, log-log rate. Adhered cells of P. lachrymans were most commonly found near veins on cucumber leaves. There appeared to be a polymeric surface layer on this bacterium on the cucumber leaf when leaves bearing bacteria were stained with ruthenium red and viewed in thin section.     

Quantitative Multi-Parametric Magnetic Resonance Imaging of Tumor Response to Photodynamic Therapy

ObjectiveThe aim of this study was to characterize response to photodynamic therapy (PDT) in a mouse cancer model using a multi-parametric quantitative MRI protocol and to identify MR parameters as potential biomarkers for early assessment of treatment outcome.MethodsCT26.WT colon carcinoma tumors were grown subcutaneously in the hind limb of BALB/c mice. Therapy consisted of intravenous injection of the photosensitizer Bremachlorin, followed by 10 min laser illumination (200 mW/cm²) of the tumor 6 h post injection. MRI at 7 T was performed at baseline, directly after PDT, as well as at 24 h, and 72 h. Tumor relaxation time constants (T₁ and T₂) and apparent diffusion coefficient (ADC) were quantified at each time point. Additionally, Gd-DOTA dynamic contrast-enhanced (DCE) MRI was performed to estimate transfer constants (K^trans) and volume fractions of the extravascular extracellular space (v_e) using standard Tofts-Kermode tracer kinetic modeling. At the end of the experiment, tumor viability was characterized by histology using NADH-diaphorase staining.ResultsThe therapy induced extensive cell death in the tumor and resulted in significant reduction in tumor growth, as compared to untreated controls. Tumor T₁ and T₂ relaxation times remained unchanged up to 24 h, but decreased at 72 h after treatment. Tumor ADC values significantly increased at 24 h and 72 h. DCE-MRI derived tracer kinetic parameters displayed an early response to the treatment. Directly after PDT complete vascular shutdown was observed in large parts of the tumors and reduced uptake (decreased K^trans) in remaining tumor tissue. At 24 h, contrast uptake in most tumors was essentially absent. Out of 5 animals that were monitored for 2 weeks after treatment, 3 had tumor recurrence, in locations that showed strong contrast uptake at 72 h.ConclusionDCE-MRI is an effective tool for visualization of vascular effects directly after PDT. Endogenous contrast parameters T₁, T₂, and ADC, measured at 24 to 72 h after PDT, are also potential biomarkers for evaluation of therapy outcome.