Studies on light absorption and photochemical activity changes in chloroplast suspensions and leaves due to light scattering and light filtration across chloroplast and vegetation layers

An experimental analysis is presented concerning the effect on relative light absorption by the two photosystems caused by (a) a highly light scattering environment (the detour effect) and (b) light filtration across successive chloroplast layers (the light attenuation effect). Both suspensions of isolated chloroplasts and leaves were employed.It is concluded that within a single spinach leaf these phenomena are likely to lead to only rather small increases in relative photosystem I absorption and activity with respect to photosystem II and will thus not exert a significant effect on non cyclic electron transport. On the contrary when light is filtrated across successive vegetation layers (shade light) significant increases in the relative PSI absorption and activity may be encountered.It is determined that the detour effect in mature leaves from a variety of plants increases overall photosynthetically useful light absorption by 35–40%.Abbreviations F_M maximal fluorescence - LHCP₂ light-harvesting chlorophyl a/b protein complex II - Q_A-primary quinone acceptor of photosystem II     

Spinach-thylakoid phosphorylation: Studies on the kinetics of changes in photosystem antenna size, spill-over and phosphorylation of light-harvesting chlorophyll a/b protein

The kinetics of LHCP phosphorylation and associated changes in photosystem cross-section and energy ‘spill-over’ from PS II to PS I have been examined in isolated spinach chloroplasts. During an initial phosphorylation period of 3–6 min, in the presence of saturating concentrations of Mg²⁺, the increase in PS I and decrease in PS II cross-section are largely completed, as judged by both measurements of the steady-state redox state of Q and fluorescence yield changes. This corresponds to a period of rapid ³²P incorporation into the low-molecular weight LHCP polypeptide. Subsequent to this initial 3–6-min period there is substantial further phosphorylation of both LHCP polypeptides, which is not accompanied by significant changes in photosystem cross-section, even after the chloroplasts had been unstacked with extensive mixing of PS I and PS II by Mg-removal. It is suggested that there exists a specific ‘mobile’ population of LHCP molecules which is rapidly phosphorylated and which may be enriched in the low-molecular-weight polypeptide. In addition, measurements of the kinetics of the ‘spill-over’ changes upon either Mg²⁺ addition or removal indicate that the continued phosphorylation of LHCP is able to increase the ‘spill-over’ process under favourable ionic conditions.     

Chemical Profile and Antioxidant Properties of Extracts and Essential Oils from Citrus × limon (L.) Burm. cv. Femminello Comune

Citrus × limon cv. Femminello Comune (Rutaceae) from Rocca Imperiale (Italy), one of the six Protected Geographical Indication (PGI) Italian lemon crops, has been recently received renewed interest. In this work, fresh and dried peels and leaves were extracted by hydrodistillation, supercritical fluid extraction (SFE), and Soxhlet apparatus. Chemical profile was assessed by gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS). Except for leaves extracts obtained by Soxhlet apparatus, the monoterpene hydrocarbons fraction dominated. Limonene, γ‐terpinene, and β‐pinene were the main identified compounds. The antioxidant activity was investigated using different in vitro assays namely 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH), ABTS, ferric reducing ability power (FRAP), and β‐carotene bleaching test. In DPPH test, the essential oil obtained by hydrodistillation of fresh peel exhibited the highest activity (IC₅₀ of 1.17 mg/ml). Leaves extracted by SFE showed a good activity in both DPPH and β‐carotene bleaching test with IC₅₀ values of 2.20 and 6.66 mg/ml, respectively. Monoterpene hydrocarbons fraction exhibited a positive Pearson's correlation coefficient with all antioxidant assays. Leaves, often considered waste material, should be considered from a different point because they represent a matrix of indisputable interest.     

Molecular Determinants of the Regioselectivity of Toluene/o-Xylene Monooxygenase from Pseudomonas sp. Strain OX1

Bacterial multicomponent monooxygenases (BMMs) are a heterogeneous family of di-iron monooxygenases which share the very interesting ability to hydroxylate aliphatic and/or aromatic hydrocarbons. Each BMM possesses defined substrate specificity and regioselectivity which match the metabolic requirements of the strain from which it has been isolated. Pseudomonas sp. strain OX1, a strain able to metabolize o-, m-, and p-cresols, produces the BMM toluene/o-xylene monooxygenase (ToMO), which converts toluene to a mixture of o-, m-, and p-cresol isomers. In order to investigate the molecular determinants of ToMO regioselectivity, we prepared and characterized 15 single-mutant and 3 double-mutant forms of the ToMO active site pocket. Using the Monte Carlo approach, we prepared models of ToMO-substrate and ToMO-reaction intermediate complexes which allowed us to provide a molecular explanation for the regioselectivities of wild-type and mutant ToMO enzymes. Furthermore, using binding energy values calculated by energy analyses of the complexes and a simple mathematical model of the hydroxylation reaction, we were able to predict quantitatively the regioselectivities of the majority of the variant proteins with good accuracy. The results show not only that the fine-tuning of ToMO regioselectivity can be achieved through a careful alteration of the shape of the active site but also that the effects of the mutations on regioselectivity can be quantitatively predicted a priori.     

Product of aromatase activity in intact LNCaP and MCF-7 human cancer cells

We investigated conversion rates of androgens to estrogens in cultured, hormone-responsive prostate (LNCaP) and breast (MCF-7) human cancer cells. For this purpose, we adopted an intact cell analysis, whereby cells were incubated for different incubation times in the presence of close-to-physiological (1 nM) or supraphysiological (1 μM) concentrations of labelled androgen precursors, i.e. testosterone (T) and androstenedione (Δ⁴Ad). The aromatase activity, as measured by estrogen formation, was detected in LNCaP cells (0.5 pmol/ml), even though to a significantly lower extent than in MCF-7 cells (5.4 pmol/ml), using 1 μM T after 72 h incubation. Surprisingly, LNCaP cells displayed a much higher aromatase activity when T was used as a substrate with respect to Δ⁴Ad. In either cell line, T transformation to Δ⁴Ad was relatively low, attaining only 2.8% in LNCaP and 7.5% MCF-7 cells. However, T was mostly converted to conjugates (over 95%), glucuronides and some sulphates, in LNCaP cells, whereas it was only partly converted to sulphates (<10%) in MCF-7 cells. Aromatase activity seems to be inconsistent in LNCaP cells, being strongly affected by culture conditions, especially by fetal calf serum (FCS). Further studies should assess the regulation of aromatase expression by serum or growth factors in different human cancer cells, also using anti-aromatase and/or anti-estrogen compounds, in different culture conditions.     

High Sodium and Low Potassium Intake among Italian Children: Relationship with Age,Body Mass and Blood Pressure

Background

Hypertension is the leading cause of death in developed countries and reduction of salt intake is recommended as a key preventive measure.

Objective

To assess the dietary sodium and potassium intakes in a national sample of Italian children and adolescents and to examine their relationships with BMI and blood pressure (BP) in the framework of the MINISAL survey, a program supported by the Italian Ministry of Health.

Population and Methods

The study population included 1424 healthy subjects (766 boys, 658 girls) aged 6-18 years (mean age: 10.1±2.9) who were consecutively recruited in participating National Health Service centers in 10 Italian regions. Electrolyte intake was estimated from 24 hour urine collections tested for completeness by the concomitant measurement of creatinine content. Anthropometric indices and BP were measured with standardized procedures.

Results

The average estimated sodium intake was 129 mmol (7.4 g of salt) per day among boys and 117 mmol (6.7 g of salt) among girls. Ninety-three percent of the boys and 89% of the girls had a consumption higher than the recommended age-specific standard dietary target. The estimated average daily potassium intakes were 39 mmol (1.53 g) and 36 mmol (1.40 g), respectively, over 96% of the boys and 98% of the girls having a potassium intake lower than the recommended adequate intake. The mean sodium/potassium ratio was similar among boys and girls (3.5 and 3.4, respectively) and over 3-fold greater than the desirable level. Sodium intake was directly related to age, body mass and BP in the whole population.

Conclusions

The Italian pediatric population is characterized by excessive sodium and deficient potassium intake. These data suggest that future campaigns should focus on children and adolescents as a major target in the framework of a population strategy of cardiovascular prevention.     

Biogenesis of linear O-alkylfuranocoumarins: A new pathway involving 5-hydroxymarmesin

adioactivity from [³H] 5-hydroxymarmesin was incorporated into 5-methoxypsoralen by administration to leaves of Ficus carica and cut ends of Ruta graveolens. No other furanocoumarins were labelled. Trapping experiments, in which [³H]marmesin together with 5-hydroxymarmesin was administered to fig leaves and to cut ends of rue, provided good evidence that 5-hydroxymarmesin is formed by hydroxylation of marmesin. These results, together with those obtained previously with 8-hydroxymarmesin demonstrate that, in addition to the pathway which involves the hydroxylation of psoralen, the O-alkylfuranocoumarins are also formed by a pathway which involves the hydroxylation of marmesin.     

Melatonin in the seasonal response of the aphid Acyrthosiphon pisum

Aphids display life cycles largely determined by the photoperiod.During the warm long-day seasons.most aphid species reproduce by viviparous parthenogenesis.The shortening of the photoperiod in autumn induces a switch to sexual reproduction.Males and sexual females mate to produce overwintering resistant eggs.In addition to this full life cycle(holocycle),there are anholocyelic lineages that do not respond to changes in photoperiod and reproduce continuously by parthenogenesis.The molecular or hormonal events that trigger the scasonal response(i.c,induction of the sexual phenotypes)are still unknown.Although circadian synthesis of melatonin is known to play a key role in vertebrate photoperiodism,the involvement of the circadian clock and/or of the hor-mone melatonin in insect seasonal responses is not so well established.Here we show that melatonin levels in the aphid Acyrthosiphon pisum are significantly higher in holocyclice aphids reared under short days than under long days,while no differences were found between anholoeyelic aphids under the same conditions.We also found that melatonin is localized in the aphid suboesophageal ganglion(SOG)and in the thoracic ganglionic mass(TGM).In analogy to vertcbrates,insect-type arylalkxylamine N-acetyltransferases(i-AANATs)are thought to play a key role in melatonin synthesis.We measured the expression of four I-AANAT genes identified in A.pisum and localized two of them in situ in the insect central nervous systems(CNS).Levels of expression of these genes were compatible with the quantities of melatonin observed.Moreover,like melatonin,expression of these genes was found in the SOG and the TGM.