Bioactive Substances in Safflower Flowers and Their Applicability in Medicine and Health-Promoting Foods.

Safflower flowers (Carthamus tinctorius) contain many natural substances with a wide range of economic uses. The most famous dye isolated from flower petals is hydroxysafflor A (HSYA), which has antibacterial, anti-inflammatory, and antioxidant properties. This review is aimed at updating the state of knowledge about their applicability in oncology, pulmonology, cardiology, gynecology, dermatology, gastrology, immunology, and suitability in the treatment of obesity and diabetes and its consequences with information published mainly in 2018-2020. They were also effective in treating obesity and diabetes and its consequences. The issues related to the possibilities of using HSYA in the production of health-promoting food were also analyzed.

Atypical skin inflammation in a 2.5-year-old girl with atopic dermatitis

No disponible

All genes encoding enzymes participating in melatonin biosynthesis in the chicken pineal gland are transcribed rhythmically.

Our recent research on the pineal gland of young chickens confirmed that three genes encoding enzymes involved in pineal melatonin biosynthesis, tryptophan hydroxylase 1 (Tph1), arylalkylamine-N-acetyltransferase (Aanat) and acetylserotonin O-methyltransferase (Asmt), are transcribed rhythmically under light:dark (L:D) 12:12 conditions in vivo. Additionally, in the pineal gland of maturing chickens, the dopa decarboxylase (Ddc) gene is transcribed rhythmically at a specific stage of the developmental process. Therefore, the aim of the present study was to verify whether all of these genes are transcribed rhythmically in vivo under constant darkness (D:D) and in pinealocyte cultures under both L:D and D:D. Experiments were performed on chickens maintained under L:D 12:12 conditions. Chickens at 15 days of age were divided into two groups; chickens from the first group remained under the same conditions, whereas those from the second group were kept in darkness. Subsequently, 16-day-old animals were sacrificed every 2 hours over a 24-h period. For the in vitro experiments, 16-day-old chickens were sacrificed at ZT 6, and their pineal glands were isolated. Pineal cultures were maintained for up to two days in L:D conditions. Then, the pinealocyte cultures were divided into two groups: the first remained under L:D conditions, whereas the second was transferred to D:D conditions. Pinealocytes were subsequently collected every 2 hours over a 24-h period. Transcription was evaluated using the RT-qPCR method, and the rhythm percentage was calculated through Cosinor analysis. The mRNA levels of all genes examined were rhythmic under all conditions. Moreover, in silico analysis of the promoters of all of the genes examined revealed the presence of enhancer box sequences in all of the promoters as well as DBP/E4BP4 binding elements in the promoters of Tph1 and Asmt. This suggests that these genes may all be regulated transcriptionally by the molecular clock mechanism and may be considered clock as controlled genes.

Season-dependent postembryonic maturation of the diurnal rhythm of melatonin biosynthesis in the chicken pineal gland.

Previously, we have demonstrated that the timing of the nocturnal peak of activity of the pineal arylalkylamine-N-acetyltransferase - a key enzyme in the melatonin biosynthesis pathway - in 3-wk-old chickens kept from the day of hatch under controlled laboratory conditions (L:D 12:12) varies depending on the season of hatch (summer vs. winter). The present study was undertaken to answer the following questions: (1) are season-related differences seen in the level of transcription of genes encoding enzymes of the melatonin biosynthesis pathway? (2) Does the pineal content of the main precursor (serotonin) and the final product (melatonin) exhibit age- and season-related changes? (3) At which step in postembryonic development are these season-related variations in pineal gland function most pronounced? Male Hy-line chickens hatched in the summer or winter, from eggs laid by hens held on L:D 16:8, were kept from the day of hatch under L:D 12:12 conditions. At the age of 2, 9, or 16 d, chickens were sacrificed every 2 h over a 24-h period and their pineal glands, isolated under dim red light, were processed for the measurement of (i) the level of Aanat and Asmt (acetylserotonin O-methyltransferase) mRNAs encoding the two last enzymes involved in melatonin biosynthesis, (ii) the activity of these enzymes, and (iii) the pineal content of serotonin and melatonin. Circadian rhythmicity of all the measured parameters was evaluated by the cosinor method. The levels of Aanat mRNA, AANAT enzymatic activity, and the pineal melatonin content changed during postembryonic development in a manner that was dependent on the season of hatch. Furthermore, the diurnal profile of Asmt mRNA was elevated during the light phase. In "winter" birds, the pattern and amplitude of the diurnal rhythm of accumulation of this transcript did not change with age, while in "summer" birds it increased in an age-related way. In contrast, the enzymatic activity of hydroxyindole-O-methyltransferase (HIOMT; encoded by the Asmt gene) did not change rhythmically, although it increased with age in a season-related way. In "winter" chickens, the pineal serotonin content was low, regardless of age, and did not change rhythmically, whereas in "summer" individuals the serotonin rhythm was already well established by day 2, with the amplitude increasing with age. These results confirm the existence of a "seasonal memory" operating within the chicken pineal gland, although the mechanism(s) underlying this phenomenon have yet to be characterized.

Light stress photodynamics of chlorophyll-binding proteins in Arabidopsis thaliana thylakoid membranes revealed by high-resolution mass spectrometric studies.

In higher plants the light energy is captured by the photosynthetic pigments that are bound to photosystem I and II and their light-harvesting complex (LHC) subunits. In this study, we examined the photodynamic changes within chlorophyll-protein complexes in the thylakoid membrane of Arabidopsis thaliana leaves adapted to low light and subsequently exposed to light stress. Chlorophyll-protein complexes were isolated using sucrose density gradient centrifugation and blue-native polyacrylamid gel electrophoresis (BN-PAGE). Proteome analysis was performed using SDS-PAGE, HPLC and high resolution mass spectrometry. We identified several rarely expressed and stress-induced chlorophyll-binding proteins, showed changes in localization of early light-induced protein family and LHC protein family members between different photosynthetic complexes and assembled/disassembled subcomplexes under light stress conditions and discuss their role in a variety of light stress-related processes.

Identification of genes expressed in response to light stress in leaves of Arabidopsis thaliana using RNA differential display.

The plant cell responds to light stress by the expression of genes encoding specific stress proteins with possible protective functions. Five genes, the mRNA levels of which increased drastically in response to light stress in mature green leaves of Arabidopsis thaliana were identified and isolated by the differential display technique. These genes were designated Lsr1-Lsr5 (light stress-regulated). Northern blot analysis demonstrated that the transcript level of Lsr1-Lsr5 increased 4- to 17-fold under light stress conditions as compared with leaves incubated at low intensity light. Further analysis of the Lsr1-Lsr5 transcript level under cold stress, heat shock, wounding, desiccation, salt stress, oxidative stress and UV-A irradiation showed that the expression of all five genes was triggered by more than one stress factor. Thus, it was expected that isolated genes encode proteins involved in general stress responses. Homology searches revealed that all of the isolated cDNAs were represented in the GenBank in genomic DNAs and expressed sequence tag (EST) cDNA clones. The Lsr1-Lsr4 genes encoded cytoplasmic proteins with assigned identities, such as ERD15 (early responsive to dehydration), ACT2 (actin 2), LEA14 (late embryogenesis abundant) and MT1a (metallothionein class 1a), respectively. Light stress had not yet been reported to induce or enhance the expression of these genes. The Lsr5 clone encoded a novel protein with high similarity to beta-1,3-galactosyltransferases from human and primates predicted to be located in the Golgi body. Three ORFs homologous to the Lsr5 gene were found on chromosome I and IV of Arabidopsis indicating that a multigene family of these proteins exists in plants. The possible role of Lsr gene products in light stress defences is discussed.

Chloroplast and mitochondrial proteases in Arabidopsis. A proposed nomenclature.

The identity and scope of chloroplast and mitochondrial proteases in higher plants has only started to become apparent in recent years. Biochemical and molecular studies suggested the existence of Clp, FtsH, and DegP proteases in chloroplasts, and a Lon protease in mitochondria, although currently the full extent of their role in organellar biogenesis and function remains poorly understood. Rapidly accumulating DNA sequence data, especially from Arabidopsis, has revealed that these proteolytic enzymes are found in plant cells in multiple isomeric forms. As a consequence, a systematic approach was taken to catalog all these isomers, to predict their intracellular location and putative processing sites, and to propose a standard nomenclature to avoid confusion and facilitate scientific communication. For the Clp protease most of the ClpP isomers are found in chloroplasts, whereas one is mitochondrial. Of the ATPase subunits, the one ClpD and two ClpC isomers are located in chloroplasts, whereas both ClpX isomers are present in mitochondria. Isomers of the Lon protease are predicted in both compartments, as are the different forms of FtsH protease. DegP, the least characterized protease in plant cells, has the most number of isomers and they are predicted to localize in several cell compartments. These predictions, along with the proposed nomenclature, will serve as a framework for future studies of all four families of proteases and their individual isomers.

A chloroplast DegP2 protease performs the primary cleavage of the photodamaged D1 protein in plant photosystem II.

Although light is the ultimate substrate in photosynthesis, it can also be harmful and lead to oxidative damage of the photosynthetic apparatus. The main target for light stress is the central oxygen-evolving photosystem II (PSII) and its D1 reaction centre protein. Degradation of the damaged D1 protein and its rapid replacement by a de novo synthesized copy represent the important repair mechanism of PSII crucial for plant survival under light stress conditions. Here we report the isolation of a single-copy nuclear gene from Arabidopsis thaliana, encoding a protease that performs GTP-dependent primary cleavage of the photodamaged D1 protein and hence catalysing the key step in the repair cycle in plants. This protease, designated DegP2, is a homologue of the prokaryotic Deg/Htr family of serine endopeptidases and is associated with the stromal side of the non-appressed region of the thylakoid membranes. Increased expression of DegP2 under high salt, desiccation and light stress conditions was measured at the protein level.

Stable insertion of the early light-induced proteins into etioplast membranes requires chlorophyll a.

Etiolated plant seedlings exposed to light respond by transient accumulation of the nucleus-encoded, plastid-located early light-inducible proteins (Elips). These proteins are distant relatives of the light-harvesting chlorophyll a/b-binding gene family and bind pigments with unusual characteristics. To investigate whether accumulation of Elips in plastid membranes is post-translationally regulated by pigments, reconstitution studies were performed, where in vitro transcribed and translated low molecular mass Elip precursors of barley were combined with lysed barley etioplasts complemented with various compositions of isolated pigments. We showed that the membrane insertion of Elips, as proven by protease protection assays and washes with a chaotropic salt or alkali, depended strictly on chlorophyll a but not on chlorophyll b or xanthophyll zeaxanthin. The amount of inserted Elips increased almost linearly with the chlorophyll a concentration, and the insertion efficiency was not significantly influenced by a light intensity between 1 and 1,000 micromol x m(-2) x s(-1). In contrast, in vitro import of Elip precursors into greening plastids was enhanced by high intensity light. Thus, we conclude that although chlorophylls bound to Elips seem to not be involved in light harvesting, they are crucial for a stable insertion of these proteins into the plastid membrane.

Proteomics of the chloroplast: systematic identification and targeting analysis of lumenal and peripheral thylakoid proteins.

The soluble and peripheral proteins in the thylakoids of pea were systematically analyzed by using two-dimensional electrophoresis, mass spectrometry, and N-terminal Edman sequencing, followed by database searching. After correcting to eliminate possible isoforms and post-translational modifications, we estimated that there are at least 200 to 230 different lumenal and peripheral proteins. Sixty-one proteins were identified; for 33 of these proteins, a clear function or functional domain could be identified, whereas for 10 proteins, no function could be assigned. For 18 proteins, no expressed sequence tag or full-length gene could be identified in the databases, despite experimental determination of a significant amount of amino acid sequence. Nine previously unidentified proteins with lumenal transit peptides are presented along with their full-length genes; seven of these proteins possess the twin arginine motif that is characteristic for substrates of the TAT pathway. Logoplots were used to provide a detailed analysis of the lumenal targeting signals, and all nuclear-encoded proteins identified on the two-dimensional gels were used to test predictions for chloroplast localization and transit peptides made by the software programs ChloroP, PSORT, and SignalP. A combination of these three programs was found to provide a useful tool for evaluating chloroplast localization and transit peptides and also could reveal possible alternative processing sites and dual targeting. The potential of proteomics for plant biology and homology-based searching with mass spectrometry data is discussed.

Light stress-regulated two-helix proteins in Arabidopsis thaliana related to the chlorophyll a/b-binding gene family.

The chlorophyll a/b, chlorophyll a/c, and chlorophyll a/a light-harvesting proteins are part of an extended gene family that also includes the transiently expressed stress proteins, the Elips (early light-induced proteins). Four Elip homologue proteins, encoded by single-copy nuclear genes, have been identified in the Arabidopsis thaliana database. These proteins were divided into two groups according to the expression pattern under light-stress conditions and the predicted secondary structure. Group one included two members of the Elip family with three predicted transmembrane helices and a gene expression strictly related to light stress. Group two included two proteins, the Seps (stress-enhanced proteins), which possessed two predicted transmembrane segments. The transcripts of Sep1 and Sep2 were present under low light conditions, but their level increased 4- to 10-fold during illumination of plants with high-intensity light. Preliminary data indicated that the induced transcripts were translated in vivo. Other physiological stress conditions, such as cold, heat, desiccation, salt, wounding, or oxidative stress, did not significantly influence the expression of Sep genes. In vitro import of radioactively labeled precursors of Seps into isolated chloroplasts confirmed the thylakoid membrane localization of these proteins. Considering the predicted protein structure and homology to other pigment-antenna proteins, the two-helix Seps might represent an evolutionary missing link between the one- and three-helix antenna proteins present in pro- and eukaryota.

Hepatitis C virus infection in hospitalized children.

AIM: The aim of the study was the estimation of the incidence of HCV infection and the analysis of risk factors for the infection in hospitalized children. MATERIAL AND METHODS: Retrospective analysis of the data of 1263 patients treated in hospital wards of Chair and Department of Gastroenterology and Paediatric Diseases from July 1995 to March 1998; all the patients had hepatitis C virus antibodies determined. Hepatitis C virus antibodies were assessed with the use of enzyme immunoassay. All the positive results were confirmed by means of Lia Tek HCV or PCR HCV RNA method. History data regarding frequency of previous hospitalizations, operations, blood transfusions and invasive diagnostic procedures patients had undergone were included in analysis. RESULTS: HCV infection was diagnosed in 47 children which accounted for 3.7% of the group enrolled in the study. The majority, that is 96.2% of seropositive children had the history of hospitalization, while 73.2% of them underwent intervention procedures disrupting tissue continuity during previous hospitalizations. Physical examination analysis indicates that all the patients with antibodies against HCV could have acquired the infection through parenteral transmission. The data indicating another route of transmission were not obtained (e.g. familial transmission). CONCLUSIONS: 1. Serological tests for HCV infection should be performed on routine basis in frequently hospitalized children. 2. HCV infection resulting from iatrogenic transmission can be suspected in the majority of seropositive children.

Isolation of pigment-binding early light-inducible proteins from pea.

The early light-inducible proteins (ELIPs) in chloroplasts possess a high sequence homology with the chlorophyll a/b-binding proteins but differ from those proteins by their substoichiometric and transient appearance. In the present study ELIPs of pea were isolated by a two-step purification strategy: perfusion chromatography in combination with preparative isoelectric focussing. Two heterogeneous populations of ELIPs were obtained after chromatographic separation of solubilized thylakoid membranes using a weak anion exchange column. One of these populations contained ELIPs in a free form providing the first isolation of these proteins. To prove whether the isolated and pure forms of ELIP bind pigments, spectroscopic and chromatographic analysis were performed. Absorption spectra and TLC revealed the presence of chlorophyll a and lutein. Measurements of steady-state fluorescence emission spectra at 77 K exhibited a major peak at 674 nm typical for chlorophyll a bound to the protein matrix. The action spectrum of the fluorescence emission measured at 674 nm showed several peaks originating mainly from chlorophyll a. It is proposed that ELIPs are transient chlorophyll-binding proteins not involved in light-harvesting but functioning as scavengers for chlorophyll molecules during turnover of pigment-binding proteins.

Developmental regulation of the PsbS gene expression in spinach seedlings: the role of phytochrome.

The PsbS gene product (PSII-S) which is an integral subunit of photosystem II has recently been reported to be a new type of pigment-binding protein [11]. The chlorophylls of the PSII-S protein exhibit weak excitonic coupling and this protein is stable also in the absence of pigments. Here we investigated the expression of the PsbS gene in etiolated spinach seedlings grown either in complete darkness or exposed to light of various qualities. The results obtained reveal that the PsbS gene expression in etiolated spinach plants is subjected to endogenous control. This developmental control occurs at different levels of gene expression and results in transient accumulation of the PsbS transcripts with progressing etiolation. During the first two days after emerging of the cotyledons from the seed coat the steady-state level of the PsbS transcripts is regulated mainly through increased transcriptional activity of the PsbS gene. Prolonged growth of the seedlings in the dark resulted in additional post-transcriptional control of the PsbS transcript level. Translational activity of PsbS mRNA estimated by an integration of mRNA into polysomal complexes shows that the translation rate of PsbS mRNA is less influenced by seedling age. The maximal rate of translation is reached at the first day after cotyledons emergence but the translational activity of PsbS mRNA remains still high (50% of maximum) in 8-day old etiolated spinach plants. In addition to the light-independent developmental control, the PsbS gene expression is positively regulated by phytochrome in etiolated seedlings exposed to light. Red light, however, negatively influences the abundance of PsbS transcripts at post-transcriptional level. Studies on blue or far-red light effects reveal that the accumulation of PsbS transcripts exhibits the characteristics of very-low-fluence responses of the phytochrome receptor.

Degradation of the light-stress protein is mediated by an ATP-independent, serine-type protease under low-light conditions.

Green plants respond to light stress by induction of the light-stress proteins (ELIPs). These proteins are stable as long as the light stress persists but are very rapidly degraded during subsequent low light conditions. Here we report that the degradation of ELIPs is mediated by an extrinsic, thylakoid-associated protease which is already present in the membranes during light stress conditions. Partial purification of the protease by perfusion chromatography indicates that this proteolytic activity may be represented by a protein with an apparent molecular mass of 65 kDa. The ELIP-directed protease is localized in the stroma lamellae of the thylakoid membranes and does not require ATP or additional stromal factors for proteolysis. The protease has an optimum activity at pH 7.5-9.5 and requires Mg2+ for its activity. The ELIP-degrading protease show an unusual temperature sensitivity and becomes reversibly inactivated at temperatures below 20 degree C and above 30 degree C. Studies with protease inhibitors indicate that this enzyme belongs to the serine class of proteases. The enhanced degradation of ELIP in isolated thylakoid membranes after addition of the ionophore nigericin suggests that a trans-thylakoid delta pH or changes in ionic strength may be involved in the mechanism of protease activation.

The nuclear-encoded chlorophyll-binding photosystem II-S protein is stable in the absence of pigments.

The 22-kDa chlorophyll a/b-binding protein (CAB) (psbS gene product) is associated with photosystem II and related to the CAB gene family. Here we report that the PSII-S protein unlike other chlorophyll-binding proteins is stable in the absence of pigments. It is present in etiolated spinach plants and accumulates in the dark progressively with the cellular development of the seedlings. Furthermore, it is present in several pigment-deficient mutants. Analysis of the pigment composition of the PSII-S protein isolated from etiolated plants suggests that neither carotenoids nor chlorophyll precursors are involved in its stabilization in the dark. Exposure of etiolated spinach to light leads to further accumulation of the PSII-S protein, which appears more early than other chlorophyll-binding proteins. Accumulation of the PSII-S protein in green plants is developmentally regulated and restricted to photosynthetic tissues. It is suggested that the function of the PSII-S protein may not be light-harvesting but it could act as a ligand chaperone required for transient binding of pigments during biogenesis or turnover of chlorophyll-binding proteins. Such function would be essential for coordination between pigment biosynthesis and ligation as well as avoiding toxic effects of non-bound chlorophyll molecules.

Regulation of Early Light-Inducible Protein Gene Expression by Blue and Red Light in Etiolated Seedlings Involves Nuclear and Plastid Factors.

Early light-inducible proteins (ELIPs) are nuclear-encoded chloroplast proteins whose genes are transiently transcribed during the greening process of etiolated plants. In the present work the regulation of ELIP gene expression by blue and red light has been investigated in plumulas of etiolated pea plants (Pisum sativum). The results show that the steady-state level of ELIP transcripts is controlled by a combined action of phytochrome and blue light receptor systems and, in addition, depends on the age of the seedlings. Both a low-light fluence system of blue and a very-low-fluence system of red light are involved in ELIP induction. The threshold for accumulation of ELIP transcripts was as low as 10-5 [mu]E m-2 s-1 for both light qualities but a different pattern of accumulation was obtained in blue and in red light. Blue light not only acts at the level of transcription but also regulates the stability of the ELIP transcripts in a light intensity-dependent manner. Moreover, it is shown that product(s) of nuclear gene(s) negatively regulate the steady-state level of ELIP transcripts during the 1st h of illumination with red light. Preillumination of seedlings with white light abolishes this repression. Accumulation of ELIP transcripts requires "plastid factors" in both blue and red light qualities.

Low temperature increases the abundance of early light-inducible transcript under light stress conditions.

Green pea plants respond to light stress by expression of a nuclear ELIP (early light-inducible protein) gene. Here we report that the accumulation of ELIP transcript in pea plants during light stress is enhanced by low temperature treatment. The enhanced level of ELIP transcript during combined light and cold stress was found to be due to an increased stability of ELIP messenger RNA under these conditions. This transcript is translatable in vitro. In vivo, however, the amount of accumulated protein in the thylakoids declines with the decrease in the temperature because the translational activity is strongly reduced already at 10 degrees C. Plants exposed to light stress at temperatures that do not allow accumulation of ELIP transcript respond by induction of ELIP mRNA and protein during recovery at low light intensity and ambient temperature. The amount of protein that accumulates as a result of this "memory effect" is, however, much lower than that which accumulates as a result of direct light stress. The memory of a perceived light stress persists in plants stored at low temperature for at least 3 h, and the stress response can be released after an increase in temperature. Prolonged cold treatment, however, has a negative effect on the translatability of the ELIP transcript that accumulates during recovery.

Early light-inducible protein in pea is stable during light stress but is degraded during recovery at low light intensity.

The nuclear-encoded, thylakoid-bound early light-inducible protein (ELIP) reported to be related to the initial stages of chloroplast differentiation is synthesized in substantial amounts in leaves of mature plants exposed to light stress conditions (Adamska, I., Ohad, I., and Kloppstech, K. (1992b) Proc. Natl. Acad. Sci. U.S.A. 89, 2610-2613). Increase in ELIP content correlates with the photoinactivation of PSII, degradation of D1 protein, and changes in the level of pigments. Inhibition of phytoene desaturase and/or zeta-carotene desaturase during light stress drastically increases accumulation of the protein. ELIP mRNA is short-lived (t1/2 = 1 h). The thylakoid bound protein is stable in high light exposed leaves and is degraded only during recovery from light stress at low light intensity (40 microE/m2s). The lifetime of the protein during the recovery process increases with the extent of initial light stress condition. We propose that ELIP synthesis and degradation is related to the process of the plant response to light stress and recovery from photoinhibition.