Are carotenoids the blue-light photoreceptor in the photoinduction of protoperithecia in Neurospora crassa?

A triple albino mutant of Neurospora crassa with a measured content of carotenoids absorbing at 470 nm less than 0.5% of that of the wild type (calculated value less than 8·10^-4%) had the same threshold for photoinduction of protoperithecia as the wild type when illuminated with monochromatic light at 471 nm. This is strong evidence against the hypothesis that the bulk of carotenoids are the blue-light photoreceptor for this phenomenon. However, it is impossible to exclude traces of carotenoids acting as the photoreceptor at less than 3·10^-12 M in a very efficient sensory transduction chain.Abbreviations A absorbance - al albino mutant - WT wild type     

Riboflavin-binding sites associated with flagella of Euglena: A candidate for blue-light photoreceptor?

The hypothesis was tested that reversible riboflavin (RF)-binding sites are part of the photoreceptor in Euglena gracilis. Published evidence shows that the phototactic stimulus — with a flavin-type action spectrum — is perceived at the paraflagellar body (PFB). Flagella with PFBs were isolated from Euglena gracilis by a combined cold and Ca²⁺ shock. Saturable binding of [¹⁴C]RF was demonstrated with such preparations, in the oxidized state as well as under reducing conditions in the presence of dithionite. Affinities for RF were high: K _D (oxidized)=0.08 M, and K _D (reduced)=0.7 M. Flavin mononucleotide and flavin adenine dinucleotide showed lower binding affinities. The in vitro RF binding per unit of protein was enriched approximately tenfold in the flagellar preparations when compared with homogenates of whole cells. The number of (reduced) binding sites per entire flagellum was determined to be in the order of 10⁶. This number is in line with published estimates of chromophores bound in or at the PFB.Abbreviations FAD flavin adenine dinucleotide - FMN flavin mononucleotide - PFB paraflagellar body - RF riboflavin This work was supported by the Deutsche Forscungsgemeinschaft.     

Boron — Key element in the actions of phytochrome and gravity?

Hypocotyl sections from dark-grown mung bean (Vigna radiata (L.) Wilczek) seedlings grown without boron exhibit little or no increase in the translocation of fluorescein in phloem cells following gravitational stimulation or red irradiation. In contrast, pretreatment of the hypocotyl sections with low concentrations of boric acid before red irradiation or gravitational stimulation results in a rapid increase in fluorescein movement. Mung bean root tips without boron fail to remain attached to a negativelycharged glass surface after several minutes of red irradiation. A low boron concentration in the solution induces tip attachment. The suggestion is made that boron is required to stabilize a positive electrostatic charge in the plasma membrane that is generated by the actions of phytochrome and gravity.     

Chemotaxis of the unicellular green algaDunaliella salina and the ciliatedTetrahymena pyriformis—effects of glycine,lysine, and alanine,and their oligopeptides

Chemotactic properties of amino acids (L-alanine, glycine and L-lysine) and their oligopeptides (10^–6M) and binding sites to these ligands were investigated in two unicellular models, the heterotrophicTetrahymena pyriformis and the auxotrophicDunaliella salina. Chemotaxis ofDunaliella induced by simple amino acids and their derivatives demonstrated that binding sites (receptors) for food molecules are not only present in the membrane but are also able to induce their basic physiological response. InTetrahymena, substances with special molecular structure and properties (polar, hydrophilic character of the signal peptide chain)-5-L-Lys, 5-Glywere required for chemoattraction, other peptides tested, lacking the required structure, were repellent. Divergences in chemotaxis and binding assays of both species suggest that trends of functional and binding parameters do not run parallel at this level of evolution.     

Uptake of HCO3? and CO2 in Cells and Chloroplasts from the Microalgae Chlamydomonas reinhardtii and Dunaliella tertiolecta

Mass-spectrometric disequilibrium analysis was applied to investigate CO₂ uptake and HCO₃⁻ transport in cells and chloroplasts of the microalgae Dunaliella tertiolecta and Chlamydomonas reinhardtii, which were grown in air enriched with 5% (v/v) CO₂ (high-Ci cells) or in ambient air (low-Ci cells). High- and low-Ci cells of both species had the capacity to transport CO₂ and HCO₃⁻, with maximum rates being largely unaffected by the growth conditions. In high- and low-Ci cells of D. tertiolecta, HCO₃⁻ was the dominant inorganic C species taken up, whereas HCO₃⁻ and CO₂ were used at similar rates by C. reinhardtii. The apparent affinities of HCO₃⁻ transport and CO₂ uptake increased 3- to 9-fold in both species upon acclimation to air. Photosynthetically active chloroplasts isolated from both species were able to transport CO₂ and HCO₃⁻. For chloroplasts from C. reinhardtii, the concentrations of HCO₃⁻ and CO₂ required for half-maximal activity declined from 446 to 33 μm and 6.8 to 0.6 μm, respectively, after acclimation of the parent cells to air; the corresponding values for chloroplasts from D. tertiolecta decreased from 203 to 58 μm and 5.8 to 0.5 μm, respectively. These results indicate the presence of inducible high-affinity HCO₃⁻ and CO₂ transporters at the chloroplast envelope membrane.     

Involvement of the Wnt/β-Catenin Signaling Pathway in the Cellular and Molecular Mechanisms of Fibrosis in Endometriosis

Background

During the development and progression of endometriotic lesions, excess fibrosis may lead to scarring, chronic pain, and altered tissue function. However, the cellular and molecular mechanisms of fibrosis in endometriosis remain to be clarified.

Objectives

The objective of the present study was to investigate whether the Wnt/β-catenin signaling pathway was involved in regulating the cellular and molecular mechanisms of fibrosis in endometriosis in vitro and to evaluate whether fibrosis could be prevented by targeting the Wnt/β-catenin pathway in a xenograft model of endometriosis in immunodeficient nude mice.

Methods

Seventy patients (40 with and 30 without endometriosis) with normal menstrual cycles were recruited. In vitro effects of small-molecule antagonists of the Tcf/β-catenin complex (PKF 115-584 and CGP049090) on fibrotic markers (alpha smooth muscle actin, type I collagen, connective tissue growth factor, fibronectin) and collagen gel contraction were evaluated in endometrial and endometriotic stromal cells from patients with endometriosis. In vitro effects of activation of the Wnt/β-catenin signaling pathway by treatment with recombinant Wnt3a on profibrotic responses were evaluated in endometrial stromal cells of patients without endometriosis. The effects of CGP049090 treatment on the fibrosis of endometriotic implants were evaluated in a xenograft model of endometriosis in immunodeficient nude mice.

Results

Treatment with PKF 115-584 and CGP049090 significantly decreased the expression of alpha smooth muscle actin, type I collagen, connective tissue growth factor and fibronectin mRNAs in both endometriotic and endometrial stromal cells with or without transforming growth factor-β1 stimulation. Both endometriotic and endometrial stromal cell-mediated contraction of collagen gels was significantly decreased by treatment with PKF 115-584 and CGP049090 as compared to that of untreated cells. The animal experiments showed that CGP049090 prevented the progression of fibrosis and reversed established fibrosis in endometriosis.

Conclusion

Aberrant activation of the Wnt/β-catenin pathway may be involved in mediating fibrogenesis in endometriosis.     

Tissular localization of coumarins in the green alga Dasycladus vermicularis (Scopoli) Krasser: a photoprotective role?

Cell distribution of coumarins, a group of UV-absorbing substances, was analysed by epifluorescence optical microscopy in the green macroalga Dasycladus vermicularis. Maximal concentration of 3,6,7-trihydroxycoumarin (THC), which corresponds to almost 100% of the total coumarins in D. vermicularis, was found in the apical part of the thallus, which is more exposed to solar radiation. At a cell level, two blue, highly fluorescent layers, corresponding to a large accumulation of THC, were found in the internal part of the cell wall and around the vacuolar membrane. The percentage of UV radiation absorbed by each THC layer could be measured from the in vitro total thallus concentration of THC and histological measurements of the layers. The THC layer close to the cell wall absorbed 88% of the incident irradiance at 346 nm (corresponding to the maximum of absorbance of THC in the UVA region), while that close to the vacuole membrane absorbed 87.5%. These results agree with the hypothesis of a natural sunscreen role, significantly reducing harmful UV radiation reaching the cell. Owing to the release of this substance into the medium under different stress conditions, its capacity as a UV filter for other macroalgae has been tested. The ecological relevance of the release process of this UV-absorbing substance in specific environments is discussed.     

Regulation of enzyme levels by phytochrome in mustard cotyledons: Multiple mechanisms?

Phytochrome controls the appearance of many enzymes in the mustard (Sinapis alba L.) cotyledons. The problem has been whether the effect of phytochrome on the appearance of enzymes in this organ is due to a common initial action of P_fr, e.g. due to the liberation of a second messenger. We have compared the modulation by light (phytochrome) of the appearance of phenylalanine ammonia lyase (PAL)⁺ and ribulosebisphosphate carboxylase (Carboxylase)⁺. PAL becomes detectable in the mustard cotyledons at 27 h after sowing while Carboxylase starts to appear only at 42 h after sowing (starting points, 25° C). The starting points cannot be shifted by light. As a major result, in the case of PAL the inductive effect of continuous red light (given from the time of sowing) remains fully reversible by 756 nm-light up to the starting point (27 h after sowing) while with Carboxylase full reversibility in continuous red light is lost at approximately 15 h after sowing. While the induction of Carboxylase is already saturated at a very low level of P_fr (e.g. continuous 756 nm-light saturates the response) and does not depend on irradiance (e.g. continuous 675 mW m^-2 red light and 67.5 mW m^-2 red light lead to the same time course), PAL induction is a graded response over a wide range of P_fr doses and depends strongly on the fluence rate (high irradiance response, HIR). It is concluded that PAL induction and Carboxylase induction are not only separated in time but differ in every regard except that both responses are mediated by phytochrome.The present data support the previous conclusion that the specification of the temporal and spatial pattern of development is independent of phytochrome even though the realization of the pattern of development can only occur in the presence of phytochrome (P_fr). It seems that there is no feedback from pattern realization to pattern specification.Abbreviations P_fr the far-red absorbing, physiologically active form of phytochrome - P_r the red absorbing physiologically inactive form of phytochrome - P_total [P_r]+[P_fr] - PAL phenylalanine ammonia-lyase (EC 4.3.1.5) - Carboxylase ribulosebisphosphate carboxylase (EC 4.1.1.39)     

Genoprotection and genotoxicity of green tea (Camellia sinensis): Are they two sides of the same redox coin?

Abstract

Objectives

Regular intake of green tea associates with lower DNA damage and increased resistance of DNA to oxidant challenge. However, in vitro pro-oxidant effects of green tea have been reported. Both effects could be mediated by hydrogen peroxide (H₂O₂) which is generated by autoxidation of tea catechins. In large amounts, H₂O₂ is genotoxic, but low concentrations could activate the redox-sensitive antioxidant response element (ARE) via the Keap-1/Nrf2 redox switch, inducing genoprotective adaptations. Our objective was to test this hypothesis.

Methods

Peripheral lymphocytes from healthy volunteers were incubated for 30 minutes at 37°C in freshly prepared tea solutions (0.005, 0.01, 0.05%w/v (7, 14, 71 µmol/l total catechins) in phosphate buffered saline (PBS), with PBS as control) in the presence and absence of catalase (CAT). H₂O₂ in tea was measured colorimetrically. Oxidation-induced DNA lesions were measured by the Fpg-assisted comet assay.

Results

H₂O₂ concentrations in 0.005, 0.01, and 0.05% green tea after 30 minutes at 37°C were, respectively, ～3, ～7, and ～52 µmol/l. Cells incubated in 0.005 and 0.01% tea showed less (P < 0.001) DNA damage compared to control cells. Cells treated with 0.05% green tea showed ～50% (P < 0.001) more DNA damage. The presence of CAT prevented this damage, but did not remove the genoprotective effects of low-dose tea. No significant changes in expression of ARE-associated genes (HMOX1, NRF2, KEAP1, BACH1, and hOGG1) were seen in cells treated with tea or tea + CAT.

Conclusion

Genoprotection by low-dose green tea could be due to direct antioxidant protection by green tea polyphenols, or to H₂O₂-independent signalling pathways.     

The fertility of women Poland in the period of trasformation of the political and economics system (the 80’s and 90’s)

Wielkopolska (western Poland) is a region, with good reason, called the main food base of Poland. All indices of agricultural production have always been there (since 19th century) much higher than elsewhere in the country (Ratajczak 1977/1978, Buszko 1984, Gorzelak 1989, Czyzewski et al. 1992; table 1). Such a situation had a beneficial effect on the amount of the income generated by a family of Wielkopolska and, thus, decided on its better financial status. Additionally, rural inhabitants of this region have always been characterised by a more advantageous educational structure in comparison with people of other Polish villages. Generally, it is easy to detect the relationship between the life standard, the level of education and family planning. The aim of this paper is to assess fertility of women from villages of Wielkopolska in the period of transformation of the political and economic system in Poland.     

Presumptive photoreceptor structures of the trochophore of Harmothoë imbricata (Polychaeta)

The behaviour of the Harmothoë trochophore changes with age, the larva being phototropic initially and later photonegative.

The trochophore possesses two ocelli midway between the prototroch and the apex in a mid‐lateral position. They appear first at the eighth day of development and grow to be kidney‐shaped structures. There is a pigment cup derived from a single cell that encloses a rhabdomeric type of photoreceptor apparatus that is also derived from a single (or rarely two) cell.

In the late trochophore (14 days old) an organ of different origin and formation but of presumed photoreceptor type begins to develop among nerve cell bodies below the apex of the animal. This structure consists of an array of membranes developed from both cilia and microvilli. The cilia are of 9 + 2 configuration.     

Effects of photoperiod and of ultraviolet radiation on the incidence of diapause in the Horn Fly, “Haematobia Irritans (L.) (Diptera: Muscidae)”

The horn fly, HAEMATOBIA IRRITANS (L.), is a bloodsucking pest of cattle found in almost all cattle-raising areas of the northern hemisphere.Predisposition to diapause in the pupal stage was determined in the adult parent. The predisposition was influenced by photoperiod which may have its effect either directly on the insect or indirectly by influencing the host blood. The percentage of the pupae that entered diapause varied inversely with the length of the photoperiod between 12 and 20 hours. Light in the visible range (7,800 to 4,000 A.U.) was provided byCool-whitefluorescent tubes. The addition of ultraviolet radiation (3,900 to 2,800 A.U.) in the 16- and 20-hour photoperiods further decreased the percentage of diapause.

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Zusammenfassung Die Hornfliege, HAEMATOBIA IRRITANS (L.), ist ein blutsaugender Parasit in fast allen Rinderzuchtgebieten der nördlichen Hemisphäre.Die Prädisposition zur Diapause im Puppenstadium wurde in den erwachsenen Eltern determiniert.Sie wurde durch Belichtung beeinflusst, die entweder direkt auf das Insekt oder indirekt über das Blut des Wirtes wirkte. Die Prozentzahl an Puppen in Diapause fiel ab, wenn die periodischen Belichtungszeiten von 12 bis 20 Stunden verlängert wurden. Das sichtbare Licht (7800–4000 Å) stammte von cool-white fluoreszierenden Röhren. Durch die periodische Zugabe einer Ultraviolett- (3900 – 2800 Å) Bestrahlung fiel der Anteil an Diapausen noch weiter.

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Résumé La mouche, HAEMATOBIA IRRITANS (L.), est un parasite hématophage du bétail répandu dans presque toutes les zones d'élevagê de l'hémisphère nord. La prédisposition à la diapause au stade de pupe a été déterminée chez les parents adultes. La prédisposition est influencée par une période d'éclairage qui peut agir soit directement sur l'insecte soit indirectement par l'intermédiaire du sang de l'hôte. Le pourcentage des pupes entrant en diapause varie inversement à la durée d'un éclairage de 12 à 20 heures. La lumière blanche (7800 – 4000 Å) est produite par des tubes fluorescents froids. L'addition de rayonnement ultra-violet (3900 à 2800 Å) dans les périodes d'éclairage de 16 et de 20 heures réduit encore le pourcentage de diapauses.

     

A Role for the PKC Signaling System in the Pathophysiology and Treatment of Mood Disorders: Involvement of a Functional Imbalance?

Mood disorders, such as bipolar and major depressive disorders, are frequent, severe, and often disabling neuropsychiatric diseases affecting millions of individuals worldwide. Available mood stabilizers and antidepressants remain unsatisfactory because of their delayed and partial therapeutic efficacy. Therefore, the development of targeted therapies, working more rapidly and being fully effective, is urgently needed. In this context, the protein kinase C (PKC) signaling system, which regulates multiple neuronal processes implicated in mood regulation, can constitute a novel therapeutic target. This paper reviews the currently available knowledge regarding the role of the PKC signaling pathway in the pathophysiology of mood disorders and the therapeutic potential of PKC modulators. Current antidepressants and mood stabilizers have been shown to modulate the PKC pathway, and the inhibition of this intracellular signaling cascade results in antimanic-like properties in animal models. Disrupted PKC activity has been found both in postmortem brains and platelet from patients with mood disorders. Finally, the PKC inhibitor tamoxifen has recently demonstrated potent antimanic properties in several clinical trials. Overall, emerging data from preclinical and clinical research suggest an imbalance of the PKC signaling system in mood disorders. Thus, PKC may be a critical molecular target for the development of innovative therapeutics.     

Begomoviruses: what is the secret(s) of their success?

Begomoviruses constitute an extremely successful group of emerging plant viruses transmitted by whiteflies of the Bemisia tabaci complex. Hosts include important vegetable, root, and fiber crops grown in the tropics and subtropics. Factors contributing to the ever-increasing diversity and success of begomoviruses include their predisposition to recombine their genomes, interaction with DNA satellites recruited throughout their evolution, presence of wild plants as a virus reservoir and a source of speciation, and extreme polyphagia and continuous movement of the insect vectors to temperate regions. These features as well as some controversial issues (replication in the insect vector, putative seed transmission, transmission by insects other than B. tabaci, and expansion of the host range to monocotyledonous plants) will be analyzed in this review.     

Involvement of glycans in the immunological cross-reaction between α-macroglobulin and hemocyanin of the gastropod Helix pomatia

By tandem-crossed immunoelectrophoresis and ELISA experiments an immunological relationship was observed between α-macroglobulin (αM) and hemocyanin (Hc) of the terrestrial snail Helix pomatia. Both glycoproteins occur in the hemolymph: αM (minor component) as a specific proteinase inhibitor, Hc (consisting of three components: α_D-HpH, α_N-HpH and β-HpH) as oxygen transport protein. The cross-reaction was found to be correlated with glycosylation. (i) With β-HpH, which is richer in carbohydrates than α_D-HpH and α_N-HpH, mainly due to a higher 3-O-methyl-d-galactose content, the cross-reaction with HpαM was highest. (ii) From the 8 functional units, designated a–h, isolated from β-HpH, two that lack carbohydrates (c and f) were not recognized by antibodies against HpαM, while the six glycosylated ones were strongly cross-reacting. The nearly complete loss of the cross-reactivity upon deglycosylation of functional units d and g and the inhibition in competitive ELISA experiments by glycopeptides isolated from both β-HpH and HpαM are further evidence that glycans are involved in the immunological relationship between HpH and HpαM. Carbohydrate analyses indicated that the glycan structures present on HpαM are very similar (or identical) to those found on HpH, suggesting that glycans are common epitopes on both proteins. Especially d-xylose and 3-O-methyl-d-galactose seem to be responsible for the cross-reactivity since the α-macroglobulin and hemocyanin of the cephalopod Sepia officinalis, which lack these two monosaccharides in their glycan structures, do not immunologically cross-react.     

Involvement of GABAergic and glutamatergic systems in the anticonvulsant activity of 3-alkynyl selenophene in 21?day-old rats

In this study, we investigated the role of GABAergic and glutamatergic systems in the anticonvulsant action of 3-alkynyl selenophene (3-ASP) in a pilocarpine (PC) model of seizures. To this purpose, 21 day-old rats were administered with an anticonvulsant dose of 3-ASP (50 mg/kg, per oral, p.o.), and [(3)H]γ-aminobutyric acid (GABA) and [(3)H]glutamate uptakes were carried out in slices of cerebral cortex and hippocampus. [(3)H]GABA uptake was decreased in cerebral cortex (64%) and hippocampus (58%) slices of 21 day-old rats treated with 3-ASP. In contrast, no alteration was observed in [(3)H]glutamate uptake in cerebral cortex and hippocampus slices of 21 day-old rats that received 3-ASP. Considering the drugs that increase synaptic GABA levels, by inhibiting its uptake or catabolism, are effective anticonvulsants, we further investigated the possible interaction between sub-effective doses of 3-ASP and GABA uptake or GABA transaminase (GABA-T) inhibitors in PC-induced seizures in 21 day-old rats. For this end, sub-effective doses of 3-ASP (10 mg/kg, p.o.) and DL-2,4-diamino-n-butyric acid hydrochloride (DABA, an inhibitor of GABA uptake--2 mg/kg, intraperitoneally; i.p.) or aminooxyacetic acid hemihydrochloride (AOAA; a GABA-T inhibitor--10 mg/kg, i.p.) were co-administrated to 21 day-old rats before PC (400 mg/kg; i.p.) treatment, and the appearance of seizures was recorded. Results demonstrated that treatment with AOAA and 3-ASP or DABA and 3-ASP significantly abolished the number of convulsing animals induced by PC. The present study indicates that 3-ASP reduced [(3)H]GABA uptake, suggesting that its anticonvulsant action is related to an increase in inhibitory tonus.     

Involvement of PKCζ and GSK3β in the stability of the metaphase spindle

In the somatic cell, the mitotic spindle apparatus is centrosomal, and several isoforms of protein kinase C (PKC) have been associated with the mitotic spindle, but their role in stabilizing the mitotic spindle is still unclear. Other protein kinases such as, glycogen synthase kinase 3β (GSK3β) have also been shown to be associated with the mitotic spindle apparatus. In this study, we show the enrichment of active (phosphorylated) PKCζ at the centrosomal region of the spindle apparatus in metaphase stage of 3T3 cells. In order to understand whether the two kinases PKC and GSK3β are associated with the mitotic spindle, first, the co-localization of phosphorylated PKC isoforms with GSK3β was studied at the poles in metaphase cells. Fluorescence resonance energy transfer (FRET) analysis was used to demonstrate close molecular proximity of phospho-PKCζ with phospho(ser9)GSK3β. Second, the involvement of inactive GSK3β in maintaining an intact mitotic spindle in 3T3 cells was shown. Third, this study also showed that addition of a phospho-PKCζ specific inhibitor to cells can disrupt the mitotic spindle microtubules and some of the proteins associated with it. The mitotic spindle at metaphase in mouse fibroblasts appears to be maintained by PKCζ acting through GSK3β. Phospho-PKCζ is in close molecular proximity to GSK3β, whereas the other isoforms of PKC such as pPKCβII, pPKCγ, pPKCμ, and pPKCθ are not close enough to have significant FRET readings. The close molecular proximity supports the idea that GSK3β may be a substrate of PKCζ.     

Involvement of the TPR2 subdomain movement in the activities of ?29 DNA polymerase

The polymerization domain of ϕ29 DNA polymerase acquires a toroidal shape by means of an arch-like structure formed by the specific insertion TPR2 (Terminal Protein Region 2) and the thumb subdomain. TPR2 is connected to the fingers and palm subdomains through flexible regions, suggesting that it can undergo conformational changes. To examine whether such changes take place, we have constructed a ϕ29 DNA polymerase mutant able to form a disulfide bond between the apexes of TPR2 and thumb to limit the mobility of TPR2. Biochemical analysis of the mutant led us to conclude that TPR2 moves away from the thumb to allow the DNA polymerase to replicate circular ssDNA. Despite the fact that no TPR2 motion is needed to allow the polymerase to use the terminal protein (TP) as primer during the initiation of ϕ29 TP–DNA replication, the disulfide bond prevents the DNA polymerase from entering the elongation phase, suggesting that TPR2 movements are necessary to allow the TP priming domain to move out from the polymerase during transition from initiation to elongation. Furthermore, the TPR2-thumb bond does not affect the equilibrium between the polymerization and exonuclease activities, leading us to propose a primer-terminus transference model between both active sites.