The peculiar NPQ regulation in the stramenopile Phaeomonas sp. challenges the xanthophyll cycle dogma.

In changing light conditions, photosynthetic organisms develop different strategies to maintain a fine balance between light harvesting, photochemistry, and photoprotection. One of the most widespread photoprotective mechanisms consists in the dissipation of excess light energy in the form of heat in the photosystem II antenna, which participates to the Non Photochemical Quenching (NPQ) of chlorophyll fluorescence. It is tightly related to the reversible epoxidation of xanthophyll pigments, catalyzed by the two enzymes, the violaxanthin deepoxidase and the zeaxanthin epoxidase. In Phaeomonas sp. (Pinguiophyte, Stramenopiles), we show that the regulation of the heat dissipation process is different from that of the green lineage: the NPQ is strictly proportional to the amount of the xanthophyll pigment zeaxanthin and the xanthophyll cycle enzymes are differently regulated. The violaxanthin deepoxidase is already active in the dark, because of a low luminal pH, and the zeaxanthin epoxidase shows a maximal activity under moderate light conditions, being almost inactive in the dark and under high light. This light-dependency mirrors the one of NPQ: Phaeomonas sp. displays a large NPQ in the dark as well as under high light, which recovers under moderate light. Our results pinpoint zeaxanthin epoxidase activity as the prime regulator of NPQ in Phaeomonas sp. and therefore challenge the deepoxidase-regulated xanthophyll cycle dogma.

Maximal cyclic electron flow rate is independent of PGRL1 in Chlamydomonas.

Cyclic electron flow (CEF) is defined as a return of the reductants from the acceptor side of Photosystem I (PSI) to the pool of its donors via the cytochrome b6f. It is described to be complementary to the linear electron flow and essential for photosynthesis. However, despite many efforts aimed to characterize CEF, its pathway and its regulation modes remain equivocal, and its physiological significance is still not clear. Here we use novel spectroscopic to measure the rate of CEF at the onset of light in the green alga Chlamydomonas reinhardtii. The initial redox state of the photosynthetic chain or the oxygen concentration do not modify the initial maximal rate of CEF (60 electrons per second per PSI) but rather strongly influence its duration. Neither the maximal rate nor the duration of CEF are different in the pgrl1 mutant compared to the wild type, disqualifying PGRL1 as the ferredoxin-plastoquinone oxidoreductase involved in the CEF mechanism.

The mechanism of cyclic electron flow.

Apart from the canonical light-driven linear electron flow (LEF) from water to CO2, numerous regulatory and alternative electron transfer pathways exist in chloroplasts. One of them is the cyclic electron flow around Photosystem I (CEF), contributing to photoprotection of both Photosystem I and II (PSI, PSII) and supplying extra ATP to fix atmospheric carbon. Nonetheless, CEF remains an enigma in the field of functional photosynthesis as we lack understanding of its pathway. Here, we address the discrepancies between functional and genetic/biochemical data in the literature and formulate novel hypotheses about the pathway and regulation of CEF based on recent structural and kinetic information.

Adducts from in vivo action of the carcinogen 4-hydroxyaminoquinoline 1-oxide in rats and from in vitro reaction of 4-acetoxyaminoquinoline 1-oxide with DNA and polynucleotides.

In vivo 4-hydroxyamino[2-3H]quinoline 1-oxide-modified DNA and in vitro 4-acetoxyamino[2-3H]quinoline 1-oxide-modified DNA were enzymatically hydrolyzed, and the hydrolysates were analyzed by high-performance liquid chromatography. The two patterns were compared, and we showed that all of the high-performance liquid chromatography peaks which were recovered from in vivo-modified DNA were present in the hydrolysate of in vitro-modified DNA. Therefore, we used the in vitro 4-acetoxyamino[2-3H]quinoline 1-oxide-modified DNA to investigate the quinoline-purine adducts which are characteristics of the mode of action of the carcinogen 4-nitroquinoline 1-oxide. By comparison with the enzymatic hydrolysates of 4-acetoxyamino[2-3H]quinoline 1-oxide-modified covalent poly(deoxyadenylate-deoxythymidylate) X poly(deoxyadenylate-deoxythymidylate) and covalent poly(deoxyguanylate-deoxycytidylate) X poly(deoxyguanylate-deoxycytidylate) three nitroquinoline adducts were enumerated on the modified DNA. One of them was previously characterized as a C8-guanyl adduct. We proved that the two other are a guanine and an adenine adduct, respectively. A quinoline derivative was identified in the hydrolysates of the in vivo- and in vitro-modified DNAs as 4-aminoquinoline 1-oxide, the origin of which was postulated to be a degradation compound of one (or more) adduct(s). Moreover, the presence of two degradation compounds of the C8-guanyl adduct was shown in mild alkaline conditions. We suspected an imidazole ring-opened form.

Adducts from the reaction of O,O'-diacetyl or O-acetyl derivatives of the carcinogen 4-hydroxyaminoquinoline 1-oxide with purine nucleosides.

The diacetyl derivative of 4-hydroxyaminoquinoline 1-oxide (4-HAQO), the proximate carcinogen of 4-nitroquinoline 1-oxide, was reacted in vitro with purine nucleosides to give five adducts (three with guanosine and two with adenosine). The same nucleoside modifications were also obtained with a monoacetyl derivative of 4-HAQO which is probably 4-acetoxyaminoquinoline 1-oxide. The structure of the major adduct (the so-called dG III) was identified as N-(deoxyguanosin-C8-yl)-4-aminoquinoline 1-oxide. The isolation of this adduct from the 4-HAQO-modified DNA in vivo provides strong support for the hypothesis that the acetyl derivatives of 4-HAQO constitute a good model for the ultimate carcinogen.

N2-guanyl and N6-adenyl arylation of chicken erythrocyte DNA by the ultimate carcinogen of 4-nitroquinoline 1-oxide.

Great quantities of chicken erythrocyte DNA with high levels of modification were obtained in vitro by reaction with 4-acetoxyaminoquinoline 1-oxide, a model ultimate carcinogen of 4-nitroquinoline 1-oxide. After enzymatic hydrolysis of the modified DNA, the three main adducts were separated and isolated by semipreparative high performance liquid chromatography. These three adducts were already characterized in vivo and in vitro in our previous work (S. Galiègue-Zouitina et al., Cancer Res., 45: 520-525, 1985). The structure of one of them was previously identified as N-(deoxyguanosin-8-yl)-4-aminoquinoline 1-oxide (B. Bailleul et al., Cancer Res., 41: 4559-4565, 1981). In this paper we have identified by mass spectroscopy and nuclear magnetic resonance the structures of the two other main adducts as 3-(deoxyguanosin-N2-yl)-4-aminoquinoline 1-oxide and 3-(deoxyadenosin-N6-yl)-4-aminoquinoline 1-oxide, respectively.

Extent of helix perturbation associated with DNA modification by the o-acetyl derivative of the carcinogen 4-hydroxyaminoquinoline-1-oxide.

Duplex unwinding associated with DNA modification by 4-acetoxyaminoquinoline-1-oxide, a model ultimate carcinogen of 4-nitroquinoline-1-oxide, has been determined by the agarose gel electrophoresis band-shift method. An average unwinding angle per stable adduct of -15.1 degrees +/- 1.5 degrees for negatively supercoiled topoisomers and -6.5 degrees +/- 1.4 degrees for positively supercoiled topoisomers was obtained. Because of the different proportion of stable adducts (dGuo-N2-AQO, dGuo-C8-AQO, dAdo-N6-AQO) between negatively (8:1.5:0.5) and positively (5:2.5:1) supercoiled topoisomers, the difference in unwinding angles is suggestive of a diverse contribution of the various adducts to the overall conformational change. Since the largest unwinding angle was coupled with the highest proportion of dGuo-N2-AQO adduct, it is likely that this adduct is the most distortive lesion. A contribution of sites of base loss to DNA unwinding was also observed.

An ATF/CREB-binding site is essential for cell-specific and inducible transcription of the murine MIP-1 beta cytokine gene.

The murine macrophage inflammatory protein 1 beta mRNA (MIP-1 beta) is rapidly and transiently induced in macrophages by lipopolysaccharide (LPS), serum or cycloheximide. Functional studies of the MIP-1 beta proximal promoter indicate that it is cell-specific, and serum- and LPS-responsive in macrophages. A 76-bp proximal promoter sequence (-51 to -127 bp) confers cell-specific and LPS-inducible activity when placed upstream from a heterologous promoter in both orientations. One essential cis-regulatory element within the enhancer-like sequence is an activating transcription factor/cAMP response element (CRE)-binding protein (ATF/CREB)-binding site, although the promoter is not cAMP responsive. Electrophoretic mobility shift assays and mutational analyses suggest that the promoter site is bound by nuclear protein complexes containing cAMP-independent members of the ATF/CREB family of proteins and c-Jun, and are functionally distinct from the AP1-related TPA-response element (TRE) binding activity.

Shift from fetal-type to Alzheimer-type phosphorylated Tau proteins in SKNSH-SY 5Y cells treated with okadaic acid.

Tau proteins are abnormally phosphorylated in Alzheimer's disease. Pathological Tau proteins named PHF-Tau 55, PHF-Tau 64, and PHF-Tau 69, are the main constituents of the paired helical filaments (PHF). When treating SKNSH-SY 5Y cells with okadaic acid (OA), Tau 55 protein was clearly induced whereas Tau 64 protein was only faintly induced. Here, we show that the absence of Tau 69 could be explained by the fact that adult isoforms containing N-terminal inserts are not detected. Phosphorylation is similar for untreated cellular Tau proteins and fetal Tau proteins, while OA cell treatment transformed fetal-type into Alzheimer-type phosphorylated proteins.

Enzymatic methylation of DNA and poly(dG-dC) X poly(dG-dC) modified by 4-acetoxyaminoquinoline-1-oxide, the ultimate carcinogen of 4-nitroquinoline-1-oxide.

Both the initial velocity and the overall methylation of Ac-4HAQO modified DNA by a calf brain DNA (cytosine-5-)-methyltransferase are increased as compared to native DNA. The affinity of the modified DNA for the enzyme decreases as a function of the extent of the modification. Heat-denatured, single-stranded DNA shows exactly the opposite results: the more it is modified, the less it is methylated. The poly(dG-dC) X poly(dG-dC) modified by 4NQO is as well methylated as the non-modified one. The carcinogen may induce a tertiary structure favouring the 'walking' of the enzyme along the DNA. The hypermethylation caused by this carcinogen could have a significance in gene activity and cellular differentiation.

Chemical induction of oncogene mutations and growth factor activity in mouse skin carcinogenesis.

The goal of understanding the molecular basis of human tumor development has been greatly facilitated by the use of animal model systems in which the etiology of tumor development can be carefully controlled. Environmental chemicals, either naturally occurring or artificially produced, are thought to make a major contribution to the human tumor burden. Many of the concepts of multistage carcinogenesis have been developed and refined using the mouse skin model system and the work described in this article has been carried out in an attempt to analyze the molecular changes that are associated with the initiation of tumor development, the selection of initiated cells to form papillomas, or the progression of premalignant tumors to carcinoma. We have analyzed a number of skin tumors induced in mice by a two-stage initiation and promotion protocol and have detected a high frequency of c-ras oncogene mutations in this system. The mutation found in each case correlates well with the known reactivity of the carcinogens used. It has also been shown that where ras activation occurs this represents an early event in the tumor model system. Transforming growth factor beta is induced in mouse skin by tumor promoter treatment and may therefore play a role in the selection of initiated cells to form papillomas. Additional events, some of which involve the loss of normal ras alleles and possibly tumor suppressor genes, appear to take place at a later stage of carcinogenesis.

B219/OB-R 5'-UTR and leptin receptor gene-related protein gene expression in mouse brain and placenta: tissue-specific leptin receptor promoter activity.

Leptin receptor (OB-R) splice variants either encode proteins with different 3' cytoplasmic domains or have different 5' untranslated regions (UTR), indicative of dual promoters. The B219/OB-R promoter transcribes only OB-R transcripts, whereas the OB-R/GRP promoter initiates transcription of both OB-R and another protein of unknown function, called the leptin receptor gene-related protein (OB-RGRP). We compared expression of B219/OB-R 5'-UTR and OB-RGRP mRNAs by in situ hybridization. We thus assessed, by inference, the contributions of the two promoters to the leptin receptor transcript pool, in murine brain or in placenta, a tissue with abundant leptin receptor mRNA. Expression of B219/OB-R 5'-UTR mRNA (and thus by inference B219/OB-R promoter activity) in brain was similar in both distribution and relative intensity to OB-R mRNA. OB-RGRP mRNA (and thus by inference OB-R/GRP promoter activity) was widely distributed in murine brain, with elevated expression in the hypothalamic regions that express the leptin receptor mRNA, including the paraventricular nucleus. B219/OB-R 5'-UTR mRNA, but not OB-RGRP mRNA, was upregulated in hypothalamus of obese ob/ob mice. In placenta, B219/OB-R 5'-UTR mRNA was restricted to the maternal interface, and transcription of both long and short leptin receptor splice variants in the main body of the tissue thus proceeds via the OB-R/GRP promoter, strongly indicative of tissue-specific promoter usage.

Polymorphism analysis of JRK/JH8, the human homologue of mouse jerky, and description of a rare mutation in a case of CAE evolving to JME.

Disruption of the function of the mouse jerky gene by transgene insertion causes generalized recurrent seizures reminiscent of human idiopathic generalized epilepsy (IGE). A human homologue, JRK/JH8, has been cloned, which maps to 8q24, a chromosomal region associated with several forms of IGE. JRK/JH8 is, therefore, a candidate locus for at least some forms of IGE. We report corrected cDNA sequences and extended open reading frames for the mouse jerky and human JRK/JH8 genes, which add 48 amino acids to the N-terminus of the Jerky protein and which extends the region of homology with the N-terminal DNA-binding domain of the centromere-binding protein, CENP-B. Systematic sequencing of the coding region of the extended JRK/JH8 gene identified single nucleotide polymorphisms that define three haplotypes, which were used for association studies in patients with idiopathic generalized epilepsy. We report one subject with childhood absence epilepsy (CAE) that evolved to juvenile myoclonic epilepsy (JME) that has a unique de novo mutation that results in a non-conservative amino acid change at a potential protein glycosylation site. Familial analysis supports a causal role for this mutation in the disease.

Identification of 4-acetoxyaminoquinoline from the hydrolysis of 1-acetoxy-4-acetoxyimino-1,4-dihydroquinoline, in vitro and in vivo properties.

4-Acetoxyaminoquinoline (Ac-4-HAQ) (1) was identified as a hydrolysis product of 1-acetoxy-4-acetoxyimino-1,4-dihydroquinoline (diAc-4-HAQO). The reaction allowing the obtention of (1) obeys to a reduction mechanism implying the N1-O cleavage. The carcinogenic properties of (1) observed by Sato et al. (Japan J. Exp. Med., 40 (1970) 475) in mice were studied in rats with the in vivo system we used previously with 4-nitroquinoline-1-oxide (4-NQO) and 4-hydroxyaminoquinoline-1-oxide (4-HAQO). In rats (1) does not covalently bind DNA. It was, therefore, possible to propose an interpretation of the results obtained by Enomoto et al. (Proc. Soc. Exp. Biol. Med., 136 (1971) 1206) who injected diAc-4-HAQO s.c. to mice and rats. Compound 1 could be responsible for the carcinogenic effects observed through the following pathway: (1) should be formed by hydrolysis of diAc-4-HAQO and reactivated by an enzymatic system to N-oxide derivative, the 4-acetoxyaminoquinoline-1-oxide (Ac-4-HAQO), which constitutes an ultimate carcinogen model of 4-NQO.

During in vivo maturation of eukaryotic nuclear mRNA, splicing yields excised exon circles.

Circular splicing has already been described on nuclear pre-mRNA for certain splice sites far apart in the multi exonic ETS-1 gene and in the single 1.2 kb exon of the Sry locus. To date, it is unclear how splice site juxtaposition occurs in normal and circular splicing. The splice site selection of an internal exon is likely to involve pairing between splice sites across that exon. Based on this, we predict that, albeit at low frequency, internal exons yield circular RNA by splicing as an error-prone mechanism of exon juxtaposition or, perhaps more interestingly, as a regulated mechanism on alternative exons. To address this question, the circular exon formation was analyzed at three ETS-1 internal exons (one alternative spliced exon and two constitutive), in human cell line and blood cell samples. Here, we show by RT-PCR and sequencing that exon circular splicing occurs at the three individual exons that we examined. RNase protection experiments suggest that there is no correlation between exon circle expression and exon skipping.

Mis-splicing yields circular RNA molecules.

We previously identified novel human ets-1 transcripts in which the normal order of exons is inverted, and demonstrated that although the order of exons is different than in the genomic DNA, splicing of these exons out of order occurs in pairs using genuine splice sites (1). Here we determine the structure of these novel transcripts, showing that they correspond to circular RNA molecules containing only exons in genomic order. These transcripts are stable molecules, localized in the cytoplasmic component of the cells. To our knowledge, this is the first case of circular transcripts being processed from nuclear pre-mRNA in eukaryotes. This new type of transcript might represent a novel aspect of gene expression and hold some interesting clues about the splicing mechanism.

The leptin receptor promoter controls expression of a second distinct protein.

The leptin receptor (OB-R) is a single membrane- spanning protein that mediates the weight-regulatory effects of leptin (OB protein). Several mRNA splice variants have been described which either encode OB-R proteins with cytoplasmic domains of different length or the OB-R and B219/OBR variants, which have different 5'-untranslated regions. Here we report evidence for the synthesis of a human mRNA splice variant of the OB-R gene that potentially encodes a novel protein, leptin receptor gene-related protein (OB-RGRP), which displays no sequence similarity to the leptin receptor itself. This OB-RGRP transcript contains the first two OB-R gene 5'-untranslated exons, but then is alternatively spliced to two novel exons which were mapped to a yeast artificial chromosome containing the leptin receptor gene. First identified by analysis of a large human expressed sequence tag database, the OB-RGRP transcript has now also been found in human and mouse tissues by the use of PCR. Preliminary experiments suggest that OB-RGRP and the OB-R variants share similar patterns of expression that are distinct from that of the B219/OBR variant. OB-RGRP is highly homologous to putative open reading frames in both yeast and Caenorhabditis elegans , suggesting a phylogenetically conserved role for this novel protein.

Regulatory elements in the first intron of the mouse Ha-ras gene.

The Ha-ras gene is one of the three oncogenes (Ha-ras, Ki-ras, and N-ras) of the ras superfamily of small G proteins. The p21ras proteins encoded by the ras genes are key proteins involved in the transduction of signals from membrane receptor-tyrosine kinases to downstream targets. The ras genes seem to play a ubiquitous role in the control of cell proliferation and cell differentiation. At the same time, ras genes may perform specific differentiated functions in certain cell types. Little is known about the regulation of expression of the Ha-ras gene. The first intron of the Ha-ras gene has been reported to be highly conserved between human and rodent. We investigated the role that this intron may play in the regulation of expression of Ha-ras. The promoter region of the Ha-ras gene exhibits characteristics of a housekeeping gene. Deletion analysis shows the existence of an enhancer-type element in the 5' region of the first intron (intron 0). DNase 1 footprinting experiments reveal five sites that interact with nuclear proteins from fibroblast and epithelial cell lines. Deletion and site-directed mutagenesis of three of these sites show that two are involved in a positive effect and one in a negative effect on the regulation of expression of the mouse Ha-ras gene.

In vitro DNA reaction with an ultimate carcinogen model of 4-nitroquinoline-1-oxide: the 4-acetoxyaminoquinoline-1-oxide. Enzymatic degradation of the modified DNA.

2-3H-Labelled 4-acetoxyaminoquinoline-1-oxide (Ac-4 HAQO), the ultimate carcinogen model of 4-nitroquinoline-1-oxide, was reacted in vitro with native and denatured DNA. We found that Ac-4 HAQO is 2- to 3-fold more reactive than diAc-4 HAQO, another ultimate carcinogen model of 4 NQO which was previously studied [Galiègue et al. (1980) Biochim. Biophys. Acta, 609, 383-391]. Ac-4 HAQO-modified DNA is thermally destabilized: when 1% of the bases of DNA were modified by Ac-4 HAQO, its melting temperature decreased 1.2 degrees C. Enzymatic degradation of Ac-4 HAQO-modified native and denatured DNA's to nucleosides was performed. The hydrolysates were analyzed, first with a simple chromatographic system, and then by h.p.l.c. The compounds recovered from the modified polymers were characterized by h.p.l.c. and a variation in their respective amounts as a function of the secondary structure of DNA was observed. Especially, the N-(deoxyguanosin-(C8-yl)-4-aminoquinoline-1-oxide, the so called dG III adduct, was recovered from DNA, and its amount was evaluated to be approximately 3.5-fold greater in the case of denatured DNA than in the case of native DNA.