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PubMed2003

Involvement of calcium/calmodulin-dependent protein kinase II in the induction of mPer1.

Recent studies suggest that CaM kinase II is involved in light-induced phase delays and induction of Per1 and Per2 genes in the hamster suprachiasmatic nucleus (SCN) (Yokota et al.,2001). We focused on intracellular mechanisms of the CaM kinase II-induced mPer1 gene expression. Immunoblotting and immunohistochemical analyses with isoform-specific antibodies against different isoforms of CaM kinase II and CaM kinase IV showed abundant expression of the delta isoform of CaM kinase II without significant expression of CaM kinase IV in the lateral ventral region of the rat SCN. We next defined the CaM kinase II-responsive region on the mPer1 promoter using a luciferase reporter gene assay. Transfection of the constitutively-active CaM kinase IIdelta greatly increased mPer1 promoter activity in NG108-15 cells and increased activity slightly but significantly in NB2A and C6 glioma cells. Similarly, transfection of a constitutively-active MEKK, an upstream kinase of mitogen-activated protein kinase (MAPK), greatly increased promoter activity in NB2A cells. Deletion and mutation analyses of the mPer1 promoter revealed that a 5'-GAGGGG-3' sequence motif near exon 1B, in which several zinc finger proteins seem to bind, was essential for the CaM kinase II-induced activation of the mPer1 promoter. These results suggest that CaM kinase IIdelta but not CaM kinase IV plays an essential role for mPer1 expression through the 5'-GAGGGG-3' motif on the mPer1 promoter.

Animals↗

PubMed1996

Nonmammalian models for sunlight carcinogenesis: genetic analysis of melanoma formation in Xiphophorus hybrid fish.

Genetic hybrids of Xiphophorus fishes have been used for decades to study heritable melanoma formation. In these models, overexpression of pigmentation patterns from melanin-producing pigment cells can lead to genetically regulated melanoma formation in backcross hybrids. In the best studied of these models, the Gordon-Kosswig hybrid melanoma, tumors form spontaneously in all individuals of a subset of backcross hybrids between the platyfish Xiphophorus maculatus Jp 163 A and the swordtail species Xiphophorus helleri. Backcross hybrids susceptible to melanoma formation inherit a sex-linked oncogene, Xmrk, associated with the spotted dorsal (Sd) pigment pattern and have lost both copies of an autosomal gene, DIFF, from the X. maculatus parent. Spontaneous melanoma formation conforms to simple, two-gene Mendelian inheritance in which DIFF behaves as a recessive tumor suppressor gene. Recently, Xiphophorus hybrids in which melanomas can be induced by UV and near-UV visible light exposure have been described. We report here results of genetic linkage analysis of one of these Xiphophorus light-inducible hybrid melanoma models, in backcross hybrids between the two platyfish species X. maculatus Jp 163 B and Xiphophorus couchianus. Our linkage results provide the first estimate of recombination between the tumor suppressor locus, DIFF, and glycerate-2-dehydrogenase (GLYDH) in Xiphophorus linkage group V. Also, they demonstrate that DIFF regulates hyperplasia of spotted side (Sp) pigment cells in this hybrid model, analogous to its regulation of hyperplasia of Sd pigment cells in the "classical" Gordon-Kosswig hybrid. Joint segregation analyses of melanoma-bearing fish indicate that segregation of DIFF is genetically linked to melanoma induction by 405 nm light in this model but that induction of melanomas by UV wavelengths apparently does not depend on segregation of the DIFF locus.

Animals↗

PubMed1991

delta-Aminolevulinic Acid Biosynthesis from Glutamatein Euglena gracilis: Photocontrol of Enzyme Levels in a Chlorophyll-Free Mutant.

Wild-type Euglena gracillis cells synthesize the key chlorophyll precursor, delta-aminolevulinic acid (ALA), from glutamate in their plastids. The synthesis requires transfer RNA(Glu) (tRNA(Glu)) and the three enzymes, glutamyl-tRNA synthetase, glutamyl-tRNA reductase, and glutamate-1-semialdehyde aminotransferase. Non-greening mutant Euglena strain W(14)ZNaIL does not synthesize ALA from glutamate and is devoid of the required tRNA(Glu). Other cellular tRNA(Glu)s present in the mutant cells were capable of being charged with glutamate, but the resulting glutamyl-tRNAs did not support ALA synthesis. Surprisingly, the mutant cells contain all three of the enzymes, and their cell extracts can convert glutamate to ALA when supplemented with tRNA(Glu) obtained from wild-type cells. Activity levels of the three enzymes were measured in extracts of cells grown under a number of light conditions. All three activities were diminished in extracts of cells grown in complete darkness, and full induction of activity required 72 hours of growth in the light. A light intensity of 4 microeinsteins per square meter per second was sufficient for full induction. Blue light was as effective as white light, but red light was ineffective, in inducing extractable enzyme activity above that of cells grown in complete darkness, indicating that the light control operates via the nonchloroplast blue light receptor in the mutant cells. Of the three enzyme activities, the one that is most acutely affected by light is glutamate-1-semialdehyde aminotransferase, as has been previously shown for wild-type Euglena cells. These results indicate that the enzymes required for ALA synthesis from glutamate are present in an active form in the nongreening mutant cells, even though they cannot participate in ALA formation in these cells because of the absence of the required tRNA(Glu), and that the activity of all three enzymes is regulated by light. Because the absence of plastid tRNA(Glu) precludes the synthesis of proteins within the plastids, the three enzymes must be synthesized in the cytoplasm and their genes encoded in the nucleus in Euglena.

Journal Article↗

PubMed1992

Critical checking of the radiological diagnosis of "complete remission" and "partial remission" following induction chemotherapy of small-cell lung cancer in the light of postoperative histological examination.

A multimodal therapy concept for small-cell lung cancer, which for patients with established pretherapeutic homolateral lymph-node metastases (N2) prescribes induction chemotherapy with subsequent resection as well as supplemental chemo- and radiotherapy, provided the opportunity to evaluate histologically the radiological diagnoses "complete remission" and "partial remission" using resection specimens. In 17 patients a 75% to 100% reduction in tumor size was achieved according to radiological diagnosis. Predictions of "no evidence of disease" or "evidence of disease" were only correct in ten cases. In the remaining seven cases, histology showed the radiological findings to be incorrect. This gives a 77% sensitivity for radiological diagnosis with no specificity. Moreover, differentiation between therapy effect on the primary tumor and on the N2 metastases gives similar results: sensitivity 64% and 67% respectively, specificity 33% and 25% respectively. It is concluded that, particularly after the tumor responds well to therapy, radiological techniques are unsuitable for establishing a diagnosis of "no evidence of disease" or "evidence of disease" in small-cell lung cancer. This is because on the one hand the radiological methods available do not permit clear differentiation between vital tumor tissue and necrosis or fibrosis, while on the other hand groups of vital tumor cells beyond the resolution power of X-ray technology will escape detection.

Antineoplastic Agents↗

PubMed1986

A labile inhibitor blocks immunoglobulin kappa-light-chain-gene transcription in a pre-B leukemic cell line.

The murine pre-B leukemic cell line 70Z/3 contains both an unrearranged immunoglobulin kappa-light-chain gene and a functionally rearranged but silent kappa-light-chain gene. Mitogenic stimulation of growing 70Z/3 cells with bacterial lipopolysaccharide (LPS) activates kappa-light-gene transcription and results in a 10- to 20-fold increase in cytoplasmic kappa-light-chain mRNA. The induction of kappa gene expression by LPS was probed by using an inhibitor of protein synthesis. Concomitant treatment of 70Z/3 cells with LPS and cycloheximide failed to block kappa-light-chain mRNA accumulation, indicating that new protein synthesis is not required for the activation of kappa-gene expression. Treatment of 70Z/3 cells with cycloheximide alone resulted in kappa-mRNA induction equivalent to those produced by LPS alone. The kappa mRNA synthesized in the presence of cycloheximide was intact and able to direct the synthesis of kappa light chains. Nuclear transcription assays revealed that cycloheximide, like LPS, activated kappa-gene transcription. These findings indicate that the trans-acting factors necessary for kappa-light-chain-gene transcription are present in pre-B cells, but their activity is blocked by short-lived inhibitory proteins.

Animals↗

PubMed1998

Inactivation of the clpP1 gene for the proteolytic subunit of the ATP-dependent Clp protease in the cyanobacterium Synechococcus limits growth and light acclimation.

ClpP functions as the proteolytic subunit of the ATP-dependent Clp protease in eubacteria, mammals and plant chloroplasts. We have cloned a clpP gene, designated clpP1, from the cyanobacterium Synechococcus sp. PCC 7942. The monocistronic 591 bp gene codes for a protein 80% similar to one of four putative ClpP proteins in another cyanobacterium, Synechocystis sp. PCC 6803. The constitutive ClpP1 content in Synechococcus cultures was not inducible by high temperatures, but it did rise fivefold with increasing growth light from 50 to 175 micromol photons m(-2) s(-1). A clpP1 inactivation strain (delta clpP1) exhibited slower growth rates, especially at the higher irradiances, and changes in the proportion of the photosynthetic pigments, chlorophyll a and phycocyanin. Many mutant cells (ca. 35%) were also severely elongated, up to 20 times longer than the wild type. The stress phenotype of delta clpP1 when grown at high light was confirmed by the induction of known stress proteins, such as the heat shock protein GroEL and the alternate form of PSII reaction center D1 protein, D1 form 2. ClpP1 content also rose significantly during short-term photoinhibition, but its loss in delta clpP1 did not exacerbate the extent of inactivation of photosynthesis, nor affect the inducible D1 exchange mechanism, indicating ClpP1 is not directly involved in D1 protein turnover.

Acclimatization↗

PubMed2003

Inactivation of the chloroplast ATP synthase gamma subunit results in high non-photochemical fluorescence quenching and altered nuclear gene expression in Arabidopsis thaliana.

The nuclear atpC1 gene encoding the gamma subunit of the plastid ATP synthase has been inactivated by T-DNA insertion mutagenesis in Arabidopsis thaliana. In the seedling-lethal dpa1 (deficiency of plastid ATP synthase 1) mutant, the absence of detectable amounts of the gamma subunit destabilizes the entire ATP synthase complex. The expression of a second gene copy, atpC2, is unaltered in dpa1 and is not sufficient to compensate for the lack of atpC1 expression. However, in vivo protein labeling analysis suggests that assembly of the ATP synthase alpha and beta subunits into the thylakoid membrane still occurs in dpa1. As a consequence of the destabilized ATP synthase complex, photophosphorylation is abolished even under reducing conditions. Further effects of the mutation include an increased light sensitivity of the plant and an altered photosystem II activity. At low light intensity, chlorophyll fluorescence induction kinetics is close to those found in wild type, but non-photochemical quenching strongly increases with increasing actinic light intensity resulting in steady state fluorescence levels of about 60% of the minimal dark fluorescence. Most fluorescence quenching relaxed within 3 min after dark incubation. Spectroscopic and biochemical studies have shown that a high proton gradient is responsible for most quenching. Thylakoids of illuminated dpa1 plants were swollen due to an increased proton accumulation in the lumen. Expression profiling of 3292 nuclear genes encoding mainly chloroplast proteins demonstrates that most organelle functions are down-regulated. On the contrary, the mRNA expression of some photosynthesis genes is significantly up-regulated, probably to compensate for the defect in dpa1.

Actins↗

PubMed1989

Cold-induced sudden reversible lowering of in vivo chlorophyll fluorescence after saturating light pulses : a sensitive marker for chilling susceptibility.

In chilling-sensitive plants (Glycine max, Saintpaulia ionantha, Saccharum officinarum) a sudden reversible drop in chlorophyll fluorescence occurs during photosynthetic induction immediately following saturating light pulses at low temperatures in the range 4 to 8 degrees C. A comparison of two soybean cultivars of different chilling sensitivities revealed that this phenomenon, termed lowwave, indicates specific thresholds of low temperature stress. Its occurrence under controlled chilling can be regarded as a quantitative marker for screening chilling susceptibility in angiosperms.

Journal Article↗

PubMed1996

Blockage of RNA polymerase as a possible trigger for u.v. light-induced apoptosis.

To study the triggering mechanism(s) of the induction of apoptosis following exposure to u.v. light, we used a genetic approach involving cell strains derived from patients with inherited deficiencies in nucleotide excision repair. It was found that cells from patients with Cockayne's syndrome, which are deficient in the processing of u.v.-induced pyrimidine dimers from the transcribed DNA strand, are induced to undergo apoptosis at much lower doses of u.v. light than cells with proficient strand-specific repair. The induction of apoptosis correlated to the induction of p53 and to the inhibition of total RNA and poly(A) mRNA synthesis. We also show that active p53 proteins accumulate following u.v.-irradiation without any apparent requirement for DNA strand breaks or excision repair intermediates. We propose that the blockage of RNA polymerases at DNA lesions in the transcribed strand triggers the induction of a pathway leading to apoptosis. These findings may help explain a long standing enigma of why, despite the DNA repair deficiency, patients with Cockayne's syndrome do not experience an elevated risk for skin cancer since potentially pre-mutagenic cells are eliminated by an easily triggered apoptotic pathway.

Apoptosis↗

PubMed1991

Do pyrimidine dimer yields correlate with erythema induction in human skin irradiated in situ with ultraviolet light (275-365 nm)?

Ultraviolet radiation produces erythema in human skin, and damages the DNA of living cells in skin. Previous work showed that broad-band UV-B (290-320 nm) radiation produced higher levels of cyclobutyl pyrimidine dimers in DNA of individuals with high UV-B sensitivity (low minimal erythema dose) than in subjects of low UV-B sensitivity [Freeman et al. (1986) J. Invest. Dermatol., 86, 34-36]. We examined the relationship between erythema induction and dimer yields in DNA of human skin irradiated in situ with narrow band radiation spanning the wavelength range 275-365 nm. We find that, in general, higher dimer yields are produced per incident photon in volunteers with higher susceptibility to erythema induced by radiation of the same wavelength.

Adult↗

PubMed1993

Effects of Anaerobiosis on Chlorophyll Fluorescence Yield in Spinach (Spinacia oleracea) Leaf Discs.

When spinach (Spinacia oleracea) leaf discs were incubated in a dark anaerobic environment, the chlorophyll fluorescence yield was much increased relative to the aerobic control. Occasionally, the fluorescence yield of the darkened anaerobic samples approached 80% of the maximum fluorescence. The anaerobic incubation period also induced in a leaf disc the capacity to exhibit a low light-mediated chlorophyll fluorescence induction phenomenon. This involved a rapid and slow increase in fluorescence yield, followed by a slow quenching. This could be induced by light levels as low as 400 [mu]W m-2. The anaerobic-dependent increase in chlorophyll fluorescence yield could be relaxed by either far-red light, O2, or a saturating pulse of white light. It was concluded that the anaerobic-dependent increase in chlorophyll fluorescence yield was due to a dark reduction of the plastoquinone pool and its relaxation by reoxidation. Darkened isolated chloroplasts did not exhibit a fluorescence yield increase under anaerobic conditions. Fluorescence slowly increased only when dithiothreitol or dithionite was added.

Journal Article↗

PubMed1996

Alpha 2-adrenergic agonists induce basic fibroblast growth factor expression in photoreceptors in vivo and ameliorate light damage.

We observed an induction of basic fibroblast growth factor (bFGF) mRNA in the rat retina after systemic administration of the alpha 2-adrenergic agonists xylazine and clonidine. A single injection of xylazine or clonidine transiently increased bFGF mRNA. Preinjection of yohimbine, an alpha 2-adrenergic antagonist, completely inhibited this increase. Higher dosage of yohimbine inhibited the baseline expression of bFGF. Of particular interest is the finding that the induced bFGF expression occurred almost exclusively in the inner segment region of photoreceptors. No increase in bFGF mRNA was found in the brain after either xylazine or clonidine injection. Xylazine or clonidine given systemically before and during constant light exposure also reduces photoreceptor degeneration in albino rats. These results indicate that regulation of bFGF expression in photoreceptors is unique in the CNS and suggest that endogenous bFGF promotes photoreceptor survival.

Adrenergic alpha-Agonists↗

PubMed2002

Induction of p21 mRNA synthesis after short-wavelength UV light visualized in individual cells by RNA FISH.

Expression of the cyclin-dependent kinase inhibitor gene p21 is induced after DNA damage and plays a role in cell survival. The exact mechanism of induction is not known, but enhancement of mRNA stability has recently been implicated as an important factor. To obtain further insight into the dynamics of p21 gene expression at the individual cell level, normal fibroblasts, GM1492 fibroblasts from a Bloom's syndrome patient, and U2OS osteosarcoma cells were UVC irradiated, fixed at different time points, and subjected to mRNA fluorescence in situ hybridization (FISH) and immunocytochemical staining. In mock-irradiated normal fibroblasts, a subfraction of cells revealed low levels of p21 mRNA synthesis. After UVC treatment, p21 transcripts accumulated over time in nuclear locations other than transcription foci. At 6 hr after irradiation, almost 50% of the cells displayed p21 mRNA in three different distribution patterns within the nuclei. The highest frequency of cells with cytoplasmic accumulation of p21 mRNA was seen at 17 hr after UVC treatment. We conclude that increased p21 gene transcription and possibly stabilization of newly synthesized p21 mRNA contribute to elevated levels of p21 protein after UVC irradiation. (J Histochem Cytochem 50:81-89, 2002)

Cell Line↗

PubMed1989

Blue-light mediated accumulation of nuclear-encoded transcripts coding for proteins of the thylakoid membrane is absent in the phytochrome-deficient aurea mutant of tomato.

Polyclonal antibodies against pea phytochrome detect 2 protein bands (about 116 and 120 kDa) on blots of crude protein extracts and protein of microsomal preparations of dark-grown tomato seedlings. Both protein bands are undetectable in Western blots of the aurea mutant extracts. Neither protein band is detectable after isogenic wild-type seedlings are illuminated with 3 h of red light, either in the crude extract or in the membrane fraction of the irradiated seedlings; this result is consistent with the hypothesis that both bands are phytochrome. When dark-grown wild-type seedlings are illuminated with 3 h of red light or blue light against a red light background, the transcript levels for chlorophyll a/b-binding proteins of photosystem I and II, plastocyanin, and the subunit II of photosystem I increase. In all cases, the same fluence rate of blue light is much more effective than red light alone, a result that indicates the involvement of a blue/UV-A light photoreceptor in addition to the involvement of the far-red-absorbing form of phytochrome, Pfr. The aurea mutant responds neither to red light nor to blue light. Thus, no Pfr-independent induction of the four transcripts by a blue/UV-A light photoreceptor can be measured in the aurea mutant.

DNA Probes↗

PubMed1994

A Light-Dependent Pathway for the Elimination of UV-Induced Pyrimidine (6-4) Pyrimidinone Photoproducts in Arabidopsis.

Light-dependent repair of UV-induced cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidinone dimers (6-4 products) was investigated in an excision repair-deficient Arabidopsis mutant. As previously described, exposure to broad-spectrum lighting was found to greatly enhance the rate of repair of CPDs. We demonstrate that 6-4 products are also efficiently eliminated in a light-dependent manner and that this photoreactivation of 6-4 products occurs independently of the previously described 6-4 product dark repair pathway. The light-dependent repair of both 6-4 products and CPDs occurs in the presence of blue light (435 nm) but not upon exposure to light of longer wavelengths. We also found that high-level expression of the CPD-specific photoreactivating activity in the Arabidopsis seedling requires induction by exposure to light prior to as well as during the period of repair while the 6-4 photoreactivating activity is constitutively expressed. This differential regulation of the photoreactivating activities suggests that the Arabidopsis seedling produces at least two distinct photolyases: one specific for CPDs and the other specific for 6-4 products.

Journal Article↗

PubMed1985

Physiological mechanism of the auxin-induced increase in light sensitivity of phytochrome-mediated growth responses in Avena coleoptile sections.

The physiology of the auxin-induced 10,000-fold increase in light sensitivity of a phytochrome-mediated growth response (Shinkle and Briggs, 1984 Proc Natl Acad Sci USA 81: 3742-3746) has been characterized in subapical coleoptile sections from dark-grown oat (Avena sativa L. cv Lodi) seedlings. Six micromolar indole-3-acetic acid (IAA) must be present for 1 hour before to 2 hour after irradiation in order to confer maximal sensitivity to light. The direct effect of IAA on growth can be separated from its effect on light sensitivity. Several classes of synthetic auxins will substitute for IAA in inducing an increase in sensitivity to light, as will both the phytotoxin fusicoccin and treatment of sections with pH 4.5 buffer. The increase in sensitivity to light induced by 6 micromolar IAA is completely inhibited by buffering the sections at pH 5.9 with 30 millimolar 2-(N-morpholino)ethanesulfonic acid. These findings suggest that the capacity to respond to very low fluences of light is regulated by extracellular pH.Between 10 and 15 millimolar K(+) will inhibit the induction of the increased sensitivity to light, independent of the mechanism of induction. The effect of K(+) appears to be specific to the process by which the sections respond to very low levels of light.

Journal Article↗

PubMed1978

The plastid membranes of barley (Hordeum vulgare). Light-induced appearance of mRNA coding for the apoprotein of the light-harvesting chlorophyll a/b protein.

Illumination of dark-grown barley plants induces a massive insertion of the light-harvesting chlorophyll a/b protein into the developing thylakoid membrane. In addition to the onset of chlorophyll synthesis, light induces specifically the appearance of a prominent mRNA species which codes for a polypeptide of Mr 29500. This component was identified as a precursor of the apoprotein of the light-harvesting chlorophyll a/b protein. The precursor has an Mr larger than the authentic protein by approximately 4000. Studies of the chlorophyll-b-less mutant chlorina f2 of barley offer the first clue to the mechanism which controls the light-dependent mRNA formation. The induction of the mRNA coding for the aproprotein of the light-harvesting chlorophyll a/b protein does not seem to be linked directly to the assembly process of the light-harvesting structure and does not require chlorophyll b. It is proposed that light exerts its influence on the mRNA formation by a reaction which is different from the phototransformation of protochlorophyll(ide) to chlorophyll(ide).

Cell Membrane↗

PubMed2000

The HSP70A promoter as a tool for the improved expression of transgenes in Chlamydomonas.

The Chlamydomonas reinhardtii HSP70A promoter can be induced by both heat shock and light. Several characteristics of this promoter suggest its usefulness as a tool for improved transgene expression in this alga. (i) It may by itself confer high inducibility to a transgene. Fusion of the HSP70A promoter to reporter genes HSP70B or ARS yields high levels of transgene product that, as shown for ARS, may accumulate when repeated cycles of heat shock induction are applied. (ii) It activates other promoters. Using HSP70B as a reporter gene, we show that the HSP70A promoter serves as a transcriptional activator when placed upstream of the promoters RBCS2, beta 2 TUB and HSP70B. Activation of these promoters was observed both under basal conditions and upon light induction. In addition, transformation rates obtained for the eubacterial resistance gene aadA were significantly increased, when expression of this gene was controlled by the HSP70A-RBCS2 promoter fusion as compared to the RBCS2 promoter alone.

Animals↗