Zebrafish visual sensitivity is regulated by a circadian clock

Carbonaceous aerosols are present in many workplace and environmental settings. Some of these aerosols are known or suspect human carcinogens and have been linked to other adverse health effects. Exposure to diesel exhaust is of particular concern because it has been classified as a probable human carcinogen and use of diesel-powered equipment is widespread in industry. Because previously used methods for monitoring exposures to particulate diesel exhaust lack adequate sensitivity and selectivity, a new method was needed. A carbon analysis technique called the thermal-optical method was evaluated for this purpose. In thermal-optical analysis, carbon in a filter sample is speciated as organic and elemental (OC and EC, respectively). When the thermal-optical method was initially evaluated, only one instrument was available, so interlaboratory variability could not be examined. More recently, additional instruments were constructed and an interlaboratory comparison was completed. Eleven laboratories participated in the study, including four in Europe that employ an alternative thermal technique based on coulometric detection of CO2. Good agreement (RSDs less than or equal to 15%) between the total carbon results reported by all laboratories was obtained. Reasonable within-method agreement was seen for EC results, but the EC content found by the two methods was differed significantly. Disagreement between th OC-EC results found by the two methods was expected because organic and elemental carbon are operationally defined. With all filter samples, results obtained with the coulometric method were positively biased relative to thermal-optical results. In addition, the alternative method gave a positive bias in the analysis of two OC standard solutions. About 52% and 70% of the carbon found in two aqueous solutions containing OC only (sucrose and EDTA, respectively) was quantified as elemental,while EC contents of about 1% and 0.1% (respectively) were found by the thermal-optical method. The positive bias in the analysis of the OC standards is attributed largely to inadequate removal of OC during the first part of the analysis; lack of correlation for pyrolytically formed carbon (char) also is a factor. Results obtained with a different thermal program having a higher maximum temperature were in better agreement with the thermal-optical method.     

The Calvin cycle enzyme sedoheptulose-1,7-bisphosphatase is encoded by a light-regulated gene in Chlamydomonas reinhardtii

OBJECTIVES: Current surgical treatment for a glottic cancer with significant subglottic extension is a total laryngectomy. The objective of this study was to expand laryngeal conservation procedures by using a reconstructive technique that allows for the repair of hemicricolaryngectomy defects. STUDY DESIGN: After resection of the ipsilateral thyroid, cricoid, and arytenoid for advanced T3 glottic cancer, the laryngeal defect was reconstructed by means of an autotransplanted segment of trachea in four patients. The reconstruction consisted of a transferable patch that was constructed from a segment of revascularized cervical trachea. METHODS: During a 14-day period, a 4-cm segment of cervical trachea was wrapped by a free radial forearm fascial flap. In the second stage, the glottic cancer was removed and the cervical trachea was isolated on its fascial blood supply and transformed into a patch that was used to repair the extended hemilaryngectomy defect. Two different patch designs were used. Two patients underwent reconstruction with a patch augmented at the glottic level (group A); two patients underwent reconstruction without glottic augmentation on the patch (group B). Tracheal continuity was restored by an end-to-end reanastomosis. The postreconstruction morphology of the two patch designs was compared with the preoperative laryngeal morphology. RESULTS: The autotransplantation technique led to complete restoration of the subglottic airway lumen in all four patients. Although the anterior-posterior glottic diameter was reduced by 36% in group A patients and by 43.5% in group B patients, a sufficient glottic airway lumen was obtained. The glottic sphincteric function was restored in both groups. CONCLUSIONS: Tracheal autotransplantation may be used reliably to repair hemicricolaryngectomy defects. Augmentation of the patch at the level of the glottis is not essential for successful rehabilitation.     

Glyceraldehyde-3-phosphate dehydrogenase is regulated on a daily basis by the circadian clock

Circadian clocks function to govern a wide range of rhythmic activities in organisms. An integral part of rhythmicity is the daily control of target genes by the clock. Here we describe the sequence and analysis of a novel clock-controlled gene, ccg-7, showing similarity to glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a glycolytic enzyme widely used as a constitutive control in a variety of systems. That ccg-7 encodes GAPDH was confirmed by demonstrating that in vitro synthesized CCG-7 possesses GAPDH activity. Rhythms in both ccg-7 mRNA accumulation and CCG-7 (GAPDH) activity are observed in a clock wild-type strain where the peak in GAPDH activity lags several hours behind the peak in ccg-7 mRNA accumulation in the late night. Together with our previous observation that ccg-7 mRNA is not developmentally regulated, we show that ccg-7 is not induced by environmental stresses such as glucose or nitrogen deprivation (which also trigger development), heat shock, or osmotic stress. Thus, the finding that GAPDH is clock-regulated points to a specific role for the circadian clock in controlling aspects of general metabolism and provides evidence for circadian regulation of a gene found in most living organisms.     

Molecular genetic analysis of plastocyanin biosynthesis in Chlamydomonas reinhardtii

Five plastocyanin-deficient mutants were identified from a population of UV-mutagenized Chlamydomonas reinhardtii cells. Genetic complementation experiments indicated that four mutants represented alleles at the PCY1 locus (pcy1-2, pcy1-3, pcy1-4, and pcy1-5). Sequence analysis confirmed that two strains, pcy1-2 and pcy1-3, carry a frameshift (-1) and a nonsense mutation, respectively, while strains pcy1-4 and pcy1-5 synthesize an extended protein as a result of read-through mutations at the stop codon. The C-terminal extension does not affect synthesis or processing of the pre-proteins, but the polypeptides are rapidly degraded after the second (lumenal) processing event. The frameshift mutation in pcy1-2 results in loss of Pcy1 mRNA, as noted previously for strain ac208 (pcy1-1), but the abundance of Pcy1 mRNA in strain pcy1-3, which carries a nonsense mutation at codon 26, is unaffected relative to wild-type cells. The decreased abundance of frameshifted Pcy1 mRNA is attributed to increased degradation rather than decreased synthesis, since the mRNAs can be stabilized by treatment of cells with cycloheximide or anisomycin. The fifth strain has a wild-type plastocyanin-encoding gene, but the strain accumulates apoplastocyanin at the expense of holoplastocyanin. We suggest that the mutation identifies a new locus (PCY2) whose function is required for normal holoplastocyanin accumulation. Like ac208 (pcy1-1), several of the new mutants were suppressed spontaneously owing to accumulation of cytochrome c6 (a functional substitute for plastocyanin). The suppressor mutation(s) displayed Mendelian inheritance and segregated independently from the PCY1 locus, which confirms that regulation of Cyc6 expression is not tightly linked to plastocyanin function.     

Temperature-sensitive mutations affecting flagellar assembly and function in Chlamydomonas reinhardtii

A series of conditional mutants of the algal, biflagellate Chlamydomonas reinhardtii with temperature-sensitive defects in flagellar assembly and function were isolated. The genetics and phenotypes of 21 mutants displaying a rapid alteration in flagellar function upon shift from the permissive (20 degrees C) to the restrictive (32 degrees C) temperatures are described. These mutants designated as "drop-down" or dd-mutants have been placed in four categories on the basis of their defective phenotypes: (a) dd-assembly mutants - the preformed flagella are resorbed at 32 degrees C and reassembly of flagella is inhibited; (b) dd-fragile flagella mutants - the flagella are lost by detachment at 32 degrees C, but can be reassembled; (c) dd-motility mutants - the flagella are retained at 32 degrees C, but are functionally defective; (d) dd-lethal mutants - display combined defects in flagellar function and cell growth. Tetrad analysis of the mutants back-crossed to wild-type, recombination analysis of intermutant crosses, and complementation tests in the construction of heterozygous diploid strains indicate that at least 14 nuclear genetic loci are represented among 21 mutants. The availability of temperature-sensitive mutations affecting the assembly and function of the flagellum suggests that the morphogenesis of this complex eukaryotic organelle is amenable to genetic dissection.     

The 1.5-A crystal structure of plastocyanin from the green alga Chlamydomonas reinhardtii

The crystal structure of plastocyanin from the green alga Chlamydomonas reinhardtii has been determined at 1.5-A resolution with a crystallographic R factor of 16.8%. Plastocyanin is a small (98 amino acids), blue copper-binding protein that catalyzes the transfer of electrons in oxygenic photosynthesis from cytochrome f in the quinol oxidase complex to P700+ in photosystem I. Chlamydomonas reinhardtii plastocyanin is an eight-stranded, antiparallel beta-barrel with a single copper atom coordinated in quasitetrahedral geometry by two imidazole nitrogens (from His-37 and His-87), a cysteine sulfur (from Cys-84), and a methionine sulfur (from Met-92). The molecule contains a region of negative charge surrounding Tyr-83 (the putative distant site of electron transfer) and an exclusively hydrophobic region surrounding His-87; these regions are thought to be involved in the recognition of reaction partners for the purpose of directing electron transfer. Chlamydomonas reinhardtii plastocyanin is similar to the other plastocyanins of known structure, particularly the green algal plastocyanins from Enteromorpha prolifera and Scenedesmus obliquus. A potential "through-bond" path of electron transfer has been identified in the protein that involves the side chain of Tyr-83, the main-chain atoms between residues 83 and 84, the side chain of Cys-84, the copper atom, and the side chain of His-87.     

Nucleus suprachiasmaticus: the biological clock in the hamster?

Destruction of the suprachiasmatic nuclei in the golden hamster by bilateral radiofrequency lesions abolishes three well-documented circadian rhythms--locomotor activity, estrous cyclicity, and photoperiodic photosensitivity. Entrainment of these rhythms by light cycles fails in lesioned hamsters; females become persistently estrous; in both sexes locomotor activity becomes sporadic, confined primarily to the light instead of darkness, and is totally arrhythmic when lesioned animals are exposed to continuous darkness; the photoperiodic gonadal response (gonadal regression induced by short day lengths) is abolished; lesioned animals remain reproductively mature irrespective of photoperiodic treatment.     

Purification and characterization of oxygen-evolving photosystem II core complexes from the green alga Chlamydomonas reinhardtii

Oxygen-evolving photosystem II complexes were isolated from the green alga Chlamydomonas reinhardtii by selective solubilization of thylakoid membranes with dodecyl maltoside followed by density gradient centrifugation and anion-exchange chromatography. In the presence of CaCl2 and K3[Fe(CN)6] the complexes evolved oxygen at rates exceeding 1000 mumol (mg of chl)-1 h-1. The particles contained 40 chlorophylls a and had properties very similar to those of PSII isolated from higher plants. Chlamydomonas reinhardtii is now the first organism which can be used for both site-directed mutagenesis and detailed biochemical and biophysical characterization of oxygen-evolving photosystem II. It seems therefore to be an ideal model organism for investigation of structure-function relationships in photosynthetic oxygen evolution.     

Periodic variations in the ratio of free to thylakoid-bound chloroplast ribosomes during the cell cycle of Chlamydomonas reinhardtii

The ratio of free to thylakoid-bound chloroplast ribosomes in Chlamydomonas reinhardtii undergoes periodic changes during the synchronous light-dark cycle. In the light, when there is an increase in the chlorophyll content and synthesis of thylakoid membrane proteins, about 20-30% of the chloroplast ribosomes are bound to the thylakoid membranes. On the other hand, only a few or no bound ribosomes are present in the dark when there is no increase in the chlorophyll content. The ribosome-membrane interaction depends not only on the developmental stage of the cell but also on light. Thus, bound ribosomes were converted to the free variety after cultures at 4 h in the light had been transferred to the dark for 10 min. Conversely, a larger number of chloroplast ribosomes became attached to the membranes after cultures at 4 h in the dark had been illuminated for 10 min. Under normal conditions, when there was slow cooling of the cultures during cell harvesting, chloroplast polysomal runoff occurred in vivo leading to low levels of thylakoid-bound ribosomes. This polysomal runoff could be arrested by either rapid cooling of the cells or the addition of chloramphenicol or erythromycin. Each of these treatments prevented polypeptide chain elongation on chloroplast ribosomes and thus allowed the polyosomes to remain bound to the thylakoids. Addition of lincomycin, an inhibitor of chain initiation on 70S ribosomes, inhibited the assembly of polysome-thylakoid membrane complex in the light. These results support a model in which initiation of mRNA translation begins in the chloroplast stroma, and the polysome subsequently becomes attached to the thylakoid membrane. Upon natural chain termination, the chloroplast ribosomes are released from the membrane into the stroma.     

Two distinct, calcium-mediated, signal transduction pathways can trigger deflagellation in Chlamydomonas reinhardtii

The molecular machinery of deflagellation can be activated in detergent permeabilized Chlamydomonas reinhardtii by the addition of Ca2+ (Sanders, M. A., and J. L. Salisbury, 1989. J. Cell Biol. 108:1751-1760). This suggests that stimuli which induce deflagellation in living cells cause an increase in the intracellular concentration of Ca2+, but this has never been demonstrated. In this paper we report that the wasp venom peptide, mastoparan, and the permeant organic acid, benzoate, activate two different signalling pathways to trigger deflagellation. We have characterized each pathway with respect to: (a) the requirement for extracellular Ca2+; (b) sensitivity to Ca2+ channel blockers; and (c) 45Ca influx. We also report that a new mutant strain of C. reinhardtii, adf-1, is specifically defective in the acid-activated signalling pathway. Both signalling pathways appear normal in another mutant, fa-1, that is defective in the machinery of deflagellation (Lewin, R. and C. Burrascano. 1983. Experientia. 39:1397-1398; Sanders, M. A., and J. L. Salisbury. 1989. J. Cell Biol. 108:1751-1760). We conclude that mastoparan induces the release of an intracellular pool of Ca2+ whereas acid induces an influx of extracellular Ca2+ to activate the machinery of deflagellation.     

Clustering of the nitrite reductase gene and a light-regulated gene with nitrate assimilation loci in Chlamydomonas reinhardtii

Degradation of extracellular matrix takes place in areas of cell-matrix contacts and is partly carried out by the action of matrix metalloproteinases (MMP). MMP-2 is a member of the MMP family that has been associated with breast-cancer metastasis. In the present study, we investigated the association of MMP-2 to the surface of breast-cancer cells and revealed an MMP-2-binding site that is expressed on sparsely plated cells and which is progressively lost as the cells approach confluence. Gelatin zymography, immunostaining and flow cytometry of MDA-MB-231 cells from sparse cultures demonstrated binding both of latent and of activated exogenous MMP-2, while little or no binding of MMP-2 was observed in confluent culture. Analysis of the expression of MTI-MMP, TIMP-2 and alpha(v) integrin, 3 proteins shown to play a role in cell-surface association of MMP-2, revealed enhanced levels of these proteins in confluent MDA-MB-231 cells. Thus, the reduced MMP-2 binding to confluent cells is not related to a deficiency in these MMP-2-binding proteins. Taken together, these studies suggest that MMP-2 binding to the surface of breast-cancer cells is regulated by cell-cell interactions and that tumor cells invading from the main tumor mass can up-regulate their MMP-2-binding capacity to acquire greater invasive capacity.