Compatible intracellular ion composition of the host improves carbon assimilation by zooxanthellae in mutualistic symbioses.

Cytosymbiotic algae within the host's plasma are exposed to completely different ionic conditions than microalgae living in the sea. The altered ionic gradients, in particular, could be the reason for higher in hospite carbon assimilation levels. To study the effect of varying extracellular ionic conditions on isolated zooxanthellae, their photosynthetic capacity in pure seawater was compared to that in a test medium in which the concentrations of the major inorganic ions, the pH and the osmolality were adjusted to the conditions measured in the host cytoplasm. In this test medium the ratio between oxygen evolution and carbon fixation was 1.2:1.0; in contrast, zooxanthellae in the hyperionic seawater medium showed a comparatively higher oxygen production (2.6:1.0). These results are attributed to a higher energy demand for ion regulation of the isolated algae in the hyperionic medium. Isolated cytosymbionts in seawater need more energy both for the readjustment to the original intracellular ion concentration within the host cell and also for the maintenance of a much steeper gradient during incubation under hyperionic conditions outside the host. The particular intracellular ion concentration of the host cells could have been a decisive evolutionary factor for the very successful establishment of the mutualistic symbioses between anthozoans and dinoflagellates more than 200 million years ago.

Improvement of photosynthesis in zooxanthellate corals by autofluorescent chromatophores.

Autofluorescent chromatophores were detected in 17 out of 71 zooxanthellate coral species studied. Chromatophores are localized either in the oral gastrodermic (endoderm) or oral epidermis (ectoderm). The pigment granules within the chromatophores (0.5-1.0 µm in diameter) show a brilliant light-blue/turquoise autofluorescence (emission between 430 and 500 nm) after excitation with light of 365-410 nm. All species where the autofluorescent gastrodermal chromatophores form a compact layer, embedding the zooxanthellae, belong to the family Agariciidae. In contrast, some species of the Faviidae (2), Pectiniidae (1) and Mussidae (1) were found to have distinct, autofluorescent chromatophores in the oral epidermis. Autofluorescent pigments of the host may enhance photosynthesis of the symbionts as in Leptoseris fragilis. Short wavelength irradiance, less suitable for photosynthesis, is transformed by host pigments into longer wavelengths which are photosynthetically more effective. Thus, host species possessing autofluorescent chromatophores might have selective advantage over non-fluorescent species, and have the potential to survive in light-limited habitats. Furthermore, the daily period of photosynthesis is extended, thus increasing the energy supply and enhancing the deposition of skeletal carbonate. The absence or presence of chromatophores may have value in taxonomy and could putatively be of plalaeontological and palaeoecological interest.

Characterization of rat liver nuclear proteins which recognize the cAMP responsive element.

1. The data herein reveal the existence of cAMP-responsive element (CRE)-binding factors (CRF) in the nuclear extracts from cAMP-treated rat liver. 2. DNAase I and DMS footprinting analysis showed that the CRFs protected the CRE (-77 to -92) in the phosphoenolpyruvate carboxykinase (PEPCK) promoter and the TGACGTCA motif in a consensus oligodeoxynucleotide based on the sequence of the CRE's of 6 cAMP-regulated genes (C32mer). 3. Competition assays indicate that the CRF(s) is a CGTCA-specific, ATF/CREB-like factor(s). 4. Southwestern (SW) blot analysis detected 2 apparent CRFs which have molecular weights of about 30 and 32 kDa, respectively. 5. Based on the comparison of the size and binding specificity of the CRFs with the CREBs reported to date, the CRFs appear to be novel CRE-binding nuclear factors.

Interaction of a nuclear factor 1-like protein with a cAMP response element-binding protein in rat liver.

1. The existence of both cAMP-responsive element binding factor and a nuclear factor 1-like (NF-1-like) protein in nuclear extracts from liver of cAMP-treated rat has been revealed. 2. Binding of these proteins to a DNA fragment containing both elements was cooperative, and 50% binding was achieved with considerably less protein than with a fragment bearing either element alone. 3. Cleavage of the fragment between the two elements abolished the apparent cooperative interaction. 4. Southwestern blot analysis showed that the NF-1-like protein has a molecular weight in the 28-30-kDa range. 5. The NF-1-like binding activity was very stable.

Functional recognition of the neuronal tyrosine hydroxylase cAMP regulatory element in different cell types.

Transient transfection of pLB2CAT constructs bearing short synthetic oligonucleotides derived either from the tyrosine hydroxylase (TH) promoter or other sources was used to examine functional cAMP regulatory element (CRE) activity in a variety of cell lines. The region containing only the putative TH CRE was found to be as or more effective in conferring cAMP responsiveness onto pLB2CAT (which employs the TK promoter) than the immediate 272 bp region of the TH promoter. Increases in CAT activity of 10- to 20-fold were observed in JEG-3 cells with a single insert of the TH CRE region (-31 to -54) in pLB2CAT, and the presence of a second insert generated only a modest further increase. This construct also responded to cAMP in 4 other cell lines tested but the degree of increase was less dramatic. Inserts containing the consensus 8 bp CRE motif embedded in other natural or artificial contexts served generally as weak functional CREs in all cell lines tested. In vitro analysis revealed that a specific protein-DNA complex apparently containing a single protein with a MW of 45-50 kDa was formed equally well with JEG-3 cell nuclear extract and CRE-bearing-TH and other fragments which produced dramatically different cAMP effects in vivo. These results suggest specificity in the effects of cAMP on different CREs which are dictated by contextual differences.

Evidence for a cAMP-dependent nuclear factor capable of interacting with a specific region of a eukaryotic gene.

Nuclear extracts prepared from the livers of rats treated with or without 8-bromo-cAMP were tested for their ability to bind to various fragments from the flanking region of the gene encoding phosphoenolpyruvate carboxykinase (GTP) [GTP: oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.32] known to contain the element that confers transcriptional regulation by cAMP. Using the nitrocellulose-filtration method, concentration-dependent, apparently saturable binding was seen that is both specific and cAMP dependent. Analysis of various fragments pinpointed the active binding region to positions within the -67 to -111 region, which coincides with the functional regulatory element as shown by recent transfection studies. Formation of an apparently single complex between a synthetic oligomer containing the region from -67 to -111 of the phosphoenolpyruvate carboxykinase gene and a factor in nuclear extracts from cAMP-treated rat liver was visualized by the gel-retardation method. Complex formation is both concentration and cAMP dependent and can be prevented by excess specific but not nonspecific competitor DNA. The congruity of the results with the two different methods suggests that the factor we have detected has properties consistent with a possible role as mediator of the transcriptional control exerted by cAMP in eukaryotic cells.

Cyclic adenonosine monophosphate does not affect the stability of the messenger ribonucleic acid for tyrosine aminotransferase in cultured hepatoma cells.

Cyclic AMP has been shown to stimulate synthesis of tyrosine aminotransferase (L-tyrosine:2-oxoglutarate aminotransferase, EC 2.6.1.5) by increasing the amount of its mRNA through an increase in initiation of transcription. However, cAMP also has posttranscriptional effects on the enzyme's synthesis, as evidenced by the 4- to 5-fold enhanced decline seen when cultured hepatoma cells are exposed to cAMP and transcription is inhibited. As a direct test of the possibility that cAMP exerts this effect by destabilizing the mRNA for tyrosine aminotransferase, we analyzed the rate of decay of the mRNA using the transcriptional inhibitor 5,6-dichlororibofuranosylbenzimidazole, Northern blot analysis, and an internal standard consisting of prelabeled rRNA. It was found that the half-life of the mRNA (2.0 +/- 0.2 h) was not changed by treatment of cultured hepatoma cells under conditions which increase intracellular cAMP levels. These mRNA half-life values were not significantly different from the decline in the rate of synthesis of the enzyme after induction in dexamethasone-treated cells. We conclude that cAMP does not affect the stability of the mRNA for tyrosine aminotransferase and discuss other possible explanations for the paradoxical effect of cAMP on deinduction of this enzyme.

Progesterone decreases DNA binding factor activity and the expression in Xenopus oocytes of a cAMP responsive gene from rat liver.

Stage VI Xenopus oocytes were injected with a plasmid (pBB0.6-CAT) which contains the cAMP regulatory element (CRE) from the rat liver phosphoenolpyruvate carboxykinase (PEPCK) gene fused upstream from a reporter gene [chloramphenicol acetyltransferase (CAT)]. Inhibition of the expression of the reporter gene (average = 51%) was observed in the presence of 10 microM progesterone, which is known to lead to inactivation of the oocyte cAMP dependent protein kinase (A kinase). In contrast, oocytes injected with a control plasmid (pSV2CAT), which contains no CRE, exhibited a variable increase (average = 31%) in CAT activity after progesterone treatment. Injection of the purified bovine cardiac A kinase catalytic subunit prior to exposure of oocytes injected with pBB0.6 CAT to progesterone prevents the loss of CAT activity generated by incubation with the steroid. Gel retardation analyses with oocyte lysates and a labeled synthetic oligonucleotide fragment containing the CRE from the PEPCK gene showed the existence of a complex with the same Rf and specificity as that formed with rat liver extracts. Subsequent exposure to progesterone, however, led to a rapid and extensive decrease in this binding activity. Taken together, these results are consistent with but do not prove the hypothesis that progesterone treatment and A kinase inactivation lead to a decrease in pBB0.6 CAT expression by virtue of a decline in the binding activity of an oocyte factor(s) to the CRE of the PEPCK fragment in pBB0.6-CAT, thereby decreasing transcription of the CAT gene.

Photoecology of the coral Leptoseris fragilis in the Red Sea twilight zone (an experimental study by submersible).

Depth-dependent photoadaptational responses of the Red Sea zooxanthellate coral (Leptoseris fragilis) were studied down to 160 m from the research submersible GEO. Light saturation curves for photosynthesis revealed, with I C=1-2, I K=10.9 and I sat=20 µE·cm-2·sec-1, the lowest values of photokinetic parameters ever reported for a symbiotic coral. In summer, positive net production occurs only around noon at approx. 100m depth. Biomass parameters of corals at 100-135 m are negatively correlated with depth in algal cell density, protein, chlorophyll and carotenoid but not in pigment ratios or cell based pigment content. Coral size decreased with depth. Corals transplanted from 110-120 m original depth to 40, 70, 90 and 160 m showed high survival after one year. O2-production and dark O2-uptake increased with decreasing transplantation depth. After one year, transplants at 70 and 90 m but not at 40 m had higher algae density and pigment concentrations. The host light-harvesting systems described by Schlichter, Fricke and Weber (1986) are partially destroyed in 40 m but not in 70 and 90 m transplants. Different light exposures alter P-I-responses (P max, I C, I K, I sat) but not biomass parameters, indicating molecular or biochemical adaptation. The coraal's optimal light fields lie between 70 to 90 m. Its exceptional bathymetric distribution is linked with the newly discovered host light-harvesting systems which probably enhance photosynthetic performance in a dim environment.

Is cyclic AMP dependent protein kinase responsible for the in vivo phosphorylation of tyrosine aminotransferase?

Undegraded tyrosine aminotransferase was purified to near homogeneity from rat liver and was confirmed to be a substrate for the beef heart cyclic AMP dependent protein kinase catalytic subunit. Specific antibody was used to quantitate the amount of phosphate incorporated into the enzyme. Phosphate incorporation was maximal at a catalytic subunit to tyrosine aminotransferase molar ratio of 7:1 using 200 microM ATP for 30 to 60 min at 30 degrees C. Phospho-peptide maps of tyrosine aminotransferase phosphorylated in vitro by the catalytic subunit were compared with those of amino-transferase immunoprecipitated from 32P labeled cells treated with and without 8-Br cAMP. Whereas the phospho-peptide maps of tyrosine aminotransferase isolated from cells treated with and without 8-Br cAMP were identical, differences were observed in the peptide map of tyrosine aminotransferase phosphorylated in vitro and in vivo. These results were taken to indicate that the catalytic subunit is not responsible for tyrosine aminotransferase phosphorylation in vivo.

In vitro phosphorylation of 4-aminobutyrate aminotransferase by cAMP dependent protein kinase.

Highly purified 4-aminobutyrate aminotransferase from pig brain is susceptible to phosphorylation by the purified cAMP-dependent protein kinase catalytic subunit. Up to 0.7 moles of phosphate from ATP-(gamma)-32P can be incorporated per mole of dimeric holoenzyme. Maximum phosphorylation was observed within about 90 minutes at 30 degrees C. Despite the extensive degree of phosphorylation observed, no kinetic property of the enzyme was perceptibly altered. Removal of cofactor had no detectable impact on the extent of phosphorylation but thermal inactivation of the enzyme increased and mild reduction with sodium borohydride decreased the phosphorylatability of the aminotransferase. It was possible to separate the enzyme into phospho and dephospho forms by the use of DEAE chromatography. Validation that the two fractions represented genuine aminotransferase was obtained by proteolytic peptide mapping. The phospho form of the enzyme was found to possess little or no aminotransferase activity while that of the dephospho form exhibited higher specific activity than the purified enzyme prior to phosphorylation. Furthermore, the dephospho form of the enzyme could not be detectably phosphorylated by reincubation with the kinase following DEAE chromatography unless it was subjected to thermal inactivation. The stoichiometry of phosphorylation of the fraction containing 32P from DEAE chromatography was approximately 1 mole/mole of dimer. These results suggest that the substrate for phosphorylation by the kinase is a form of the aminotransferase which is somehow inactivated during routine purification even when extensive precautions are taken to maximally preserve catalytic activity.

On the role of protein kinase subunits in the control of eukaryotic gene expression.

Transcriptional regulation by cAMP has been demonstrated for several eukaryotic genes; however, the identity of the protein kinase subunit involved has been a source of debate. Based on homologies with the procaryotic cAMP-binding catabolite activator protein, a recent hypothesis has invoked the regulatory protein RII as the mediator. The evidence currently available on the effects of microinjected kinase subunits suggests, however, that the catalytic subunit is the active factor. Moreover, the proposed homologies between the catabolite activator protein and RII are difficult to reconcile with its proposed mediatory role. We suggest as an alternative hypothesis that a phosphoprotein other than RII may mediate the effects of cAMP on eukaryotic gene expression.

Direct evidence that the protein kinase catalytic subunit mediates the effects of cAMP on tyrosine aminotransferase synthesis.

The effect of purified beef heart cAMP-dependent protein kinase catalytic subunit on tyrosine aminotransferase activity in intact cultured rat H35 hepatoma cells was directly tested by micro-injection using human red blood cell ghosts as vehicles. Although the micro-injection procedure itself produced temporary fluctuations in protein synthesis and in tyrosine aminotransferase activity in H35 cells, after a recovery period of 8-12 h, these parameters returned to normal in parallel with restoration of full inducibility of the aminotransferase by both 8-Br-cAMP and dexamethasone. Eight to sixteen hours after fusion of H35 cells with unloaded ghosts, ghosts loaded with bovine serum albumin or mock-loaded with the partially purified protein kinase catalytic subunit, no significant change in the activity of the aminotransferase was detected. In contrast, fusion with ghosts loaded with the catalytic subunit at concentrations between 0.1-2 mg/ml caused reproducible 2-3-fold increases in enzyme activity. Homogeneous preparations of the catalytic subunit exhibited even greater potency as an inducer. The effect was both time- and concentration-dependent and was abolished by inactivation of the catalytic subunit with N-ethylmaleimide prior to loading. The partially purified inhibitor of protein kinase from beef heart, while not affecting basal tyrosine aminotransferase activity, selectively inhibited the ability of 8-Br-cAMP but not that of dexamethasone to stimulate the activity of this enzyme. In addition, micro-injection of the pure regulatory subunit of the kinase blocked the response of the aminotransferase to low concentrations of 8-Br-cAMP. These results provide strong support for the proposition that the catalytic subunit of protein kinase mediates the effects of cAMP on the synthesis of tyrosine aminotransferase.

Cyclic nucleotide-dependent protein kinases and some major substrates in the rat cerebellum after neonatal X-irradiation.

The levels of cAMP-dependent protein kinase (type I), or cGMP-dependent protein kinase, or protein I, and of a 23,000 MW substrate for the cGMP-dependent protein kinase were measured in cerebella from normal rats and in the cerebella from rats in which a selective loss of interneurons in the cerebellar cortex had been produced by X-irradiation. A decrease was observed in the concentrations of cAMP-dependent protein kinase and of protein I, whereas an increase was observed in the concentrations of cGMP-dependent protein kinase and of the 23,000 MW substrate. The data, taken together with the results of other studies, support the interpretation that cAMP-dependent protein kinase and protein I are distributed throughout the cerebellum, but that cGMP-dependent protein kinase and the 23,000 MW substrate are highly concentrated in Purkinje cells.

Localization of cyclic GMP-dependent protein kinase and substrate in mammalian cerebellum.

The regional and cellular distribution of guanosine 3',5'-cyclic monophosphate (cGMP)-dependent protein kinase (ATP:protein phosphotransferase,EC 2.7.1.37) in mammalian brain was examined by use of the photoaffinity label 8-azidoinosine 3',5'-cyclic monophosphate. Of the regions examined, cerebellum had by far the highest concentration of this enzyme. The cellular localization of cGMP-dependent protein kinase within the cerebellum was determined by examination of mutant mice missing specific types of cerebellar neurons. Mutant mice lacking Purkinje cells had greatly reduced amounts of cGMP-dependent protein kinase, whereas the loss of another cell type, granule cells, did not reduce cGMP-dependent protein kinase levels. By using the same strains of mutant mice, a 23,000-dalton soluble cerebellar substrate for cGMP-dependent protein kinase was also shown to be enriched in Purkinje cells. In contrast, the concentration of type I 3',5'-cyclic AMP-dependent protein kinase in the cerebellum was unaffected by the absence of Purkinje cells and only slightly reduced by the absence of granule cells. The enrichment in Purkinje cells of the cGMP-dependent protein kinase and its substrate suggests an important role for cGMP and cGMP-dependent protein phosphorylation in the function of this type of neuronal cell.

Interference of inhibitors of dopamine-beta-hydroxylase with uptake of monoamines by chromaffin granular membranes.

Disulfiram and bis-(4-methyl-1-homopiperazinylthiocarbonyl)-disulphide (FLA 63) markedly inhibited the Mg2+/ATP-dependent uptake of various monoamines, e.g. dopamine (DA), by isolated membranes of bovine adrenal chromaffin granules. Both compounds affected DA-beta-hydroxylase (DBH) activity more markedly than the uptake of DA. Other inhibitors of DBH, e.g. fusaric acid and diethyldithiocarbaminate (DDC), did not interfere with DA uptake. Disulfiram and FLA 63, in contrast to fusaric acid and DDC, also caused a partial inhibition of Mg2+-dependent ATPase. It is concluded that the inhibition of monoamine uptake by disulfiram and FLA 63 is not related to their effect on DBH.