Metabolism of delta-Aminolevulinic Acid in Red and Blue-Green Algae

δ-Aminolevulinic acid was incorporated in vivo into C-phycocyanin and B-phycoerythrin in two species of the Rhodophyta (Cyanidium caldarium, Porphyridium cruentum) and three species of the Cyanophyta (Anacystis nidulans, Plectonema boryanum, Phormidium luridum). Amino acid analysis of phycocyanin-¹⁴C from C. caldarium cells which had been incubated with δ-aminolevulinate-4-¹⁴C showed that 84% of the radioactivity incorporated was present in the phycocyanobilin chromophore and less than 16% of the radioactivity cochromatographed with amino acids. These results indicate that δ-aminolevulinate is utilized predominantly via the porphyrin pathway in C. caldarium. Conversely, analysis of phycocyanin-¹⁴C prepared from cells of A. nidulans, P. boryanum, and P. luridum which had been incubated with radiolabeled δ-aminolevulinate demonstrated that 85%, 81%, and 93%, respectively, of the radioactivity incorporated cochromatographed with amino acids. The ratio of incorporated radioactivity in amino acids and phycoerythrobilin was 40:60 in P. cruentum phycoerythrin obtained from cells which had been incubated with δ-aminolevulinate-4-¹⁴C. Succinate-2-3-¹⁴C appeared to be as good a carbon source of amino acids as did C₄ and C₅ of δ-aminolevulinate. These data demonstrate a major alternate route (other than the porphyrin pathway) of δ-aminolevulinate metabolism in red and blue-green algae. The factors responsible for the extent to which δ-aminolevulinate is utilized for synthesis of porphyrins and their derivatives and routes of δ-aminolevulinate catabolism in the organisms employed are discussed.     

Mode of Action of the Massively Accumulated beta-Carotene of Dunaliella bardawil in Protecting the Alga against Damage by Excess Irradiation

When grown under defined conditions Dunaliella bardawil accumulates a high concentration of β-carotene, which is composed primarily of two isomers, all-trans and 9-cis β-carotene. The high β-carotene alga is substantially resistant to photoinhibition of photosynthetic oxygen evolution when compared with low β-carotene D. bardawil or with Dunaliella salina which is incapable of accumulating β-carotene. Protection against photoinhibition in the high β-carotene D. bardawil is very strong when blue light is used as the photoinhibitory agent, intermediate with white light, and nonexistent with red light. These observations suggest that the massively accumulated β-carotene in D. bardawil protects the alga against damage by high irradiation by screening through absorption of the blue region of the spectrum. Irradiation of D. bardawil by high intensity blue light results in the following temporal sequence of events: photoinhibition of oxygen evolution, photodestruction of 9-cis β-carotene, photodestruction of all-trans β-carotene, photodestruction of chlorophyll and cell death.     

Oxygen Isotope Variability within Nautilus Shell Growth Bands

Nautilus is often used as an analogue for the ecology and behavior of extinct externally shelled cephalopods. Nautilus shell grows quickly, has internal growth banding, and is widely believed to precipitate aragonite in oxygen isotope equilibrium with seawater. Pieces of shell from a wild-caught Nautilus macromphalus from New Caledonia and from a Nautilus belauensis reared in an aquarium were cast in epoxy, polished, and then imaged. Growth bands were visible in the outer prismatic layer of both shells. The thicknesses of the bands are consistent with previously reported daily growth rates measured in aquarium reared individuals. In situ analysis of oxygen isotope ratios using secondary ion mass spectrometry (SIMS) with 10 μm beam-spot size reveals inter- and intra-band δ¹⁸O variation. In the wild-caught sample, a traverse crosscutting 45 growth bands yielded δ¹⁸O values ranging 2.5‰, from +0.9 to -1.6 ‰ (VPDB), a range that is larger than that observed in many serial sampling of entire shells by conventional methods. The maximum range within a single band (~32 μm) was 1.5‰, and 27 out of 41 bands had a range larger than instrumental precision (±2 SD = 0.6‰). The results from the wild individual suggest depth migration is recorded by the shell, but are not consistent with a simple sinusoidal, diurnal depth change pattern. To create the observed range of δ¹⁸O, however, this Nautilus must have traversed a temperature gradient of at least ~12°C, corresponding to approximately 400 m depth change. Isotopic variation was also measured in the aquarium-reared sample, but the pattern within and between bands likely reflects evaporative enrichment arising from a weekly cycle of refill and replacement of the aquarium water. Overall, this work suggests that depth migration behavior in ancient nektonic mollusks could be elucidated by SIMS analysis across individual growth bands.     

Mercury Analysis of Acid- and Alkaline-Reduced Biological Samples: Identification of meta-Cinnabar as the Major Biotransformed Compound in Algae

The biotransformation of Hg^II in pH-controlled and aerated algal cultures was investigated. Previous researchers have observed losses in Hg detection in vitro with the addition of cysteine under acid reduction conditions in the presence of SnCl₂. They proposed that this was the effect of Hg-thiol complexing. The present study found that cysteine-Hg, protein and nonprotein thiol chelates, and nucleoside chelates of Hg were all fully detectable under acid reduction conditions without previous digestion. Furthermore, organic (R-Hg) mercury compounds could not be detected under either the acid or alkaline reduction conditions, and only β-HgS was detected under alkaline and not under acid SnCl₂ reduction conditions. The blue-green alga Limnothrix planctonica biotransformed the bulk of Hg^II applied as HgCl₂ into a form with the analytical properties of β-HgS. Similar results were obtained for the eukaryotic alga Selenastrum minutum. No evidence for the synthesis of organomercurials such as CH₃Hg⁺ was obtained from analysis of either airstream or biomass samples under the aerobic conditions of the study. An analytical procedure that involved both acid and alkaline reduction was developed. It provides the first selective method for the determination of β-HgS in biological samples. Under aerobic conditions, Hg^II is biotransformed mainly into β-HgS (meta-cinnabar), and this occurs in both prokaryotic and eukaryotic algae. This has important implications with respect to identification of mercury species and cycling in aquatic habitats.     

Photosynthetic action spectra of marine algae

A polarographic oxygen determination, with tissue in direct contact with a stationary platinum electrode, has been used to measure the photosynthetic response of marine algae. These were exposed to monochromatic light, of equal energy, at some 35 points through the visible spectrum (derived from a monochromator). Ulva and Monostroma (green algae) show action spectra which correspond very closely to their absorption spectra. Coilodesme (a brown alga) shows almost as good correspondence, including the spectral region absorbed by the carotenoid, fucoxanthin. In green and brown algae, light absorbed by both chlorophyll and carotenoids seems photosynthetically effective, although some inactive absorption by carotenoids is indicated. Action spectra for a wide variety of red algae, however, show marked deviations from their corresponding absorption spectra. The photosynthetic rates are high in the spectral regions absorbed by the water-soluble "phycobilin" pigments (phycoerythrin and phycocyanin), while the light absorbed by chlorophyll and carotenoids is poorly utilized for oxygen production. In red algae containing chiefly phycoerythrin, the action spectrum closely resembles that of the water-extracted pigment, with peaks corresponding to its absorption maxima (495, 540, and 565 mµ). Such algae include Delesseria, Schizymenia, and Porphyrella. In the genus Porphyra, there is a series P. nereocystis, P. naiadum, and P. perforata, with increasingly more phycocyanin and less phycoerythrin: the action spectra reflect this, with increasing activity in the orange-red region (600 to 640 mµ) where phycocyanin absorbs. In all these red algae, photosynthesis is almost minimal at 435 mµ and 675 mµ, where chlorophyll shows maximum absorption. Although the chlorophylls (and carotenoids) are present in quantities comparable to the green algae, their function is apparently not that of a primary light absorber; this role is taken over by the phycobilins. In this respect the red algae (Rhodophyta) appear unique among photosynthetic plants.     

Changes in Chlorophyll a/b Ratio and Products of CO(2) Fixation by Algae Grown in Blue or Red Light

Chlamydomonas and Chlorella were grown for 10 days in white light. 955 μw/cm² blue light (400-500 mμ) or 685 μw/cm² red light (above 600 mμ). Rates of growth in blue or red light were initially slow, but increased over a period of 5 days until normal growth rates were reestablished. During this adaptation period in blue light, total chlorophyll per volume of algae increased 20% while the chlorophyll a/b ratio decreased. In red light no change was observed in the total amount of chlorophyll or in the chlorophyll a/b ratio. After adaptation to growth in blue light and upon exposure to ¹⁴CO₂ with either blue or white light for 3 to 10 minutes, 30 to 36% of the total soluble fixed ¹⁴C accumulated in glycolate-¹⁴C which was the major product. However, with 1 minute experiments, it was shown that phosphate esters of the photosynthetic carbon cycle were labeled before the glycolate. Glycolate accumulation by algae grown in blue light occurred even at low light intensity. After growth of the algae in red light, ¹⁴C accumulated in malate, aspartate, glutamate and alanine, whereas glycolate contained less than 3% of the soluble ¹⁴C fraction.     

Primary Events, Energy Transfer, and Reactions in Photosynthetic Units: Lifetime of the Excited State In Vivo: I. Chlorophyll a in Algae, at Room and at Liquid Nitrogen Temperatures; Rate Constants of Radiationless Deactivation and Trapping

Using a mode-locked laser (λ, 632.8 nm), fluorescence decay of chlorophyll (Chl) a in the green alga Chlorella pyrenoidosa, the red alga Porphyridium cruentum, and the blue-green alga Anacystis nidulans was measured by the phase-shift method under conditions when photosynthesis was not operative (3-(3,4-dichlorophenyl)-1,1-dimethylurea [DCMU] poisoning, or cooling to 77°K). In the presence of 10^-5 M DCMU, the lifetime of Chl a fluorescence (τ) at room temperature is about 1.7 nsec in Chlorella, 1.0 nsec in Porphyridium, and 0.7 nsec in Anacystis. At 77°K, τ is 1.4 nsec (for fluorescence at about 685 nm, F-685) and 2.3 nsec (for F-730) in Chlorella, 0.9 nsec (F-685) and 1.2 nsec (F-730) in Porphyridium, and 0.8 nsec (F-685 and F-730) in Anacystis. From the above measurement, and the assumption that τ₀ (the intrinsic fluorescence lifetime) for Chl a in all three algae is 15.2 nsec, we have calculated the rate constants of radiationless transition (that includes energy transfer to weakly fluorescent system I) processes competing with fluorescence at room temperature to be about 5 × 10⁸ sec^-1 in Chlorella, 9 × 10⁸ sec^-1 in Porphyridium, and 13 × 10⁸ sec^-1 in Anacystis. At 77°K, this rate constant for Chl a that fluoresces at 685 nm remains, in the first approximation, the same as at room temperature. From the τ data, the rate constant for the trapping of excitation energy is calculated to be about 1.2 × 10⁹ sec^-1 for Chlorella, 2 × 10⁹ sec^-1 for Porphyridium, and 2 × 10⁹ sec^-1 for Anacystis. The efficiency of trapping is calculated to be about 66% (Chlorella), 68% (Porphyridium), and 60% (Anacystis). (It is recognized that variations in the above values are to be expected if algae grown under different conditions are used for experimentation.) The maximum quantum yield of Chl a fluorescence for system II (λ, 632.8 nm), calculated from τ measurements, is about 10% in Chlorella, 6-7% in Porhyridium, and 5% in Anacystis under conditions when photosynthesis is not operative; the values at 77°K appear to be very close to those with DCMU added at room temperature. ø for F-730 at 77°K, however, is somewhat higher than for F-685. The predicted quantum yields of fluorescence for Chl a in intact cells (both systems I and II) at low intensities of 632.8 nm light are about 2-3, 1-2, and 1% for Chlorella, Porphyridium, and Anacystis, respectively.     

Respiratory Chain of Colorless Algae II. Cyanophyta

Whole cell difference spectra of the blue-green algae, Saprospira grandis, Leucothrix mucor, and Vitreoscilla sp. have one, or at the most 2, broad α-bands near 560 mμ. At −190° these bands split to give 4 peaks in the α-region for b and c-type cytochromes, but no α-band for a-type cytochromes is visible. The NADH oxidase activity of these organisms was shown to be associated with particulate fractions of cell homogenates. The response of this activity to inhibitors differed from the responses of the NADH oxidase activities of particulate preparations from the green algae and higher plants to the same inhibitors, but is more typical of certain bacteria. No cytochrome oxidase activity was present in these preparations. The respiration of Saprospira and Vitreoscilla can be light-reversibly inhibited by CO, and all 3 organisms have a CO-binding pigment whose CO complex absorbs near 570, 535, and 417 mμ. The action spectrum for the light reversal of CO-inhibited Vitreoscilla respiration shows maxima at 568, 534, and 416 mμ. The results suggest that the terminal oxidase in these blue-greens is an o-type cytochrome.     

Stereoisomers of beta-Carotene and Phytoene in the Alga Dunaliella bardawil

Dunaliella bardawil, a halotolerant green alga, was previously shown to accumulate high concentrations of β-carotene when grown outdoors under defined conditions. The β-carotene of algae cultivated under high light intensity in media containing a high salt concentration is composed of approximately 50% all-trans β-carotene and 40% 9-cis β-carotene. We show here that the 9-cis to all-trans ratio is proportional to the integral light intensity to which the algae are exposed during a division cycle. In cells grown under a continuous white light of 2000 microeinsteins per square meter per second, the ratio reached a value of around 1.5, while in cells grown under a light intensity of 50 microeinsteins per square meter per second, the ratio was around 0.2. As previously shown, algae treated with the herbicide norflurazon accumulate phytoene in place of β-carotene. Electron micrographs showed that the phytoene is accumulated in many distinct globules located in the interthylakoid spaces of the chloroplast. Here too, two isomers are present, apparently all-trans and 9-cis phytoene, and their ratio is dependent upon the integral light intensity to which the algae are exposed during a division cycle. In the presence of norflurazon, Dunaliella bardawil grown under a light intensity of 2000 microeinsteins per square meter per second contained about 8% phytoene with a 9-cis to all-trans ratio of about 1.0. This ratio decreased to about 0.1 when the light intensity was reduced to 50 microeinsteins per square meter per second. These data suggest that the isomerization reaction which leads to the production of the 9-cis isomer occurs early in the path of carotene biosynthesis, at or before the formation of all-trans phytoene. The presence of the 9-cis isomer of β-carotene and the dependence of its preponderance on light intensity seem to be a common feature of many plant parts. Thus carrots which are exposed to minimal light contain no 9-cis isomer while sun-exposed leaves, fruits, and flowers contain 20 to 50% of the 9-cis isomer.     

CpeS Is a Lyase Specific for Attachment of 3Z-PEB to Cys82 of β-phycoerythrin from Prochlorococcus marinus MED4

In contrast to the majority of cyanobacteria, the unicellular marine cyanobacterium Prochlorococcus marinus MED4 uses an intrinsic divinyl-chlorophyll-dependent light-harvesting system for photosynthesis. Despite the absence of phycobilisomes, this high-light adapted strain possesses β-phycoerythrin (CpeB), an S-type lyase (CpeS), and enzymes for the biosynthesis of phycoerythrobilin (PEB) and phycocyanobilin. Of all linear tetrapyrroles synthesized by Prochlorococcus including their 3Z- and 3E-isomers, CpeS binds both isomers of PEB and its biosynthetic precursor 15,16-dihydrobiliverdin (DHBV). However, dimerization of CpeS is independent of bilins, which are tightly bound in a complex at a ratio of 1:1. Although bilin binding by CpeS is fast, transfer to CpeB is rather slow. CpeS is able to attach 3E-PEB and 3Z-PEB to dimeric CpeB but not DHBV. CpeS transfer of 3Z-PEB exclusively yields correctly bound βCys⁸²-PEB, whereas βCys⁸²-DHBV is a side product of 3E-PEB transfer. Spontaneous 3E- and 3Z-PEB addition to CpeB is faulty, and products are in both cases βCys⁸²-DHBV and likely a PEB bound at βCys⁸² in a non-native configuration. Our data indicate that CpeS is specific for 3Z-PEB transfer to βCys⁸² of phycoerythrin and essential for the correct configuration of the attachment product.     

Characterization of cryptomonad phycoerythrin and phycocyanin

Phycoerythrin, a chromoprotein, from the cryptomonad alga Rhodomonas lens is composed of two pairs of nonidentical polypeptides (α₂β₂). This structure is indicated by a molecular weight of 54,300, calculated from osmotic pressure measurements and by sodium dodecyl sulfate (SDS) gel electrophoresis, which showed bands with molecular weights of 9800 and 17,700 in a 1:1 molar ratio. The s_20,w⁰ of 4.3S is consistent with a protein of this molecular weight. Similar results were obtained with another cryptomonad phycoerythrin and a cryptomonad phycocyanin. Electrophoresis after partial cross-linking by dimethyl suberimidate revealed seven bands for the cryptomonad phycocyanin and six bands for cryptomonad phycoerythrin and confirmed the proposed structure. Spectroscopic studies on α and β subunits of cryptomonad phycocyanin and phycoerythrin were carried out on the separated bands in SDS gels. The individual polypeptides possessed a single absorption band with the following maxima: phycoerythrin (R. lens), α at 565 nm, β at 531 nm; phycocyanin (Chroomonas sp.), α at 644 nm, β at 566 nm. Fluorescence polarization was not constant across the visible absorption band regions of phycoerythrin (R. lens and C. ovata) with higher polarizations located at higher wavelengths, as had also been previously shown for cryptomonad phycocyanin (Chroomonas sp.). Combining the absorption spectra and the polarization results indicates that in each case the β subunit contains sensitizing chromophores and the α subunit fluorescing chromophores. The CD spectra of cryptomonad phycocyanin and both phycoerythrins were similar and were related to the spectra of the individual subunits. In Ouchterlony double-diffusion experiments the cryptomonad phycoerythrins and phycocyanins cross-reacted, with spurring, with phycoerythrin isolated from a red alga. The cryptomonad phycoerythrins were immunochemically very similar to each other and to cryptomonad phycocyanin, with little spurring detected.     

Detection and Quantification of Snow Algae with an Airborne Imaging Spectrometer

We describe spectral reflectance measurements of snow containing the snow alga Chlamydomonas nivalis and a model to retrieve snow algal concentrations from airborne imaging spectrometer data. Because cells of C. nivalis absorb at specific wavelengths in regions indicative of carotenoids (astaxanthin esters, lutein, β-carotene) and chlorophylls a and b, the spectral signature of snow containing C. nivalis is distinct from that of snow without algae. The spectral reflectance of snow containing C. nivalis is separable from that of snow without algae due to carotenoid absorption in the wavelength range from 0.4 to 0.58 μm and chlorophyll a and b absorption in the wavelength range from 0.6 to 0.7 μm. The integral of the scaled chlorophyll a and b absorption feature (I_0.68) varies with algal concentration (C_a). Using the relationship C_a = 81019.2 I_0.68 + 845.2, we inverted Airborne Visible Infrared Imaging Spectrometer reflectance data collected in the Tioga Pass region of the Sierra Nevada in California to determine algal concentration. For the 5.5-km² region imaged, the mean algal concentration was 1,306 cells ml⁻¹, the standard deviation was 1,740 cells ml⁻¹, and the coefficient of variation was 1.33. The retrieved spatial distribution was consistent with observations made in the field. From the spatial estimates of algal concentration, we calculated a total imaged algal biomass of 16.55 kg for the 0.495-km² snow-covered area, which gave an areal biomass concentration of 0.033 g/m².     

Triparanol Inhibition of Sterol Biosynthesis in Chlorella ellipsoidea

The sterol composition of C. ellipsoidea was markedly changed when this alga was grown in the presence of 1 μg/g triparanol. Triparanol appears to inhibit the removal of 14α-methyl group, the second alkylation at C-24, Δ⁷-reductase, and Δ⁸ → Δ⁷-isomerase. The effect of triparanol in Chlorella is much more diversified than the specific effect originally assigned to it in animals.     

Effects of Agronomic Treatments on Structure and Function of Ammonia-Oxidizing Communities

The aim of this study was to determine the effects of different agricultural treatments and plant communities on the diversity of ammonia oxidizer populations in soil. Denaturing gradient gel electrophoresis (DGGE), coupled with specific oligonucleotide probing, was used to analyze 16S rRNA genes of ammonia oxidizers belonging to the β subgroup of the division Proteobacteria by use of DNA extracted from cultivated, successional, and native deciduous forest soils. Community profiles of the different soil types were compared with nitrification rates and most-probable-number (MPN) counts. Despite significant variation in measured nitrification rates among communities, there were no differences in the DGGE banding profiles of DNAs extracted from these soils. DGGE profiles of DNA extracted from samples of MPN incubations, cultivated at a range of ammonia concentrations, showed the presence of bands not amplified from directly extracted DNA. Nitrosomonas-like bands were seen in the MPN DNA but were not detected in the DNA extracted directly from soils. These bands were detected in some samples taken from MPN incubations carried out with medium containing 1,000 μg of NH₄⁺-N ml⁻¹, to the exclusion of bands detected in the native DNA. Cell concentrations of ammonia oxidizers determined by MPN counts were between 10- and 100-fold lower than those determined by competitive PCR (cPCR). Although no differences were seen in ammonia oxidizer MPN counts from the different soil treatments, cPCR revealed higher numbers in fertilized soils. The use of a combination of traditional and molecular methods to investigate the activities and compositions of ammonia oxidizers in soil demonstrates differences in fine-scale compositions among treatments that may be associated with changes in population size and function.     

Inter-Species Complementation of the Translocon Beta Subunit Requires Only Its Transmembrane Domain

In eukaryotes, proteins enter the secretory pathway through the translocon pore of the endoplasmic reticulum. This protein translocation channel is composed of three major subunits, called Sec61α, β and γ in mammals. Unlike the other subunits, the β subunit is dispensable for translocation and cell viability in all organisms studied. Intriguingly, the knockout of the Sec61β encoding genes results in different phenotypes in different species. Nevertheless, the β subunit shows a high level of sequence homology across species, suggesting the conservation of a biological function that remains ill-defined. To address its cellular roles, we characterized the homolog of Sec61β in the fission yeast Schizosaccharomyces pombe (Sbh1p). Here, we show that the knockout of sbh1 ⁺ results in severe cold sensitivity, increased sensitivity to cell-wall stress, and reduced protein secretion at 23°C. Sec61β homologs from Saccharomyces cerevisiae and human complement the knockout of sbh1 ⁺ in S. pombe. As in S. cerevisiae, the transmembrane domain (TMD) of S. pombe Sec61β is sufficient to complement the phenotypes resulting from the knockout of the entire encoding gene. Remarkably, the TMD of Sec61β from S. cerevisiae and human also complement the gene knockouts in both yeasts. Together, these observations indicate that the TMD of Sec61β exerts a cellular function that is conserved across species.     

Characteristics of an R-Phycoerythrin with Two γ Subunits Prepared from Red Macroalga Polysiphonia urceolata

An R-phycoerythrin (R-PE) was isolated by gel filtrations on Sepharose CL-4B and Sephadex G-150 from the phycobiliprotein extract of the marine red macroalga Polysiphonia urceolata Grev and further purified by ion exchange chromatography on DEAE-Sepharose Fast Flow. The purified R-PE showed three absorption peaks at 498 nm, 538 nm, 566 nm and one fluorescent emission maximum at 577 nm. Although the R-PE showed a single band on the examination by native PAGE, it exhibited two very close bands at pH about 4.7 in native isoelectric focusing (IEF). Polypeptide analysis of the R-PE demonstrated that it contained four chromophore-carrying subunits, α^18.2, β^20.6, γ^31.6 (γ''), γ^34.6 (γ), and no colorless polypeptide; its subunit composition was 6α^18.2:6β^20.6:1 γ^31.6:2γ^34.6. The α and β subunits were distributed within a acidic pH range from 5.0 to 6.0 in denaturing IEF and the γ subunits were in a basic pH range from 7.6 to 8.1. These results reveal that the prepared R-PE may exist in two hexamers of γ (αβ)₃ γ (αβ)₃γ'' and γ (αβ)₃ γ''(αβ)₃ γ and that the R-PE participate in the rod domain assembly of P. urceolata phycobilisomes by stacking each of its trimer (αβ)₃ face-to-face with the aid of one γ subunit (γ or γ'').     

Trophic Relationships and Habitat Preferences of Delphinids from the Southeastern Brazilian Coast Determined by Carbon and Nitrogen Stable Isotope Composition

To investigate the foraging habitats of delphinids in southeastern Brazil, we analyzed stable carbon (δ¹³C) and nitrogen (δ¹⁵N) isotopes in muscle samples of the following 10 delphinid species: Sotalia guianensis, Stenella frontalis, Tursiops truncatus, Steno bredanensis, Pseudorca crassidens, Delphinus sp., Lagenodelphis hosei, Stenella attenuata, Stenella longirostris and Grampus griseus. We also compared the δ¹³C and δ¹⁵N values among four populations of S. guianensis. Variation in carbon isotope results from coast to ocean indicated that there was a significant decrease in δ¹³C values from estuarine dolphins to oceanic species. S. guianensis from Guanabara Bay had the highest mean δ¹³C value, while oceanic species showed significantly lower δ¹³C values. The highest δ¹⁵N values were observed for P. crassidens and T. truncatus, suggesting that these species occupy the highest trophic position among the delphinids studied here. The oceanic species S. attenuata, G. griseus and L. hosei had the lowest δ¹⁵N values. Stable isotope analysis showed that the three populations of S. guianensis in coastal bays had different δ¹³C values, but similar δ¹⁵N results. Guiana dolphins from Sepetiba and Ilha Grande bays had different foraging habitat, with specimens from Ilha Grande showing more negative δ¹³C values. This study provides further information on the feeding ecology of delphinids occurring in southeastern Brazil, with evidence of distinctive foraging habitats and the occupation of different ecological niches by these species in the study area.     

Photoinduction of Massive beta-Carotene Accumulation by the Alga Dunaliella bardawil: Kinetics and Dependence on Gene Activation

The massive accumulation of β-carotene by the halotolerant micro alga Dunaliella bardawil, in response to high light intensity and several other environmental factors, has been studied so far under different sets of fixed conditions. To determine the kinetics and characteristics of the induction of β-carotene accumulation, cells continuously grown under white light of approximately 27 microeinsteins per square meter per second were exposed to light of approximately 1650 microeinsteins per square meter per second. The exposed cells accumulate β-carotene in two stages: the first stage, lasting for 24 hours, starts shortly after exposure, whereas the second stage starts concomitantly with the onset of the stationary phase and persists until the cells collapse. Actinomycin D, chloramphenicol, or cycloheximide added to low-illuminated cultures abolish the subsequent induction of β-carotene accumulation by high light intensity. These results, together with the early exponential kinetics of accumulation, point to the role of gene activation in the process. In vivo labeling of proteins and in vitro translation of poly(A)⁺ mRNA revealed several pronounced differences between low-illuminated and high-illuminated cells. A strongly light-induced protein of approximately 55 kilodaltons, as well as other light-induced proteins could possibly fulfill a carotenogenic function.     

Ca2+-binding Motif of βγ-Crystallins

βγ-Crystallin-type double clamp (N/D)(N/D)XX(S/T)S motif is an established but sparsely investigated motif for Ca²⁺ binding. A βγ-crystallin domain is formed of two Greek key motifs, accommodating two Ca²⁺-binding sites. βγ-Crystallins make a separate class of Ca²⁺-binding proteins (CaBP), apparently a major group of CaBP in bacteria. Paralleling the diversity in βγ-crystallin domains, these motifs also show great diversity, both in structure and in function. Although the expression of some of them has been associated with stress, virulence, and adhesion, the functional implications of Ca²⁺ binding to βγ-crystallins in mediating biological processes are yet to be elucidated.     

Positive and Negative Allosteric Modulation of an α1β3γ2 γ-Aminobutyric Acid Type A (GABAA) Receptor by Binding to a Site in the Transmembrane Domain at the γ+-β? Interface

In the process of developing safer general anesthetics, isomers of anesthetic ethers and barbiturates have been discovered that act as convulsants and inhibitors of γ-aminobutyric acid type A receptors (GABA_ARs) rather than potentiators. It is unknown whether these convulsants act as negative allosteric modulators by binding to the intersubunit anesthetic-binding sites in the GABA_AR transmembrane domain (Chiara, D. C., Jayakar, S. S., Zhou, X., Zhang, X., Savechenkov, P. Y., Bruzik, K. S., Miller, K. W., and Cohen, J. B. (2013) J. Biol. Chem. 288, 19343–19357) or to known convulsant sites in the ion channel or extracellular domains. Here, we show that S-1-methyl-5-propyl-5-(m-trifluoromethyl-diazirynylphenyl) barbituric acid (S-mTFD-MPPB), a photoreactive analog of the convulsant barbiturate S-MPPB, inhibits α1β3γ2 but potentiates α1β3 GABA_AR responses. In the α1β3γ2 GABA_AR, S-mTFD-MPPB binds in the transmembrane domain with high affinity to the γ⁺-β⁻ subunit interface site with negative energetic coupling to GABA binding in the extracellular domain at the β⁺-α⁻ subunit interfaces. GABA inhibits S-[³H]mTFD-MPPB photolabeling of γ2Ser-280 (γM2–15′) in this site. In contrast, within the same site GABA enhances photolabeling of β3Met-227 in βM1 by an anesthetic barbiturate, R-[³H]methyl-5-allyl-5-(m-trifluoromethyl-diazirynylphenyl)barbituric acid (mTFD-MPAB), which differs from S-mTFD-MPPB in structure only by chirality and two hydrogens (propyl versus allyl). S-mTFD-MPPB and R-mTFD-MPAB are predicted to bind in different orientations at the γ⁺-β⁻ site, based upon the distance in GABA_AR homology models between γ2Ser-280 and β3Met-227. These results provide an explanation for S-mTFD-MPPB inhibition of α1β3γ2 GABA_AR function and provide a first demonstration that an intersubunit-binding site in the GABA_AR transmembrane domain binds negative and positive allosteric modulators.