Sedimentation of chlorophylls in an Arctic fjord under freshwater discharge

Sedimentation of chlorophylls was studied during summer 1997 in Adventfjorden (Spitsbergen, Arctic). During the period of study, the water column was found to be well stratified by a freshened surface layer (salinity <31 PSS). A high load of suspended particulate matter from riverine discharge reduced the euphotic zone to an interval of 0.4–1.1m. Total particulate matter sedimentation rates were about twice as high in June as in July. The following chlorophylls were distinguished in the sedimented particles: chl a and its degradation products (allomer chl a, phaeophytin a, phaeophorbide a, chlorophyllide a), chl b and chl c ₁+c ₂. The quantitatively most important derivative of chl a was phaeophorbide a (31--41% of porphyrin a). Generally, the sedimentation rate of chlorophylls increased with depth. Linear relationships between concentrations of chl a and phaeophorbide a (r ²=0.92), as well as between concentrations of chl a and phaeophytin a (r ²=0.90) indicated a strong connection between phytoplankton abundance and zooplankton grazing. The significant correlation between chl a and chlorophyllide a concentrations (r ²=0.82) showed that most of the sinking chl a belonged primarily to diatoms, and low chlorophyllide a:chl a ratio (0.03) indicated that cellular senescence was not an important reason for the sinking of chl a. Moreover, very low chl b:chl a ratios (about 0.05 calculated for samples where chl b was detectable) suggest that contributions of green algae and/or higher plant detritus were negligible in sinking particles. The ratio of chl c ₁+c ₂:chl a was 0.85 indicating that chl c-containing algae were dominating.     

Nucleotide sequence of two cDNAs encoding fucoxanthin chlorophyll a/c proteins in the diatom Odontella sinensis

Two cDNA clones encoding fucoxanthin chlorophyll a/c-binding proteins (FCP) in the diatom Odontella sinensis have been cloned and sequenced. The derived amino acid sequences of both clones are identical, comparison of the corresponding nucleic acids reveals differences only in the third codon position, suggesting a recent gene duplication. The derived proteins are similar to the chlorophyll a/b-binding proteins of higher plants. The presequences for plastid import resemble signal sequences for cotranslational import rather than transit peptides of higher plants. They are very similar to the presequences of FCP proteins in the diatom Phaeodactylum, but different from the presequences of the -subunit of CF₀CF₁ of Odontella and the peridinin chlorophyll a binding proteins (PCP) of the dinoflagellate Symbiodinium.Abbreviations CAB chlorophyll a/b-binding protein - FCP fucoxanthin chlorophyll a/c-binding protein - fcp the respective FCP genes - LHC light-harvesting complex - PCP peridinin chlorophyll a-binding protein - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate     

Semiquantitative paper chromatographic separation of chlorophylls a and b

[¹⁴C]Chlorophyll (chl) a has been utilized to demonstrate the contamination of chl b by (probably) oxidation products of chl a in thin-layer or paper chromatography. By circular chromatography of both chlorophylls as their pheophytins, the contamination of chl a (as pheophytin a) in chl b (as pheophytin b) may be reduced to 0.15–0.35.     

Mutations of the Calcium Channel Gene cacophony Suppress Seizures in Drosophila

Bang sensitive (BS) Drosophila mutants display characteristic seizure-like phenotypes resembling, in some aspects, those of human seizure disorders such as epilepsy. The BS mutant para^bss1, caused by a gain-of-function mutation of the voltage-gated Na⁺ channel gene, is extremely seizure-sensitive with phenotypes that have proven difficult to ameliorate by anti-epileptic drug feeding or by seizure-suppressor mutation. It has been presented as a model for intractable human epilepsy. Here we show that cacophony (cac^TS2), a mutation of the Drosophila presynaptic Ca⁺⁺ channel α₁ subunit gene, is a particularly potent seizure-suppressor mutation, reverting seizure-like phenotypes for para^bss1 and other BS mutants. Seizure-like phenotypes for para^bss1 may be suppressed by as much as 90% in double mutant combinations with cac^TS2. Unexpectedly, we find that para^bss1 also reciprocally suppresses cac^TS2 seizure-like phenotypes. The cac^TS2 mutant displays these seizure-like behaviors and spontaneous high-frequency action potential firing transiently after exposure to high temperature. We find that this seizure-like behavior in cac^TS2 is ameliorated by 85% in double mutant combinations with para^bss1.     

Discovery of new low-molecular-weight p53–Mdmx disruptors and their anti-cancer activities

Although several p53–Mdm2-binding disruptors have been identified to date, few studies have been published on p53–Mdmx-interaction inhibitors. In the present study, we demonstrated that o-aminothiophenol derivatives with molecular weights of 200–300 selectively inhibited the p53–Mdmx interaction. S-2-Isobutyramidophenyl 2-methylpropanethioate (K-178) (1c) activated p53, up-regulated the expression of its downstream genes such as p21 and Mdm2, and preferentially inhibited the growth of cancer cells with wild-type p53 over those with mutant p53. Furthermore, we found that the S-isobutyryl-deprotected forms 1b and 3b of 1c and S-2-benzamidophenyl 2-methylpropanethioate (K-181) (3c) preferentially inhibited the p53–Mdmx interaction over the p53–Mdm2 interaction, respectively, by using a Flag-p53 and glutathione S-transferase (GST)-fused protein complex (Mdm2, Mdmx, DAPK1, or PPID). In addition, the interaction of p53 with Mdmx was lost by replacing a sulfur atom with an oxygen atom in 1b and 1c. These results suggest that sulfides such as 1b, 3b, 4b, and 5b interfere with the binding of p53–Mdmx, resulting in the dissociation of the two proteins. Furthermore, the results of oral administration experiments using xenografts in nude mice indicated that 1c reduced the volume of tumor masses to 49.0% and 36.6% that of the control at 100 mg/kg and 150 mg/kg, respectively, in 40 days.     

A c Subunit with Four Transmembrane Helices and One Ion (Na+)-binding Site in an Archaeal ATP Synthase: IMPLICATIONS FOR c RING FUNCTION AND STRUCTURE*

The ion-driven membrane rotors of ATP synthases consist of multiple copies of subunit c, forming a closed ring. Subunit c typically comprises two transmembrane helices, and the c ring features an ion-binding site in between each pair of adjacent subunits. Here, we use experimental and computational methods to study the structure and specificity of an archaeal c subunit more akin to those of V-type ATPases, namely that from Pyrococcus furiosus. The c subunit was purified by chloroform/methanol extraction and determined to be 15.8 kDa with four predicted transmembrane helices. However, labeling with DCCD as well as Na⁺-DCCD competition experiments revealed only one binding site for DCCD and Na⁺, indicating that the mature c subunit of this A₁A_O ATP synthase is indeed of the V-type. A structural model generated computationally revealed one Na⁺-binding site within each of the c subunits, mediated by a conserved glutamate side chain alongside other coordinating groups. An intriguing second glutamate located in-between adjacent c subunits was ruled out as a functional Na⁺-binding site. Molecular dynamics simulations indicate that the c ring of P. furiosus is highly Na⁺-specific under in vivo conditions, comparable with the Na⁺-dependent V₁V_O ATPase from Enterococcus hirae. Interestingly, the same holds true for the c ring from the methanogenic archaeon Methanobrevibacter ruminantium, whose c subunits also feature a V-type architecture but carry two Na⁺-binding sites instead. These findings are discussed in light of their physiological relevance and with respect to the mode of ion coupling in A₁A_O ATP synthases.     

Sequence conservation of light-harvesting and stress-response proteins in relation to the three-dimensional molecular structure of LHCII

The structure of pea light-harvesting complex LHCII determined to 3.4 Å resolution by electron crystallography (Kühlbrandt, Wang and Fujiyoshi (1994) Nature 367: 614–621) was examined to determine the relationship between structural elements and sequence motifs conserved in the extended family of light-harvesting antennas (Chl a/b, fucoxanthin Chl a/c proteins) and membrane-intrinsic stress-induced proteins (ELIPs) to which LHCII belongs. It is predicted that the eukaryotic ELIPs can bind at least four molecules of Chl. The one-helix prokaryotic ELIP of Synechococcus was modelled as a homodimer based on the high degree of conservation of residues involved in the interactions of the first (B) and third (A) helices of LHCII.Abbreviations CAB Chl a/b-binding - ELIP early light-inducible protein - FCP fucoxanthin-Chl a/c protein - Lut1, Lut2 lutein molecules 1 and 2     

The oxidation-reduction characteristics of cytochrome b5 in hen liver microsomes.

Hen liver microsomes contained 0.20 nmol of cytochromeb₅ per mg of protein. Upon addition of NADH about 95% cytochrome b₅ was reduced very fast with a rate constant of 206 s^?1When ferricyanide was added to the reaction system the cytochrome stayed in the oxidized form until the ferricyanide reduction was almost completed. The reduced cytochrome b₅ in microsomes was oxidized very rapidly by ferricyanide. The rate constant of 4.5 × 10⁸m^?1 s^?1, calculated on the basis of assumption that ferricyanide reacts directly with the cytochrome, was found to be more than 100 times higher than that of the reaction between ferricyanide and soluble cytochrome b₅. To explain the results, therefore, the reverse electron flow from cytochrome b₅ to the flavin coenzyme in microsomes was assumed.By three independent methods the specific activity of the microsomes was measured at about 20 nmol of NADH oxidized per s per mg of protein and it was concluded that the reduction of the flavin coenzyme of cytochrome b₅ reductase by NADH is rate-limiting in the NADH-cytochrome b₅ and NADH-ferricyanide reductase reactions of hen liver microsomes. In the NADH-ferricyanide reductase reaction the apparent Michaelis constant for NADH was 2.8 μm and that for ferricyanide was too low to be measured. In the NADH-cytochrome c reductase reaction the maximum velocity was 2.86 nmol of cytochrome c reduced per s per mg of protein and the apparent Michaelis constant for cytochrome c was 3.8 μm.     

Genetics and biochemistry of the peptidoglycan-associated proteins b and c of Escherichia coli K12.

Summary To collect information on synthesis and regulation of the peptidoglycan-associated pore-forming outer membrane proteins b and c, mutants resistant to phages Mel and TuIa were analyzed. Genetic analysis showed three linkage groups, corresponding with the genes tolF (phenotype b^-c⁺), meo A (phenotype b⁺c^-) and ompB (phenotypes b^-c^-, b^- c⁺, b⁺⁺ c^- and b⁺⁺ c^±). It has recently been described that also a b⁺ c^- phenotype can occur in the latter linkage group [Chai, T., Foulds, J., J. Bacteriol. 130, 781–786 (1977)]. Among ompB (b^- c⁺)/meoA (b⁺ c^-) double mutants strains were found with the b⁺ c^- phenotype, showing that ompB is not the structural gene for protein b. Studies on purified proteins b and c showed profound differences between the two proteins with respect to the electrophoretic mobility of fragments obtained by treatment with cyanogen bromide, trypsin and chymotrypsin. The amino acid in position three of the amino-termini of proteins b and c, isolated from isogenic strains, were identified as isoleucine and valine respectively. Both the genetic and biochemical results are consistent with a model recently published [Ichihara, S., Mizushima, S., J. Biochem. (Japan) 83, 1095–1100 (1978)] which predicts that tolF and meoA are the structural genes for the proteins b and c respectively and that ompB is a regulatory gene whose product regulates the levels of both proteins.     

Polypeptide sequence of the chlorophyll a/b/c-binding protein of the prasinophycean alga Mantoniella squamata

The primary structure of the Chla/b/c-binding protein from Mantoniella squamata is determined. This is the first report that protein sequencing reveals one modified amino acid resulting in a LHCP-specific TFA-cleavage site. The comparison of the sequence of Mantoniella with other Chla/b-and Chla/c-binding proteins shows that the modified amino acid is located in a region which is highly conserved in all these proteins. The alignment also reveals that the LHCP of Mantoniella is related to the Chla/b-binding proteins. Finally, possible Chl-binding regions are discussed.Abbreviations a.m.u. atomic mass unit - LHC light-harvesting complex - LHC II major LHC of Photosystem II - LHCP light-harvesting chlorophyll-binding protein - LSIMS liquid secondary ion mass spectrometry - TFA trifluoroacetic acid     

Hypothesis for the evolution of three-helix Chl a/b and Chl a/c light-harvesting antenna proteins from two-helix and four-helix ancestors

The nuclear-encoded Chl a/b and Chl a/c antenna proteins of photosynthetic eukaryotes are part of an extended family of proteins that also includes the early light-induced proteins (ELIPs) and the 22 kDa intrinsic protein of PS II (encoded by psbS gene). All members of this family have three transmembrane helices except for the psbS protein, which has four. The amino acid sequences of these proteins are compared and related to the three-dimensional structure of pea LHC II Type I (Kühlbrandt and Wang, Nature 350: 130–134, 1991). The similarity of psbS to the three-helix members of the family suggests that the latter arose from a four-helix ancestor that lost its C-terminal helix by deletion. Strong internal similarity between the two halves of the psbS protein suggests that it in turn arose as the result of the duplication of a gene encoding a two-helix protein. Since psbS is reported to be present in at least one cyanobacterium, the ancestral four-helix protein may have been present prior to the endosymbiotic event or events that gave rise to the photosynthetic eukaryotes. The Chl a/b and Chl a/c antenna proteins, and the immunologically-related proteins in the rhodophytes may have had a common ancestor which was present in the early photosynthetic eukaryotes, and predated their division into rhodophyte, chromophyte and chlorophyte lineages. The LHC I-LHC II divergence probably occurred before the separation of higher plants from chlorophyte algae and euglenophytes, and the different Types of LHC I and LHC II proteins arose prior to the separation of angiosperms and gymnosperms.Abbreviations CAB Chl a/b-binding - ELIP early light-induced protein - FCP fucoxanthin-Chl a/c protein - PCR polymerase chain reaction - TMH trans-membrane helix     

Protective immunity against lethal anaphylactic reaction in Toxoplasma gondii-infected mice by DNA vaccination with T. gondii-derived heat shock protein 70 gene

Toxoplasma gondii-derived heat shock protein 70 (T.g.HSP70) was proven to induce lethal anaphylactic reaction in T. gondii-infected mice through platelet-activating factor (PAF)-mediated, but not classical IgE-dependent, pathway via TLR4/MyD88 signal pathway. The effector cells generating PAF and causing T.g.HSP70-induced anaphylactic reaction were CD11b⁺ and CD11c⁺ cells, although the reaction was enhanced by marked IFN-γ production by CD11b⁺, CD11c⁺, CD4⁺ and CD8⁺ splenocytes. In the present study, the effects of T.g.HSP70 gene vaccine targeting peripheral dendritic cells were evaluated against T.g.HSP70-induced anaphylactic reaction in T. gondii-infected mice. C57BL/6 mice receiving T.g.HSP70 gene vaccine showed prolonged survival. Platelets of peripheral blood, which completely disappeared during the T.g.HSP70-induced anaphylactic reaction, were partially restored with the T.g.HSP70 gene vaccination. The T.g.HSP70-induced marked production of PAF and IFN-γ from splenocytes of infected mice during the T.g.HSP70-induced anaphylactic reaction was shown to decrease after the T.g.HSP70 gene vaccination. Thus, T.g.HSP70 gene vaccine induced protective immunity against T.g.HSP70-induced PAF-mediated lethal anaphylactic reaction in T. gondii-infected mice.     

The functional differentiation of four smad4 paralogs in TGF-β signaling pathway of Japanese flounder (Paralichthys olivaceus)

As a classical signaling pathway, transforming growth factor β (TGF-β) has been studied in various animals for more than decade years. However, the members of TGF-β were markedly expanded in teleost specific third and fourth rounds of whole genome duplication (WGD). Here, four smad4s named Posmad4a, Posmad4b, Posmad4c and Posmad4d were identified in Japanese flounder. Our study showed that four flounder smad4s had distinct properties in terms of their protein structure, expression pattern, protein interaction and subcellular localization. PoSMAD4a/b were mainly located in the cytoplasm, and could co-localize in the nucleus with PoSMAD3a after TGF-β activator stimulation. PoSMAD4c was mainly located in nucleus, whereas PoSMAD4d distributed in the whole cell. Both PoSMAD4c and PoSMAD4d could co-localize in the nucleus with PoSMAD3b after TGF-β activator stimulation. Furthermore, Posmad4c responded most strongly to TGF-β signal stimulation. Dual-luciferase reporter assay also showed that Posmad4c could specifically up-regulate the TGF-β signal luciferase reporter gene, Posmad4b could enhance Wnt signal luciferase reporter gene, while both Posmad4b and Posmad4d could markedly up-regulate Notch signal reporter gene. All results indicated that Posmad4a/b/c/d had significantly functional differences among TGF-β, Notch and Wnt signaling pathways. Our study provided important understanding to the biology of smad4s and its pathway crosstalk in teleost.     

PHOTOSYNTHESIS AND COLD ACCLIMATION: MOLECULAR EVIDENCE FROM A POLAR DIATOM1

The psychrophilic diatom Fragilariopsis cylindrus (Grunow) Krieger in Helmcke & Krieger was used to investigate photosynthesis and growth under freezing temperatures. Gene expression during a temperature shift from +5° C to ?1.8° C was studied under 3 and 35 μmol photons·m^?2·s^?1 by using a macroarray. These measurements were paralleled by determination of fluorescence induction at PSII and pigment analysis. The shift to ?1.8° C at 35 μmol photons·m^?2·s^?1 caused a marginal decrease of photosynthetic quantum yield (F_v/F_m) from 0.61 to 0.52 with fast recovery after 1 day. The ratio of chl c to chl a increased from 3.1 to 5.5, and the ratio of diatoxanthin to diadinoxanthin increased from 0.7 to 5.0. Genes encoding proteins of PSII (psbA, psbC) and for carbon fixation (rbcL) were down‐regulated, whereas genes encoding chaperons (hsp70) and genes for plastid protein synthesis and turnover (elongation factor EfTs, ribosomal protein rpS4, ftsH protease) were up‐regulated. In contrast, cold exposure at 3 μmol photons·m^?2·s^?1 induced a marginal increase in F_v/F_m from 0.61 to 0.63 and a strong increase in fucoxanthin concentrations from 0.04 up to 0.12 pg·cell^?1. This was paralleled by up‐regulation of fcp genes. The ratio of chl c to chl a also increased from 3.1 to 4.2, as did the ratio of diatoxanthin to diadinoxanthin from 0.7 to 2.2. Down‐regulation of psbA, psbC, and rbcL could also be measured but not up‐regulation of hsp70, EfTs, rpS4, and the ftsH protease. The latter genes are probably necessary to avoid cold shock photoinhibition only at higher light intensities.     

Mitochondrial cytochrome b-c complex: its oxidation-reduction components and their stoichiometry.

A cytochrome b-c₁ complex was isolated from pigeon breast muscle mitochondria and purified to a content of 3 nmol of cytochrome c₁ per milligram of protein. Anaerobic suspensions of the preparation were titrated with reducing equivalents (NADH) and oxidizing equivalents (ferricyanide). The oxidation-reduction components of the complex were measured by the number of reducing equivalents accepted or donated per cytochrome c₁ and compared with the stoichiometries of the known redox components as measured by independent methods. The preparation accepts or donates 5.2 ± 0.3 equivalents per cytochrome c₁, while the measured content of cytochrome c₁, cytochrome b₅₆₁, cytochrome b₅₆₅, Rieske iron-sulfur protein, ubiquinone, and succinate dehydrogenase accounts for 5.0 ± 0.2 equivalents per cytochrome c₁. It is concluded that there are no unknown redox components in the cytochrome b-c₁ complex. The cytochrome b-c₁ complex (energy transduction site 2) appears to be a structural unit containing equal amounts of cytochrome c₁, cytochrome b₅₆₁, cytochrome b₅₆₆, and the Rieske iron-sulfur protein.     

Cloning and nucleotide sequence of a cDNA encoding a major fucoxanthin-, chlorophylla/c-containing protein from the chrysophyteIsochrysis galbana: implications for evolution of thecab gene family

We investigated the primary structure of a cDNA encoding a light-harvesting protein from the marine chrysophyteIsochrysis galbana. Antibodies raised against the major fucoxanthin, chlorophylla/c-binding light-harvesting protein (FCP) ofI. galbana were used to select a cDNA clone encoding one of the FCP apoproteins. The nucleic acid and deduced amino acid sequences reveal conserved regions within the first and third transmembrane spans with Chla/b-binding proteins and with FCPs of another chromophyte. However, the amino acid identity betweenI. galbana FCP and othercab genes of FCPs is only ca. 30%. Phylogenetic analyses demonstrated that the FCP genes of both diatoms and chrysophytes sequenced to date are more closely related tocab genes encoding LHC I, CP 29, and CP 24 of higher plants than tocab genes encoding LHC II of chlorophytes. We propose that LHC I, CP 24 and CP 29 and FCP might have originated from a common ancestral chl binding protein and that the major LHC II of Chla/b-containing organisms arose after the divergence between the chromophytes and the chlorophytes.     

Modification of the catalytic function of the mitochondrial cytochrome b-c1 complex by dicyclohexylcarbodiimide

N,N′-Dicyclohexylcarbodiimide (DCCD) induces a complex set of effects on the succinate-cytochrome c span of the mitochondrial respiratory chain. At concentrations below 1000 mol per mol of cytochrome c₁, DCCD is able to block the proton-translocating activity associated to succinate or ubiquinol oxidation without inhibiting the steady-state redox activity of the b-c₁ complex either in intact mitochondrial particles or in the isolated ubiquinol-cytochrome c reductase reconstituted in phospholipid vesicles. In parallel to this, DCCD modifies the redox responses of the endogenous cytochrome b, which becomes more rapidly reduced by succinate, and more slowly oxidized when previously reduced by substrates. At similar concentrations the inhibitor apparently stimulates the redox activity of the succinate-ubiquinone reductase. Moreover, DCCD, at concentrations about one order of magnitude higher than those blocking proton translocation, produces inactivation of the redox function of the b-c₁ complex. The binding of [¹⁴C]DCCD to the isolated b-c₁ complex has shown that under conditions leading to the inhibition of the proton-translocating activity of the enzyme, a subunit of about 9500 Da, namely Band VIII, is the most heavily labelled polypeptide of the complex. The possible correlations between the various effects of DCCD and its modification of the b-c₁ complex are discussed.