Venom Peptides From Cone Snails: Pharmacological Probes for Voltage-Gated Sodium Channels.

The venoms of cone snails provide a rich source of neuroactive peptides (conotoxins). Several venom peptide families have been identified that are either agonists (ι- and δ-conotoxins) or antagonists (µ- and µO-conotoxins) of voltage-gated sodium channels (VGSCs). Members of these conotoxin classes have been integral in identifying and characterizing specific neurotoxin binding sites on the channel. Furthermore, given the specificity of some of these peptides for one sodium channel subtype over another, conotoxins have also proven useful in exploring differences between VGSC subtypes. This chapter summarizes the current knowledge of the structure and function based on the results of conotoxin interactions with VGSCs and correlates the peptides with the phylogeny of the Conus species from which they were derived.

Mesenteric fat necrosis after recent surgery causing bowel obstruction: a case report and review of the literature.

Mesenteric fat necrosis causing bowel obstruction is a rare occurrence with only one case reported in humans. It is due to accidental or surgical trauma to the adipose tissue with extracellular liberation of fat or enzymatic lysis of fat due to the release of lipases resulting in fibrosis. Preoperative imaging may often be misleading and fail to identify fat necrosis as the cause of bowel obstruction. As surgical intervention is the only suitable treatment option in cases of failed conservative treatment, the diagnosis is made postoperatively. There is no published advice on the management of mesenteric fat necrosis. We recommend safe operating techniques to minimize the risk of developing fat necrosis and its potential harmful consequences.

A family of selfish minicircular chromosomes with jumbled chloroplast gene fragments from a dinoflagellate.

Chloroplast genes of several dinoflagellate species are located on unigenic DNA minicircular chromosomes. We have now completely sequenced five aberrant minicircular chromosomes from the dinoflagellate Heterocapsa triquetra. These probably nonfunctional DNA circles lack complete genes, with each being composed of several short fragments of two or three different chloroplast genes and a common conserved region with a tripartite 9G-9A-9G core like the putative replicon origin of functional single-gene circular chloroplast chromosomes. Their sequences imply that all five circles evolved by differential deletions and duplications from common ancestral circles bearing fragments of four genes: psbA, psbC, 16S rRNA, and 23S rRNA. It appears that recombination between separate unigenic chromosomes initially gave intermediate heterodimers, which were subsequently stabilized by deletions that included part or all of one putative replicon origin. We suggest that homologous recombination at the 9G-9A-9G core regions produced a psbA/psbC heterodimer which generated two distinct chimeric circles by differential deletions and duplications. A 23S/16S rRNA heterodimer more likely formed by illegitimate recombination between 16S and 23S rRNA genes. Homologous recombination between the 9G-9A-9G core regions of both heterodimers and additional differential deletions and duplications could then have yielded the other three circles. Near identity of the gene fragments and 9G-9A-9G cores, despite diverging adjacent regions, may be maintained by gene conversion. The conserved organization of the 9G-9A-9G cores alone favors the idea that they are replicon origins and suggests that they may enable the aberrant minicircles to parasitize the chloroplast's replication machinery as selfish circles.

Phylogeny of ultra-rapidly evolving dinoflagellate chloroplast genes: a possible common origin for sporozoan and dinoflagellate plastids.

Complete chloroplast 23S rRNA and psbA genes from five peridinin-containing dinoflagellates (Heterocapsa pygmaea, Heterocapsa niei, Heterocapsa rotun-data, Amphidinium carterae, and Protoceratium reticulatum) were amplified by PCR and sequenced; partial sequences were obtained from Thoracosphaera heimii and Scrippsiella trochoidea. Comparison with chloroplast 23S rRNA and psbA genes of other organisms shows that dinoflagellate chloroplast genes are the most divergent and rapidly evolving of all. Quartet puzzling, maximum likelihood, maximum parsimony, neighbor joining, and LogDet trees were constructed. Intersite rate variation and invariant sites were allowed for with quartet puzzling and neighbor joining. All psbA and 23S rRNA trees showed peridinin-containing dinoflagellate chloroplasts as monophyletic. In psbA trees they are related to those of chromists and red algae. In 23S rRNA trees, dinoflagellates are always the sisters of Sporozoa (apicomplexans); maximum likelihood analysis of Heterocapsa triquetra 16S rRNA also groups the dinoflagellate and sporozoan sequences, but the other methods were inconsistent. Thus, dinoflagellate chloroplasts may actually be related to sporozoan plastids, but the possibility of reproducible long-branch artifacts cannot be strongly ruled out. The results for all three genes fit the idea that dinoflagellate chloroplasts originated from red algae by a secondary endosymbiosis, possibly the same one as for chromists and Sporozoa. The marked disagreement between 16S rRNA trees using different phylogenetic algorithms indicates that this is a rather poor molecule for elucidating overall chloroplast phylogeny. We discuss possible reasons why both plastid and mitochondrial genomes of alveolates (Dinozoa, Sporozoa and Ciliophora) have ultra-rapid substitution rates and a proneness to unique genomic rearrangements.

Single gene circles in dinoflagellate chloroplast genomes.

Photosynthetic dinoflagellates are important aquatic primary producers and notorious causes of toxic 'red tides'. Typical dinoflagellate chloroplasts differ from all other plastids in having a combination of three envelope membranes and peridinin-chlorophyll a/c light-harvesting pigments. Despite evidence of a dinoflagellete satellite DNA containing chloroplast genes, previous attempts to obtain chloroplast gene sequences have been uniformly unsuccessful. Here we show that the dinoflagellate chloroplast DNA genome structure is unique. Complete sequences of chloroplast ribosomal RNA genes and seven chloroplast protein genes from the dinoflagellate Heterocapsa triquetra reveal that each is located alone on a separate minicircular chromosome: 'one gene-one circle'. The genes are the most divergent known from chloroplast genomes. Each circle has an unusual tripartite non-coding region (putative replicon origin), which is highly conserved among the nine circles through extensive gene conversion, but is very divergent between species. Several other dinoflagellate species have minicircular chloroplast genes, indicating that this type of genomic organization may have evolved in ancestral peridinean dinoflagellates. Phylogenetic analysis indicates that dinoflagellate chloroplasts are related to chromistan and red algal chloroplasts and supports their origin by secondary symbiogenesis.

A phylogenetic assessment of the eukaryotic light-harvesting antenna proteins, with implications for plastid evolution.

The light-harvesting complexes (LHCs) are a superfamily of chlorophyll-binding proteins present in all photosynthetic eukaryotes. The Lhc genes are nuclear-encoded, yet the pigment-protein complexes are localized to the thylakoid membrane and provide a marker to follow the evolutionary paths of plastids with different pigmentation. The LHCs are divided into the chlorophyll a/b-binding proteins of the green algae, euglenoids, and higher plants and the chlorophyll a/c-binding proteins of various algal taxa. This work examines the phylogenetic position of the LHCs from three additional taxa: the rhodophytes, the cryptophytes, and the chlorarachniophytes. Phylogenetic analysis of the LHC sequences provides strong statistical support for the clustering of the rhodophyte and cryptomonad LHC sequences within the chlorophyll a/c-binding protein lineage, which includes the fucoxanthin-chlorophyll proteins (FCP) of the heterokonts and the intrinsic peridinin-chlorophyll proteins (iPCP) of the dinoflagellates. These associations suggest that plastids from the heterokonts, haptophytes, cryptomonads, and the dinoflagellate, Amphidinium, evolved from a red algal-like ancestor. The Chlorarachnion LHC is part of the chlorophyll a/b-binding protein assemblage, consistent with pigmentation, providing further evidence that its plastid evolved from a green algal secondary endosymbiosis. The Chlorarachnion LHC sequences cluster with the green algal LHCs that are predominantly associated with photosystem II (LHCII). This suggests that the green algal endosymbiont that evolved into the Chlorarachnion plastid was acquired following the emergence of distinct LHCI and LHCII complexes.

The 38 kDa chlorophyll a/b protein of the prokaryote Prochlorothrix hollandica is encoded by a divergent pcb gene.

The chlorophyll (Chl) a/b proteins of the photosynthetic prokaryotes appear to have evolved by gene duplication and divergence of the core Chl a antenna family, which also includes CP43 and CP47 and the iron-stress induced Chl a-binding IsiA proteins. We show here that Prochlorothrix hollandica has a cluster of three pcb (prochlorophyte chlorophyll b) genes which are co-transcribed. The major antenna polypeptides of 32 and 38 kDa are encoded by pcbA and pcbC respectively. The pcbC gene is significantly divergent from the other two and may have originated by a gene duplication independent of the one that led to isiA and the other prochlorophyte pcb genes. The distant relatedness of the three prochlorophyte genera implies that not only the ability to make Chl b and use it for light-harvesting arose independently in the three lineages, but also that the pcb genes may have arisen as the result of independent gene duplications in each lineage.

hcf5, a nuclear photosynthetic electron transport mutant of Arabidopsis thaliana with a pleiotropic effect on chloroplast gene expression.

A photosynthetic mutant of Arabidopsis thaliana, hcf5, was isolated by screening M2 seedlings for high chlorophyll fluorescence. Thylakoid morphology was strikingly abnormal, with large grana stacks and almost no stroma lamellae. Fluorescence induction kinetics, activity assays, and immunoblotting showed that photosystem II was absent. Polypeptides of the photosystem I complex, the Cyt b6/f complex, coupling factor, and the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase were also severely depleted. However, the nuclear-encoded chlorophyll a/b light-harvesting complex polypeptides were unaffected. The rbcL transcript was present at very low levels, the pattern of transcripts from the polycistronic psbB-psbH-petB-petD operon was abnormal, and the mature psbH message was almost completely lacking. This suggests that the hcf5 locus may encode a product required for the correct expression of several chloroplast genes.

Independent evolution of the prochlorophyte and green plant chlorophyll a/b light-harvesting proteins.

The prochlorophytes are oxygenic prokaryotes differing from other cyanobacteria by the presence of a light-harvesting system containing both chlorophylls (Chls) a and b and by the absence of phycobilins. We demonstrate here that the Chl a/b binding proteins from all three known prochlorophyte genera are closely related to IsiA, a cyanobacterial Chl a-binding protein induced by iron starvation, and to CP43, a constitutively expressed Chl a antenna protein of photosystem II. The prochlorophyte Chl a/b protein (pcb) genes do not belong to the extended gene family encoding eukaryotic Chl a/b and Chl a/c light-harvesting proteins. Although higher plants and prochlorophytes share common pigment complements, their light-harvesting systems have evolved independently.

The fucoxanthin-chlorophyll proteins from a chromophyte alga are part of a large multigene family: structural and evolutionary relationships to other light harvesting antennae.

A fucoxanthin-chlorophyll protein (FCP) cDNA from the raphidophyte Heterosigma carterae encodes a 210-amino acid polypeptide that has similarity to other FCPs and to the chlorophyll a/b-binding proteins (CABs) of terrestrial plants and green algae. The putative transit sequence has characteristics that resemble a signal sequence. The Heterosigma fcp genes are part of a large multigene family which includes members encoding at least two significantly different polypeptides (Fcp1, Fcp2). Comparison of the FCP sequences to the recently determined three-dimensional structure of the pea LHC II complex indicates that many of the key amino acids thought to participate in the binding of chlorophyll and the formation of complex-stabilizing ionic interactions are well conserved. Phylogenetic analyses of sequences of light-harvesting proteins shows that the FCPs of several chromophyte phyla form a natural group separate from the intrinisic peridinin-chlorophyll proteins (iPCPs) of the dinoflagellates: Although the FCP and CAB genes shared a common ancestor, these lineages diverged from each other prior to the separation of the CAB LHC I and LHC II sequences in the green algae and terrestrial plants.

THE CHLOROPHYLL-CAROTENOID PROTEINS OF OXYGENIC PHOTOSYNTHESIS.

The chlorophyll-carotenoid binding proteins responsible for absorption and conversion of light energy in oxygen-evolving photosynthetic organisms belong to two extended families: the Chl a binding core complexes common to cyanobacteria and all chloroplasts, and the nuclear-encoded light-harvesting antenna complexes of eukaryotic photosynthesizers (Chl a/b, Chl a/c, and Chl a proteins). There is a general consensus on polypeptide and pigment composition for higher plant pigment proteins. These are reviewed and compared with pigment proteins of chlorophyte, rhodophyte, and chromophyte algae. Major advances have been the determination of the structures of LHCII (major Chl a/b complex of higher plants), cyanobacterial Photosystem I, and the peridinen-Chl a protein of dinoflagellates to atomic resolution. Better isolation methods, improved transformation procedures, and the availability of molecular structure models are starting to provide insights into the pathways of energy transfer and the macromolecular organization of thylakoid membranes.

The nuclear-encoded chlorophyll-binding photosystem II-S protein is stable in the absence of pigments.

The 22-kDa chlorophyll a/b-binding protein (CAB) (psbS gene product) is associated with photosystem II and related to the CAB gene family. Here we report that the PSII-S protein unlike other chlorophyll-binding proteins is stable in the absence of pigments. It is present in etiolated spinach plants and accumulates in the dark progressively with the cellular development of the seedlings. Furthermore, it is present in several pigment-deficient mutants. Analysis of the pigment composition of the PSII-S protein isolated from etiolated plants suggests that neither carotenoids nor chlorophyll precursors are involved in its stabilization in the dark. Exposure of etiolated spinach to light leads to further accumulation of the PSII-S protein, which appears more early than other chlorophyll-binding proteins. Accumulation of the PSII-S protein in green plants is developmentally regulated and restricted to photosynthetic tissues. It is suggested that the function of the PSII-S protein may not be light-harvesting but it could act as a ligand chaperone required for transient binding of pigments during biogenesis or turnover of chlorophyll-binding proteins. Such function would be essential for coordination between pigment biosynthesis and ligation as well as avoiding toxic effects of non-bound chlorophyll molecules.

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.

The intrinsic 22 kDa protein is a chlorophyll-binding subunit of photosystem II.

The intrinsic 22 kDa polypeptide associated with photosystem II (psbS protein) was found to be able to bind chlorophyll. Extraction of isolated photosystem II membranes with octyl-thioglucopyranoside, followed by repetitive electrophoresis under partially denaturing conditions gave only one green band. It contained both chlorophyll a and chlorophyll b, exhibited an absorption maximum at 674 nm and a 77 K fluorescence peak at 675 nm. The chlorophyll-protein band contained a single polypeptide of 22 kDa. Based on these results and on previous protein sequence comparisons, it is suggested that the psbS protein is a chlorophyll a/b binding polypeptide and should thus be denoted CP22.

Separation of closely related intrinsic membrane polypeptides of the photosystem II light-harvesting complex (LHC II) by reversed-phase high-performance liquid chromatography on a poly(styrene-divinylbenzene) column.

The three closely related intrinsic membrane polypeptides of the photosystem II light-harvesting complex (LHC II) were successfully resolved on a PRP-1 poly(styrene-divinylbenzene) column using a three-stage linear water-acetonitrile gradient containing 0.1% trifluoroacetic acid. The hydrophobic proteins of photosystem I (PS I-200) and photosystem II core particles were also separated by this method, showing that membrane proteins of different sizes and hydrophobicities can be resolved in this system.

A nuclear photosynthetic electron transport mutant of Arabidopsis thaliana with altered expression of the chloroplast petA gene.

The nuclear photosynthetic mutant, hcf2, of Arabidopsis thaliana was isolated by screening M2 seedlings for abnormally-high chlorophyll fluorescence (hcf), indicative of a block in photosynthetic electron transport. Fluorescence induction kinetics, photosynthetic electron transport activity assays and immunoblotting revealed that all the complexes involved in photosynthetic electron transport were affected to some extent. The most striking effect of the mutation was on the relative steady state levels of the petA transcript (encoding the apoprotein of cytochrome f) which were more than five-times higher in the mutant plants than in their wild-type siblings.