Impaired Epidermal Permeability Barrier in Mice Lacking Elovl1, the Gene Responsible for Very-Long-Chain Fatty Acid Production

The sphingolipid backbone ceramide (Cer) is a major component of lipid lamellae in the stratum corneum of epidermis and has a pivotal role in epidermal barrier formation. Unlike Cers in other tissues, Cers in epidermis contain extremely long fatty acids (FAs). Decreases in epidermal Cer levels, as well as changes in their FA chain lengths, cause several cutaneous disorders. However, the molecular mechanisms that produce such extremely long Cers and determine their chain lengths are poorly understood. We generated mice deficient in the Elovl1 gene, which encodes the FA elongase responsible for producing C₂₀ to C₂₈ FAs. Elovl1 knockout mice died shortly after birth due to epidermal barrier defects. The lipid lamellae in the stratum corneum were largely diminished in these mice. In the epidermis of the Elovl1-null mice, the levels of Cers with ≥C₂₆ FAs were decreased, while those of Cers with ≤C₂₄ FAs were increased. In contrast, the levels of C₂₄ sphingomyelin were reduced, accompanied by an increase in C₂₀ sphingomyelin levels. Two ceramide synthases, CerS2 and CerS3, expressed in an epidermal layer-specific manner, regulate Elovl1 to produce acyl coenzyme As with different chain lengths. Elovl1 is a key determinant of epidermal Cer chain length and is essential for permeability barrier formation.     

On the validation of crystallographic symmetry and the quality of structures

In 2008, Zwart and colleagues observed that the fraction of the structures deposited in the PDB alleged to have “pseudosymmetry” or “special noncrystallographic symmetry” (NCS) was about 6%, and that this percentage was rising annually. A few years later, Poon and colleagues found that 2% of all the crystal structures in the PDB belonged to higher symmetry space groups than those assigned to them. Here, I report an analysis of the X-ray diffraction data deposited for this class of structures, which shows that most of the “pseudosymmetry” and “special NCS” that has been reported is in fact true crystallographic symmetry (CS). This distinction is important because the credibility of crystal structures depends heavily on quality control statistics such as R_free that are unreliable when they are computed incorrectly, which they often are when CS is misidentified as “special NCS” or “pseudosymmetry”. When mistakes of this kind are made, artificially low values of R_free can give unjustified confidence in the accuracy of the reported structures.     

Changes in membrane biophysical properties induced by sphingomyelinase depend on the sphingolipid N-acyl chain

Ceramide (Cer) is involved in the regulation of several cellular processes by mechanisms that depend on Cer-induced changes on membrane biophysical properties. Accumulating evidence shows that Cers with different N-acyl chain composition differentially impact cell physiology, which may in part be due to specific alterations in membrane biophysical properties. We now address how the sphingolipid (SL) N-acyl chain affects membrane properties in cultured human embryonic kidney cells by overexpressing different Cer synthases (CerSs). Our results show an increase in the order of cellular membranes in CerS2-transfected cells caused by the enrichment in very long acyl chain SLs. Formation of Cer upon treatment of cells with bacterial sphingomyelinase promoted sequential changes in the properties of the membranes: after an initial increase in the order of the fluid plasma membrane, reorganization into domains with gel-like properties whose characteristics are dependent on the acyl chain structure of the Cer was observed. Moreover, the extent of alterations of membrane properties correlates with the amount of Cer formed. These data reinforce the significance of Cer-induced changes on membrane biophysical properties as a likely molecular mechanism by which different acyl chain Cers exert their specific biological actions.     

Blood sphingolipidomics in healthy humans: impact of sample collection methodology

We used a HPLC-MS/MS methodology for determination of a basic metabolomic profile (18:1,18:0 sphingoid backbone, C₁₄-C₂₆ N-acyl part) of “normal” sphingolipid levels in human serum and plasma. Blood was collected from healthy males and nonpregnant females under fasting and nonfasting conditions with and without anticoagulants. Sphingolipids analyzed included sphingoid bases, sphingosine and dihydrosphingosine, their 1-phosphates (S1P and dhS1P), molecular species (C_n-) of ceramide (Cer), sphingomyelin (SM), hexosylceramide (HexCer), lactosylceramide (LacCer), and Cer 1-phosphate (Cer1P). SM, LacCer, HexCer, Cer, and Cer1P constituted 87.7, 5.8, 3.4, 2.8, and 0.15% of total sphingolipids, respectively. The abundant circulating SM was C₁₆-SM (64.0 µM), and it increased with fasting (100 µM). The abundant LacCer was C₁₆-LacCer (10.0 µM) and the abundant HexCer was C₂₄-HexCer (2.5 µM). The abundant Cer, C₂₄-Cer (4.0 µM), was not influenced by fasting; however, levels of C₁₆-C₂₀ Cers were decreased in response to fasting. S1P levels were higher in serum than plasma (0.68 µM vs. 0.32 µM). We also determined levels of sphingoid bases and SM species in isolated lipoprotein classes. HDL₃ was the major carrier of S1P, dhS1P, and Sph, and LDL was the major carrier of Cer and dhSph. Per particle, VLDL contained the highest levels of SM, Cer, and S1P. HPLC-MS/MS should provide a tool for clinical testing of circulating bioactive sphingolipids in human blood.     

PDMP Blocks Brefeldin A–induced Retrograde Membrane Transport from Golgi to ER: Evidence for Involvement of Calcium Homeostasis and Dissociation from Sphingolipid Metabolism

In this study, we show that an inhibitor of sphingolipid biosynthesis, d,l-threo-1-phenyl-2- decanoylamino-3-morpholino-1-propanol (PDMP), inhibits brefeldin A (BFA)-induced retrograde membrane transport from Golgi to endoplasmic reticulum (ER). If BFA treatment was combined with or preceded by PDMP administration to cells, disappearance of discrete Golgi structures did not occur. However, when BFA was allowed to exert its effect before PDMP addition, PDMP could not “rescue” the Golgi compartment.Evidence is presented showing that this action of PDMP is indirect, which means that the direct target is not sphingolipid metabolism at the Golgi apparatus. A fluorescent analogue of PDMP, 6-(N-[7-nitro-2,1,3-benzoxadiazol-4-yl]amino)hexanoyl-PDMP (C₆-NBD-PDMP), did not localize in the Golgi apparatus. Moreover, the effect of PDMP on membrane flow did not correlate with impaired C₆-NBD-sphingomyelin biosynthesis and was not mimicked by exogenous C₆-ceramide addition or counteracted by exogenous C₆-glucosylceramide addition. On the other hand, the PDMP effect was mimicked by the multidrug resistance protein inhibitor MK571.The effect of PDMP on membrane transport correlated with modulation of calcium homeostasis, which occurred in a similar concentration range. PDMP released calcium from at least two independent calcium stores and blocked calcium influx induced by either extracellular ATP or thapsigargin. Thus, the biological effects of PDMP revealed a relation between three important physiological processes of multidrug resistance, calcium homeostasis, and membrane flow in the ER/ Golgi system.     

Arginine methylation in subunits of mammalian pre-mRNA cleavage factor I

Mammalian cleavage factor I (CF I_m) is composed of two polypeptides of 25 kDa and either a 59 or 68 kDa subunit (CF I_m25, CF I_m59, CF I_m68). It is part of the cleavage and polyadenylation complex responsible for processing the 3′ ends of messenger RNA precursors. To investigate post-translational modifications in factors of the 3′ processing complex, we systematically searched for enzymes that modify arginines by the addition of methyl groups. Protein arginine methyltransferases (PRMTs) are such enzymes that transfer methyl groups from S-adenosyl methionine to arginine residues within polypeptide chains resulting in mono- or dimethylated arginines. We found that CF I_m68 and the nuclear poly(A) binding protein 1 (PABPN1) were methylated by HeLa cell extracts in vitro. By fractionation of these extracts followed by mass spectral analysis, we could demonstrate that the catalytic subunit PRMT5, together with its cofactor WD45, could symmetrically dimethylate CF I_m68, whereas pICln, the third polypeptide of the complex, was stimulatory. As sites of methylation in CF I_m68 we could exclusively identify arginines in a GGRGRGRF or “GAR” motif that is conserved in vertebrates. Further in vitro assays revealed a second methyltransferase, PRMT1, which modifies CF I_m68 by asymmetric dimethylation of the GAR motif and also weakly methylates the C-termini of both CF I_m59 and CF I_m68. The results suggest that native—as compared with recombinant—protein substrates may contain additional determinants for methylation by specific PRMTs. A possible involvement of CF I_m methylation in the context of RNA export is discussed.     

Beetroot juice reduces infarct size and improves cardiac function following ischemia–reperfusion injury: Possible involvement of endogenous H2S

Ingestion of high dietary nitrate in the form of beetroot juice (BRJ) has been shown to exert antihypertensive effects in humans through increasing cyclic guanosine monophosphate (cGMP) levels. Since enhanced cGMP protects against myocardial ischemia–reperfusion (I/R) injury through upregulation of hydrogen sulfide (H₂S), we tested the hypothesis that BRJ protects against I/R injury via H₂S. Adult male CD-1 mice received either regular drinking water or those dissolved with BRJ powder (10 g/L, containing ∼0.7 mM nitrate). Seven days later, the hearts were explanted for molecular analyses. Subsets of mice were subjected to I/R injury by occlusion of the left coronary artery for 30 min and reperfusion for 24 h. A specific inhibitor of H₂S producing enzyme – cystathionine-γ-lyase (CSE), dl-propargylglycine (PAG, 50 mg/kg) was given i.p. 30 min before ischemia. Myocardial infarct size was significantly reduced in BRJ-fed mice (15.8 ± 3.2%) versus controls (46.5 ± 3.5%, mean ± standard error [SE], n = 6/group, P < .05). PAG completely blocked the infarct-limiting effect of BRJ. Moreover, BRJ significantly preserved ventricular function following I/R. Myocardial levels of H₂S and its putative protein target – vascular endothelial growth factor receptor 2 (VEGFR2) were significantly increased by BRJ intake, whereas CSE mRNA and protein content did not change. Interestingly, the BRJ-induced cardioprotection was not associated with elevated blood nitrate–nitrite levels following I/R nor induction of cardiac peroxiredoxin 5, a mitochondrial antioxidant enzyme previously linked to nitrate-induced cardioprotection. We conclude that BRJ ingestion protects against post-I/R myocardial infarction and ventricular dysfunction possibly through CSE-mediated endogenous H₂S generation. BRJ could be a promising natural and inexpensive nutraceutical supplement to reduce cardiac I/R injury in patients.     

A Proposed Mechanism for the Promotion of Prion Conversion Involving a Strictly Conserved Tyrosine Residue in the β2-α2 Loop of PrPC

The transmission of infectious prions into different host species requires compatible prion protein (PrP) primary structures, and even one heterologous residue at a pivotal position can block prion infection. Mapping the key amino acid positions that govern cross-species prion conversion has not yet been possible, although certain residue positions have been identified as restrictive, including residues in the β₂-α₂ loop region of PrP. To further define how β₂-α₂ residues impact conversion, we investigated residue substitutions in PrP^C using an in vitro prion conversion assay. Within the β₂-α₂ loop, a tyrosine residue at position 169 is strictly conserved among mammals, and transgenic mice expressing mouse PrP having the Y169G, S170N, and N174T substitutions resist prion infection. To better understand the structural requirements of specific residues for conversion initiated by mouse prions, we substituted a diverse array of amino acids at position 169 of PrP. We found that the substitution of glycine, leucine, or glutamine at position 169 reduced conversion by ∼75%. In contrast, replacing tyrosine 169 with either of the bulky, aromatic residues, phenylalanine or tryptophan, supported efficient prion conversion. We propose a model based on a requirement for tightly interdigitating complementary amino acid side chains within specific domains of adjacent PrP molecules, known as “steric zippers,” to explain these results. Collectively, these studies suggest that an aromatic residue at position 169 supports efficient prion conversion.     

Lamellar Thickness and Stretching Temperature Dependency of Cavitation in Semicrystalline Polymers

Polybutene-1 (PB-1), a typical semicrystalline polymer, in its stable form I shows a peculiar temperature dependent strain-whitening behavior when being stretched at temperatures in between room temperature and melting temperature of the crystallites where the extent of strain-whitening weakens with the increasing of stretching temperature reaching a minima value followed by an increase at higher stretching temperatures. Correspondingly, a stronger strain-hardening phenomenon was observed at higher temperatures. The strain-whitening phenomenon in semicrystalline polymers has its origin of cavitation process during stretching. In this work, the effect of crystalline lamellar thickness and stretching temperature on the cavitation process in PB-1 has been investigated by means of combined synchrotron ultrasmall-angle and wide-angle X-ray scattering techniques. Three modes of cavitation during the stretching process can be identified, namely “no cavitation” for the quenched sample with the thinnest lamellae where only shear yielding occurred, “cavitation with reorientation” for the samples stretched at lower temperatures and samples with thicker lamellae, and “cavitation without reorientation” for samples with thinner lamellae stretched at higher temperatures. The mode “cavitation with reorientation” occurs before yield point where the plate-like cavities start to be generated within the lamellar stacks with normal perpendicular to the stretching direction due to the blocky substructure of the crystalline lamellae and reorient gradually to the stretching direction after strain-hardening. The mode of “cavitation without reorientation” appears after yield point where ellipsoidal shaped cavities are generated in those lamellae stacks with normal parallel to the stretching direction followed by an improvement of their orientation at larger strains. X-ray diffraction results reveal a much improved crystalline orientation for samples with thinner lamellae stretched at higher temperatures. The observed behavior of microscopic structural evolution in PB-1 stretched at different temperatures explains above mentioned changes in macroscopic strain-whitening phenomenon with increasing in stretching temperature and stress-strain curves.     

Regio- and Stereospecificity of Filipin Hydroxylation Sites Revealed by Crystal Structures of Cytochrome P450 105P1 and 105D6 from Streptomyces avermitilis

The polyene macrolide antibiotic filipin is widely used as a probe for cholesterol and a diagnostic tool for type C Niemann-Pick disease. Two position-specific P450 enzymes are involved in the post-polyketide modification of filipin during its biosynthesis, thereby providing molecular diversity to the “filipin complex.” CYP105P1 and CYP105D6 from Streptomyces avermitilis, despite their high sequence similarities, catalyze filipin hydroxylation at different positions, C26 and C1′, respectively. Here, we determined the crystal structure of the CYP105P1-filipin I complex. The distal pocket of CYP105P1 has the second largest size among P450 hydroxylases that act on macrolide substrates. Compared with previously determined substrate-free structures, the FG helices showed significant closing motion on substrate binding. The long BC loop region adopts a unique extended conformation without a B′ helix. The binding site is essentially hydrophobic, but numerous water molecules are involved in recognizing the polyol side of the substrate. Therefore, the distal pocket of CYP105P1 provides a specific environment for the large filipin substrate to bind with its pro-S side of position C26 directed toward the heme iron. The ligand-free CYP105D6 structure was also determined. A small sub-pocket accommodating the long alkyl side chain of filipin I was observed in the CYP105P1 structure but was absent in the CYP105D6 structure, indicating that filipin cannot bind to CYP105D6 with a similar orientation due to steric hindrance. This observation can explain the strict regiospecificity of these enzymes.     

Procoagulant Platelets Form an α-Granule Protein-covered “Cap” on Their Surface That Promotes Their Attachment to Aggregates

Strongly activated “coated” platelets are characterized by increased phosphatidylserine (PS) surface expression, α-granule protein retention, and lack of active integrin α_IIbβ₃. To study how they are incorporated into thrombi despite a lack of free activated integrin, we investigated the structure, function, and formation of the α-granule protein “coat.” Confocal microscopy revealed that fibrin(ogen) and thrombospondin colocalized as “cap,” a single patch on the PS-positive platelet surface. In aggregates, the cap was located at the point of attachment of the PS-positive platelets. Without fibrin(ogen) retention, their ability to be incorporated in aggregates was drastically reduced. The surface fibrin(ogen) was strongly decreased in the presence of a fibrin polymerization inhibitor GPRP and also in platelets from a patient with dysfibrinogenemia and a fibrinogen polymerization defect. In contrast, a fibrinogen-clotting protease ancistron increased the amount of fibrin(ogen) and thrombospondin on the surface of the PS-positive platelets stimulated with collagen-related peptide. Transglutaminases are also involved in fibrin(ogen) retention. However, platelets from patients with factor XIII deficiency had normal retention, and a pan-transglutaminase inhibitor T101 had only a modest inhibitory effect. Fibrin(ogen) retention was normal in Bernard-Soulier syndrome and kindlin-3 deficiency, but not in Glanzmann thrombasthenia lacking the platelet pool of fibrinogen and α_IIbβ₃. These data show that the fibrin(ogen)-covered cap, predominantly formed as a result of fibrin polymerization, is a critical mechanism that allows coated (or rather “capped”) platelets to become incorporated into thrombi despite their lack of active integrins.     

The Structure of a Full-length Membrane-embedded Integrin Bound to a Physiological Ligand

Increased ligand binding to integrin (“activation”) underpins many biological processes, such as leukocyte trafficking, cell migration, host-pathogen interaction, and hemostasis. Integrins exist in several conformations, ranging from compact and bent to extended and open. However, the exact conformation of membrane-embedded, full-length integrin bound to its physiological macromolecular ligand is still unclear. Integrin α_IIbβ₃, the most abundant integrin in platelets, has been a prototype for integrin activation studies. Using negative stain electron microscopy and nanodisc-embedding to provide a membrane-like environment, we visualized the conformation of full-length α_IIbβ₃ in both a Mn²⁺-activated, ligand-free state and a Mn²⁺-activated, fibrin-bound state. Activated but ligand-free integrins exist mainly in the compact conformation, whereas fibrin-bound α_IIbβ₃ predominantly exists in a fully extended, headpiece open conformation. Our results show that membrane-embedded, full-length integrin adopts an extended and open conformation when bound to its physiological macromolecular ligand.     

New distinct compartments in the G2 phase of the cell cycle defined by the levels of γH2AX.

Induction of DNA double strand breaks leads to phosphorylation and focus-formation of H2AX. However, foci of phosphorylated H2AX (γH2AX) appear during DNA replication also in the absence of exogenously applied injury. We measured the amount and the number of foci of γH2AX in different phases of the cell cycle by flow cytometry, sorting and microscopy in 4 malignant B-lymphocyte cell lines. There were no detectable γH2AX and no γH2AX-foci in G₁ cells in exponentially growing cells and cells treated with PARP inhibitor (PARPi) for 24 h to create damage and reduce DNA repair. The amount of γH2AX increased immediately upon S phase entry, and about 10 and 30 γH2AX foci were found in mid-S phase control and PARPi-treated cells, respectively. The γH2AX-labeled damage caused by DNA replication was not fully repaired before entry into G₂. Intriguingly, G₂ cells populated a continuous distribution of γH2AX levels, from cells with a high content of γH2AX and the same number of foci as S phase cells (termed “G₂H” compartment), to cells that there were almost negative and had about 2 foci (termed “G₂L” compartment). EdU-labeling of S phase cells revealed that G₂H was directly populated from S phase, while G₂L was populated from G₂H, but in control cells also directly from S phase. The length of G₂H in particular increased after PARPi treatment, compatible with longer DNA-repair times. Our results show that cells repair replication-induced damage in G₂H, and enter mitosis after a 2–3 h delay in G₂L.     

Integrity and barrier function of the epidermis critically depend on glucosylceramide synthesis

Ceramides are vital components of the water barrier in mammalian skin. Epidermis-specific, a major ceramide portion contains omega-hydroxy very long chain fatty acids (C30-C36). These omega-hydroxy ceramides (Cers) are found in the extracellular lamellae of the stratum corneum either as linoleic acyl esters or protein bound. Glucosylceramide is the major glycosphingolipid of the epidermis. Synthesized from ceramide and UDP-glucose, it is thought to be itself an intracellular precursor and carrier for extracellular omega-hydroxy ceramides. To investigate whether GlcCer is an obligatory intermediate in ceramide metabolism to maintain epidermal barrier function, a mouse with an epidermis-specific glucosylceramide synthase (Ugcg) deficiency has been generated. Four days after birth animals devoid of GlcCer synthesis in keratinocytes showed a pronounced desquamation of the stratum corneum and extreme transepidermal water loss leading to death. The stratum corneum appeared as a thick unstructured mass. Lamellar bodies of the stratum granulosum did not display the usual ordered inner structure and were often irregularly arranged. Although the total amount of epidermal protein-bound ceramides remained unchanged, epidermal-free omega-hydroxy ceramides increased 4-fold and omega-hydroxy sphingomyelins, almost not detectable in wild type epidermis, emerged in quantities comparable with lost GlcCer. We conclude that the transient formation of GlcCer is vital for a regular arrangement of lipids and proteins in lamellar bodies and for the maintenance of the epidermal barrier.     

Structure and mechanism of E. coli RNA 2′,3′-cyclic phosphodiesterase

2H (two-histidine) phosphoesterase enzymes are distributed widely in all domains of life and are implicated in diverse RNA and nucleotide transactions, including the transesterification and hydrolysis of cyclic phosphates. Here we report a biochemical and structural characterization of the Escherichia coli 2H protein YapD, which was identified originally as a reversible transesterifying “nuclease/ligase” at RNA 2′,5′-phosphodiesters. We find that YapD is an “end healing” cyclic phosphodiesterase (CPDase) enzyme that hydrolyzes an _HORNA>p substrate with a 2′,3′-cyclic phosphodiester to a _HORNAp product with a 2′-phosphomonoester terminus, without concomitant end joining. Thus we rename this enzyme ThpR (two-histidine 2′,3′-cyclic phosphodiesterase acting on RNA). The 2.0 Å crystal structure of ThpR in a product complex with 2′-AMP highlights the roles of extended histidine-containing motifs ⁴³HxTxxF⁴⁸ and ¹²⁵HxTxxR¹³⁰ in the CPDase reaction. His43-Nε makes a hydrogen bond with the ribose O3′ leaving group, thereby implicating His43 as a general acid catalyst. His125-Nε coordinates the O1P oxygen of the AMP 2′-phosphate (inferred from geometry to derive from the attacking water nucleophile), pointing to His125 as a general base catalyst. Arg130 makes bidentate contact with the AMP 2′-phosphate, suggesting a role in transition-state stabilization. Consistent with these inferences, changing His43, His125, or Arg130 to alanine effaced the CPDase activity of ThpR. Phe48 makes a π–π stack on the adenine nucleobase. Mutating Phe28 to alanine slowed the CPDase by an order of magnitude. The tertiary structure and extended active site motifs of ThpR are conserved in a subfamily of bacterial and archaeal 2H enzymes.     

The role of sphingolipid metabolism in cutaneous permeabilitybarrier formation

The epidermal permeability barrier of mammalian skin is localized in the stratum corneum. Corneocytes are embedded in an extracellular, highly ordered lipid matrix of hydrophobic lipids consisting of about 50% ceramides, 25% cholesterol and 15% long and very long chain fatty acids. The most important lipids for the epidermal barrier are ceramides. The scaffold of the lipid matrix is built of acylceramides, containing ω-hydroxylated very long chain fatty acids, acylated at the ω-position with linoleic acid. After glucosylation of the acylceramides at Golgi membranes and secretion, the linoleic acid residues are replaced by glutamate residues originating from proteins exposed on the surface of corneocytes. Removal of their glucosyl residues generates a hydrophobic surface on the corneocytes used as a template for the formation of extracellular lipid layers of the water permeability barrier. Misregulation or defects in the formation of extracellular ceramide structures disturb barrier function. Important anabolic steps are the synthesis of ultra long chain fatty acids, their ω-hydroxylation, and formation of ultra long chain ceramides and glucosylceramides. The main probarrier precursor lipids, glucosylceramides and sphingomyelins, are packed in lamellar bodies together with hydrolytic enzymes such as glucosylceramide-β-glucosidase and acid sphingomyelinase and secreted into the intercelullar space between the stratum corneum and stratum granulosum. Inherited defects in the extracellular hydrolytic processing of the probarrier acylglucosylceramides impair epidermal barrier formation and cause fatal diseases: such as prosaposin deficiency resulting in lack of lysosomal lipid binding and transfer proteins, or the symptomatic clinical picture of the “collodion baby” in the absence of glucocerebrosidase. This article is part of a Special Issue entitled The Important Role of Lipids in the Epidermis and their Role in the Formation and Maintenance of the Cutaneous Barrier. Guest Editors: Kenneth R. Feingold and Peter Elias.     

Subtle Population Genetic Structure in Yelloweye Rockfish (Sebastes ruberrimus) Is Consistent with a Major Oceanographic Division in British Columbia,Canada

The boundaries between oceanographic domains often function as dispersal barriers for many temperate marine species with a dispersive pelagic larval phase. Yelloweye rockfish (Sebastes ruberrimus, YR) are widely distributed across the northeastern Pacific Ocean, inhabiting coastal rocky reefs from the Aleutian Islands in Alaska through southern California. This species exhibits an extended pelagic larval duration and has the capacity for long distance larval transport. We assayed 2,862 YR individuals from 13 general areas in the northeast Pacific Ocean for allelic variation at nine microsatellite loci. Bayesian model-based clustering analyses grouped individuals from the Strait of Georgia (SG) into a distinct genetic cluster, while individuals from outer coastal water locations (OCLs) were partitioned equally across two genetic clusters, including the cluster associated with the SG fish. Pairwise F_ST values were consistently an order of magnitude higher for comparisons between the SG and OCLs than they were for all OCL-OCL comparisons (∼0.016 vs. ∼0.001). This same pattern was observed across two time points when individuals were binned into an “old” and “young” group according to birth year (old: ∼0.020 vs. 0.0003; young: ∼0.020 vs. ∼0.004). Additionally, mean allelic richness was markedly lower within the SG compared to the OCLs (8.00 vs. 10.54–11.77). These results indicate that the Strait of Georgia “deep-basin” estuary oceanographic domain acts as a dispersal barrier from the outer coastal waters via the Juan de Fuca Strait. Alternatively, selection against maladapted dispersers across this oceanographic transition may underlie the observed genetic differentiation between the Georgia basin and the outer coastal waters, and further work is needed to confirm the SG-OCL divide acts as a barrier to larval dispersal.     

The Nature of the Intramolecular Charge Transfer State in Peridinin

Experimental and theoretical evidence is presented that supports the theory that the intramolecular charge transfer (ICT) state of peridinin is an evolved state formed via excited-state bond-order reversal and solvent reorganization in polar media. The ICT state evolves in <100 fs and is characterized by a large dipole moment (∼35 D). The charge transfer character involves a shift of electron density within the polyene chain, and it does not involve participation of molecular orbitals localized in either of the β-rings. Charge is moved from the allenic side of the polyene into the furanic ring region and is accompanied by bond-order reversal in the central portion of the polyene chain. The electronic properties of the ICT state are generated via mixing of the “1¹B_u⁺” ionic state and the lowest-lying “2¹A_g^–” covalent state. The resulting ICT state is primarily ¹B_u⁺-like in character and exhibits not only a large oscillator strength but an unusually large doubly excited character. In most solvents, two populations exist in equilibrium, one with a lowest-lying ICT ionic state and a second with a lowest-lying “2¹A_g^–” covalent state. The two populations are separated by a small barrier associated with solvent relaxation and cavity formation.