α<Subscript>2A</Subscript>- and α<Subscript>2C</Subscript>-Adrenoceptors as Potential Targets for Dopamine and Dopamine Receptor Ligands

The poor norepinephrine innervation and high density of Gi/o-coupled α_2A- and α_2C-adrenoceptors in the striatum and the dense striatal dopamine innervation have prompted the possibility that dopamine could be an effective adrenoceptor ligand. Nevertheless, the reported adrenoceptor agonistic properties of dopamine are still inconclusive. In this study, we analyzed the binding of norepinephrine, dopamine, and several compounds reported as selective dopamine D₂-like receptor ligands, such as the D₃ receptor agonist 7-OH-PIPAT and the D₄ receptor agonist RO-105824, to α₂-adrenoceptors in cortical and striatal tissue, which express α_2A-adrenoceptors and both α_2A- and α_2C-adrenoceptors, respectively. The affinity of dopamine for α₂-adrenoceptors was found to be similar to that for D₁-like and D₂-like receptors. Moreover, the exogenous dopamine receptor ligands also showed high affinity for α_2A- and α_2C-adrenoceptors. Their ability to activate Gi/o proteins through α_2A- and α_2C-adrenoceptors was also analyzed in transfected cells with bioluminescent resonance energy transfer techniques. The relative ligand potencies and efficacies were dependent on the Gi/o protein subtype. Furthermore, dopamine binding to α₂-adrenoceptors was functional, inducing changes in dynamic mass redistribution, adenylyl cyclase activity, and ERK1/2 phosphorylation. Binding events were further studied with computer modeling of ligand docking. Docking of dopamine at α_2A- and α_2C-adrenoceptors was nearly identical to its binding to the crystallized D₃ receptor. Therefore, we provide conclusive evidence that α_2A- and α_2C-adrenoceptors are functional receptors for norepinephrine, dopamine, and other previously assumed selective D₂-like receptor ligands, which calls for revisiting previous studies with those ligands.     

Cooperation of adenosine and prostaglandin E<Subscript>2</Subscript> (PGE<Subscript>2</Subscript>) in amplification of cAMP–PKA signaling and immunosuppression

INTRODUCTION: We hypothesize that adenosine and PGE(2) could have a complementary immunosuppressive effect that is mediated via common cAMP-PKA signaling. MATERIALS AND METHODS: To test this hypothesis, the effect of adenosine and PGE(2) on the cytotoxic activity and cytokine production of lymphokine activated killer (LAK) cells was investigated. RESULTS: PGE(2) and adenosine inhibited LAK cells cytotoxic activity and production of INF-gamma, GM-CSF and TNF-alpha. In combination they showed substantially higher inhibition than each modality used alone. Using agonists and antagonists specific for PGE(2) and adenosine receptors we found that cooperation of PGE(2) and adenosine in their inhibitory effects are mediated via EP(2) and A(2A) receptors, respectively. LAK cells have 35-fold higher expression of EP(2) than A(2A). Combined PGE(2) and adenosine treatment resulted in augmentation of cAMP production, PKA activity, CREB phosphorylation and inhibition of Akt phosphorylation. Wortmannin and LY294002 enhanced the suppressive effects of adenosine and PGE(2). In contrast, Rp-8-Br-cAMPS, an inhibitor of PKA type I blocked their immunosuppressive effects, suggesting that the inhibitory effects of PGE(2) and adenosine are mediated via common pathway with activation of cAMP-PKA and inhibition of Akt. CONCLUSION: In comparison to other immunosuppressive molecules (TGF-beta and IL-10), adenosine and PGE(2) are unique in their ability to inhibit the executive function of highly cytotoxic cells. High intratumor levels of adenosine and PGE(2) could protect tumor from immune-mediated destruction by inactivation of the tumor infiltrating functionally active immune cells.     

β<Subscript>2</Subscript>-Integrin-Mediated Adhesion and Intracellular Ca<Superscript>2+</Superscript> Release in Human Eosinophils

Human eosinophils spontaneously adhere to various substrates in the absence of exogenously added activators. In the present study a method was developed for characterizing eosinophil adhesion by measuring changes in impedance. Impedance measurements were performed in HCO₃-buffered HybriCare medium maintained in a humidified 5% CO₂ incubator at 37°C. Impedance increased by more than 1 kΩ within minutes after eosinophils made contact with the substrate, reaching a peak within 20 min. Blocking mobilization of intracellular [Ca²⁺] that precedes adhesion with BAPTA-AM (10 μM) completely inhibited the rise in impedance as well as the changes in cell shape typically observed in adherent cells. However, lowering the extracellular [Ca²⁺] with 2.5 mM EGTA did not inhibit the increase in impedance. Pretreatment with anti-CD18 antibody to block substrate interactions with β₂-integrins, or jasplakinolide (2 μM) to block actin reorganization, abolished the increase in impedance and adherent morphology of the cells. Exposure of eosinophils to the phosphatidylinositol 3 kinase inhibitor LY294002 (5 μM) or treatment with protein kinase C zeta pseudosubstrate to competitively inhibit activity of the enzyme significantly reduced the increase in impedance and inhibited the cell spreading associated with adhesion. These results demonstrate a novel method for measuring eosinophil adhesion and showed that, following formation of a tethered attachment, a rapid increase in intracellular [Ca²⁺] precedes the cytoskeletal rearrangements required for cell shape changes and plasma membrane-substrate interactions associated with adhesion.     

Spectroscopic Behavior and Biological Activity of K<Subscript>2</Subscript>[VO(O<Subscript>2</Subscript>)NTA]·2H<Subscript>2</Subscript>O

The dihydrated potassium salt of the complex anion [VO(O₂)NTA]²⁻ (NTA = nitrilotriacetate anion, [N(CH₂-COO)₃]³⁻) was thoroughly characterized by electronic and vibrational (infrared and Raman) spectroscopies. The bioactivity of the complex on the cell proliferation was tested on three cell lines in culture (UMR106 rat osteosarcoma-derived cells, Caco-2 derived from a human colon adenocarcinoma, and RAW 264.7, a macrophage murine cell line).     

Pen2 and Presenilin-1 Modulate the Dynamic Equilibrium of Presenilin-1 and Presenilin-2 ��-Secretase Complexes

γ-Secretase is known to play a pivotal role in the pathogenesis of Alzheimer disease through production of amyloidogenic Aβ42 peptides. Early onset familial Alzheimer disease mutations in presenilin (PS), the catalytic core of γ-secretase, invariably increase the Aβ42:Aβ40 ratio. However, the mechanism by which these mutations affect γ-secretase complex formation and cleavage specificity is poorly understood. We show that our in vitro assay system recapitulates the effect of PS1 mutations on the Aβ42:Aβ40 ratio observed in cell and animal models. We have developed a series of small molecule affinity probes that allow us to characterize active γ-secretase complexes. Furthermore we reveal that the equilibrium of PS1- and PS2-containing active complexes is dynamic and altered by overexpression of Pen2 or PS1 mutants and that formation of PS2 complexes is positively correlated with increased Aβ42:Aβ40 ratios. These data suggest that perturbations to γ-secretase complex equilibrium can have a profound effect on enzyme activity and that increased PS2 complexes along with mutated PS1 complexes contribute to an increased Aβ42:Aβ40 ratio.β-Amyloid (Aβ)⁵ peptides are believed to play a causative role in Alzheimer disease (AD). Aβ peptides are generated from the processing of the amyloid precursor protein (APP) by two proteases, β-secretase and γ-secretase. Although γ-secretase generates heterogenous Aβ peptides ranging from 37 to 46 amino acids in length, significant work has focused mainly on the Aβ40 and Aβ42 peptides that are the major constituents of amyloid plaques. γ-Secretase is a multisubunit membrane aspartyl protease comprised of at least four known subunits: presenilin (PS), nicastrin (Nct), anterior pharynx-defective (Aph), and presenilin enhancer 2 (Pen2). Presenilin is thought to contain the catalytic core of the complex (1–4), whereas Aph and Nct play critical roles in the assembly, trafficking, and stability of γ-secretase as well as substrate recognition (5, 6). Lastly Pen2 facilitates the endoproteolysis of PS into its N-terminal (NTF) and C-terminal (CTF) fragments thereby yielding a catalytically competent enzyme (5, 7–10). All four proteins (PS, Nct, Aph1, and Pen2) are obligatory for γ-secretase activity in cell and animal models (11, 12). There are two homologs of PS, PS1 and PS2, and three isoforms of Aph1, Aph1aS, Aph1aL, and Aph1b. At least six active γ-secretase complexes have been reported (two presenilins × three Aph1s) (13, 14). The sum of apparent molecular masses of the four proteins (PS1-NTF/CTF ≈ 53 kDa, Nct ≈ 120 kDa, Aph1 ≈ 30 kDa, and Pen2 ≈ 10kDa) is ∼200 kDa. However, active γ-secretase complexes of varying sizes, ranging from 250 to 2000 kDa, have been reported (15–19). Recently a study suggested that the γ-secretase complex contains only one of each subunit (20). Collectively these studies suggest that a four-protein complex around 200–250 kDa may be the minimal functional γ-secretase unit with additional cofactors and/or varying stoichiometry of subunits existing in the high molecular weight γ-secretase complexes. CD147 and TMP21 have been found to be associated with the γ-secretase complex (21, 22); however, their role in the regulation of γ-secretase has been controversial (23, 24).Mutations of PS1 or PS2 are associated with familial early onset AD (FAD), although it is debatable whether these familial PS mutations act as “gain or loss of function” alterations in regard to γ-secretase activity (25–27). Regardless the overall outcome of these mutations is an increased ratio of Aβ42:Aβ40. Clearly these mutations differentially affect γ-secretase activity for the production of Aβ40 and Aβ42. Despite intensive studies of Aβ peptides and γ-secretase, the molecular mechanism controlling the specificity of γ-secretase activity for Aβ40 and Aβ42 production has not been resolved. It has been found that PS1 mutations affect the formation of γ-secretase complexes (28). However, the precise mechanism by which individual subunits alter the dynamics of γ-secretase complex formation and activity is largely unresolved. A better mechanistic understanding of γ-secretase activity associated with FAD mutations has been hindered by the lack of suitable assays and probes that are necessary to recapitulate the effect of these mutations seen in cell models and to characterize the active γ-secretase complex.In our present studies, we have determined the overall effect of Pen2 and PS1 expression on the dynamics of PS1- and PS2-containing complexes and their association with γ-secretase activity. Using newly developed biotinylated small molecular probes and activity assays, we revealed that expression of Pen2 or PS1 FAD mutants markedly shifts the equilibrium of PS1-containing active complexes to that of PS2-containing complexes and results in an overall increase in the Aβ42:Aβ40 ratio in both stable cell lines and animal models. Our studies indicate that perturbations to the equilibrium of active γ-secretase complexes by an individual subunit can greatly affect the activity of the enzyme. Moreover they serve as further evidence that there are multiple and distinct γ-secretase complexes that can exist within the same cells and that their equilibrium is dynamic. Additionally the affinity probes developed here will facilitate further study of the expression and composition of endogenous active γ-secretase from a variety of model systems.     

Repeated Stress Down-Regulates β<Subscript>2</Subscript>- and α<Subscript>2C</Subscript>-Adrenergic Receptors and Up-Regulates Gene Expression of IL-6 in the Rat Spleen

Catecholamines are among first compounds released during stress, and they regulate many functions of the organism, including immune system, via adrenergic receptors (ARs). Spleen, as an immune organ with high number of macrophages, possesses various ARs, from which β₂-ARs are considered to be the most important for the modulation of immune functions. Nevertheless, little is known about the regulation and involvement of ARs in the splenic function by stress. Therefore, the aim of this work was to measure the gene expression of ARs and several cytokines in the spleen of rats exposed to a single and repeated (14×) immobilization stress (IMO). We have found a significant increase in β₂-AR mRNA after a single IMO, but a significant decrease in β₂-AR mRNA and protein level after repeated (14×) IMO. The most prominent decrease was detected in the gene expression of the α_2A- and α_2C-AR after repeated IMO. However, changes in mRNA were translated into protein levels only for the α_2C-subtype. Other types of ARs remained unchanged during the stress situation. Since we proposed that these ARs might affect production of cytokines, we measured gene expression of pro-inflammatory (TNF-α, IL-1β, IL-6 and IL-18) and anti-inflammatory (IL-10 and TGF-β1) cytokines. We detected changes only in IL-6 and IL-10 mRNA levels. While IL-6 mRNA was increased, IL-10 mRNA dropped after repeated IMO. According to these results we suggest that changes of β₂- and α_2C-ARs participate in IL-6-mediated processes in the spleen, especially during chronic stress situations.     

Cyclooxygenase-2 169C > G and 8473T > C gene polymorphisms and prostaglandin E2 level in breast cancer: A case–control study

Aims

Cyclooxygenase 2 (COX-2) with the resulting prostaglandin E2 (PGE2) is linked to increased risk of human breast cancer (BC). The aim of this study was to determine COX-2 169C > G and 8473T > C gene polymorphisms and PGE2 level at various stages of BC clarifying the role of COX-2 gene polymorphism and PGE2 in relation to BC.

Methods

The study population comprised 160 women at different stages of BC and 150 gender- and age-matched healthy control subjects. Plasma PGE2 was measured by ELISA, the COX-2 gene polymorphisms were determined using PCR-RFLP.

Results

The variant alleles COX-2 169G and 8473C were significantly associated with BC susceptibility [OR = 3.1, 95% CI (2.2–4.4), P < 0.001 for 169C>G and OR = 1.74, 95%CI (1.3–2.4), P = 0.005 for 8473C]. However, both COX-2 gene polymorphisms were not associated with breast cancer stage. Plasma PGE2 levels were significantly increased in patients compared to the controls. In early and late stages of BC, there was a significant increase in the plasma PGE2 levels towards the presence of homozygous GG compared with homozygous CC (P < 0.001) for 169 C>G, also towards the presence of CC than TT (P < 0.001) for 8473T>C SNP.

Conclusion

The 169C>G and 8473T>C polymorphisms of the COX-2 gene were associated with the BC in Egyptian women. Furthermore, individuals with COX-2 169GG and 8473CC genotypes showed significant increase in plasma PGE2 levels. PGE2 levels may serve as a predictor of poor prognosis in patients with BC.     

DFT conformation and energies of amylose fragments at atomic resolution. Part 2: ‘band-flip’ and ‘kink’ forms of α-maltotetraose

In Part 2 of this series of DFT optimization studies of α-maltotetraose, we present results at the B3LYP/6-311++G∗∗ level of theory for conformations denoted ‘band-flips’ and ‘kinks’. Recent experimental X-ray studies have found examples of amylose fragments with conformations distorted from the usual syn forms, and it was of interest to examine these novel structural motifs by the same high-level DFT methods used in Part 1. As in Part 1, we have examined numerous hydroxymethyl rotamers (gg, gt, and tg) at different locations in the residue sequence, and include the two hydroxyl rotamers, the clockwise ‘c’ and counterclockwise ‘r’ forms. A total of fifty conformations were calculated and energy differences were found to attempt to identify those sources of electronic energy that dictate stressed amylose conformations. Most stressed conformations were found to have relative energies considerably greater (i.e., ∼4 to 12 kcal/mol) than the lowest energy syn forms. Relative energy differences between ‘c’ and ‘r’ forms are somewhat mixed with some stressed conformations being ‘c’ favored and some ‘r’ favored, with the lowest energy ‘kink’ form being an all-gg-r conformation with the ‘kink’ in the bc glycosidic dihedral angles. Comparison of our calculated structures with experimental results shows very close correspondence in dihedral angles.     

Effects of Prostaglandins F<Subscript>2α</Subscript>, E<Subscript>2</Subscript> and E<Subscript>1</Subscript> on Fertility in Mice

PROSTAGLANDIN (PG) F_2αhas antifertility effects in many species^1–3 but there are conflicting suggestions as to its mechanism of action. For example, it may cause the degeneration of the corpus luteum by decreasing blood flow in the uteroovarian vein⁴; alternatively, its action may be due to a hypersecretion of luteinizing hormone (LH) by the pituitary^3,5. I have investigated the effects of PGF_2α, E₂ and E₁ on pregnancy in mice and examined the mechanism of action of PGF_2α.     

Different effects of SNP and GSNO on mitochondrial O<Stack><Subscript>2</Subscript><Superscript>.−</Superscript></Stack>/H<Subscript>2</Subscript>O<Subscript>2</Subscript> production

Sodium Nitroprusside (SNP) and S-Nitrosoglutathione (GSNO) differently affect mitochondrial H₂O₂ release at Complex-I. mM SNP increases while GSNO decreases the release induced by succinate alone or added on top of NAD-linked substrates. Stimulation likely depends on Nitric Oxide ( ^. NO) (released by SNP but not by GSNO) inhibiting cytochrome oxidase and mitochondrial respiration. Preincubations with SNP or high GSNO (10 mM plus DTE to increases its ^. NO release) induces an inhibition of the succinate dependent H₂O₂ production consistent with a ^. NO dependent covalent modification. However maximal inhibition of the succinate dependent H₂O₂ release is obtained in the presence of low GSNO (20–100 μM), but not with SNP. This inhibition appears independent of ^. NO release since μM GSNO does not affect mitochondrial respiration, or the H₂O₂ detection systems and its effect is very rapid. Inhibition may be partly due to an increased removal of O₂^.− since GSNO chemically competes with NBT and cytochrome C in O₂^.− detection.     

Interactions between oestrogen and 1α,25(OH)<Subscript>2</Subscript>-vitamin D<Subscript>3</Subscript> signalling and their roles in spermatogenesis and spermatozoa functions

Oestrogens and 1α,25(OH)₂-vitamin D₃ (1,25-D₃) are steroids that can provide effects by binding to their receptors localised in the cytoplasm and in the nucleus or the plasma membrane respectively inducing genomic and non-genomic effects. As confirmed notably by invalidation of the genes, coding for their receptors as tested with mice with in vivo and in vitro treatments, oestrogens and 1,25-D₃ are regulators of spermatogenesis. Moreover, some functions of ejaculated spermatozoa as viability, DNA integrity, motility, capacitation, acrosome reaction and fertilizing ability are targets for these hormones. The studies conducted on their mechanisms of action, even though not completely elicited, have allowed the demonstration of putative interactions between their signalling pathways that are worth examining more closely. The present review focuses on the elements regulated by oestrogens and 1,25-D₃ in the testis and spermatozoa as well as the interactions between the signalling pathways of both hormones.     

<Superscript>2</Superscript>H–<Superscript>13</Superscript>C correlation solid-state NMR for investigating dynamics and water accessibilities of proteins and carbohydrates

Site-specific determination of molecular motion and water accessibility by indirect detection of ²H NMR spectra has advantages over dipolar-coupling based techniques due to the large quadrupolar couplings and the ensuing high angular resolution. Recently, a Rotor Echo Short Pulse IRrAdiaTION mediated cross polarization (^RESPIRATIONCP) technique was developed, which allowed efficient transfer of ²H magnetization to ¹³C at moderate ²H radiofrequency field strengths available on most commercial MAS probes. In this work, we investigate the ²H–¹³C magnetization transfer characteristics of one-bond perdeuterated CD _n spin systems and two-bond H/D exchanged C–(O)–D and C–(N)–D spin systems in carbohydrates and proteins. Our results show that multi-bond, broadband ²H–¹³C polarization transfer can be achieved using ²H radiofrequency fields of ~50 kHz, relatively short contact times of 1.3–1.7 ms, and with sufficiently high sensitivity to enable 2D ²H–¹³C correlation experiments with undistorted ²H spectra in the indirect dimension. To demonstrate the utility of this ²H–¹³C technique for studying molecular motion, we show ²H–¹³C correlation spectra of perdeuterated bacterial cellulose, whose surface glucan chains exhibit a motionally averaged C6 ²H quadrupolar coupling that indicates fast trans-gauche isomerization about the C5–C6 bond. In comparison, the interior chains in the microfibril core are fully immobilized. Application of the ²H–¹³C correlation experiment to H/D exchanged Arabidopsis primary cell walls show that the O–D quadrupolar spectra of the highest polysaccharide peaks can be fit to a two-component model, in which 74% of the spectral intensity, assigned to cellulose, has a near-rigid-limit coupling, while 26% of the intensity, assigned to matrix polysaccharides, has a weakened coupling of 50 kHz. The latter O–D quadrupolar order parameter of 0.22 is significantly smaller than previously reported C–D dipolar order parameters of 0.46–0.55 for pectins, suggesting that additional motions exist at the C–O bonds in the wall polysaccharides. ²H–¹³C polarization transfer profiles are also compared between statistically deuterated and H/D exchanged GB1.     

Effect of ACTH and CRH on Plasma Levels of Cortisol and Prostaglandin F<Subscript>2α</Subscript> Metabolite in Cycling Gilts and Castrated Boars

The present study was designed to evaluate the effects of synthetic ACTH (1–24, tetracosactid) and porcine CRH on the plasma levels of cortisol and PGF_2α metabolite in cycling gilts (n = 3) and castrated boars (n = 3). The experiments were designed as crossover studies for each gender separately. Each animal received, during three consecutive days; 1) ACTH (Synacthen^® Depot) at a dose of 10 μg/kg body weight in 5 ml physiological saline, 2) porcine CRH at a dose 0.6 μg/kg body weight in 5 ml physiological saline or 3) physiological saline (5 ml). The test substances were administered via an indwelling jugular cannula in randomized order according to a Latin square. The administration of ACTH to cycling gilts resulted in concomitant elevations of cortisol and PGF_2α metabolite with peak levels reached at 70.0 ± 10.0 and 33.3 ± 6.7 min, respectively. Similarly, the administration of ACTH to castrated boars resulted in concomitant elevation of cortisol and PGF_2α metabolite with peak levels reached at 60.0 ± 0.0 and 20.0 ± 0.0 min, respectively. Cortisol peaked at 20 min after administration of CRH in both cycling gilts and castrated boars with maximum levels of 149.3 ± 16.5 nmol/1 and 138.3 ± 10.1 nmol/1, respectively. It can be concluded that administration of synthetic ACTH (tetracosactid) to pigs caused a concomitant elevation of cortisol and PGF_2α metabolite levels in both cycling gilts as well as castrated boars. The administration of CRH to pigs resulted in an elevation of cortisol levels in both cycling gilts and castrated boars. Conversely, PGF_2α metabolite levels were not influenced by the administration of CRH either in cycling gilts or in castrated boars.     

Membranotropic effects of ω-hydroxypalmitic acid and Ca<Superscript>2+</Superscript> on rat liver mitochondria and lecithin liposomes. Aggregation and membrane permeabilization

The paper examines membranotropic Ca²⁺-dependent effects of ω-hydroxypalmitic acid (HPA), a product of ω-oxidation of fatty acids, on the isolated rat liver mitochondria and artificial membrane systems (liposomes). It was established that in the presence of Ca²⁺, HPA induced aggregation of liver mitochondria, which was accompanied by the release of cytochrome c from the organelles. It was further demonstrated that the addition of Ca²⁺ to HPA-containing liposomes induced their aggregation and/or fusion. Ca²⁺ also caused the release of the fluorescent dye sulforhodamine B from liposomes, indicating their permeabilization. HPA was shown to induce a high-amplitude swelling of Ca²⁺-loaded mitochondria, to decrease their membrane potential, to induce the release of Ca²⁺ from the organelles and to result in the oxidation of the mitochondrial NAD(P)H pool. Those effects of HPA were not blocked by the MPT pore inhibitor CsA, but were suppressed by the mitochondrial calcium uniporter inhibitor ruthenium red. The effects of HPA were also observed when Ca²⁺ was replaced with Sr²⁺ (but not with Ba²⁺ or Mg²⁺). A supposition is made that HPA can induce a Ca²⁺-dependent aggregation of mitochondria, as well as Ca²⁺dependent CsA-insensitive permeabilization of the inner mitochondrial membrane – with the subsequent lysis of the organelles.     

Investigation of the encapsulation of metal cations (Cu<Superscript>2+</Superscript>, Zn<Superscript>2+</Superscript>, Ca<Superscript>2+</Superscript> and Ba<Superscript>2+</Superscript>) by the dipeptide Phe–Phe using natural bond orbital theory and molecular dynamics simulation

Complexes of the dipeptide phenylalanine–phenylalanine (Phe–Phe) with divalent metal cations (Cu²⁺, Zn²⁺, Ca²⁺ and Ba²⁺) were studied at the B3LYP and MP2 levels of theory with the basis sets 6-311++G(d,p) and 6-31 + G(d) in the gas phase. The relative energies of these complexes indicated that cation–π bidentate/tridentate conformations are more favourable than other conformations with uncoordinated rings. These findings were confirmed by the calculated values of thermodynamic parameters such as the Gibbs free energy. Natural bond orbital (NBO) analysis was carried out to explore the metal–ligand coordination in Phe–Phe–Cu²⁺/Zn²⁺ complexes. Possible orbital transitions, types of orbitals and their occupancies were determined for a range of Phe–Phe–Cu²⁺/Zn²⁺ complexes. The charge transfer involved in various orbital transitions was explored by considering the second-order perturbation energy. NBO analysis revealed that the change transfer is stronger when the metal cation uses both the 4s + 4p subshells rather than just its 4p subshell. We also performed molecular dynamics (MD) simulations to check the stability and consistency of the most favourable binding motifs of Cu²⁺, Zn²⁺, Ca²⁺ and Ba²⁺ with Phe–Phe over time. The structures of the Phe–Phe–Cu²⁺/Zn²⁺/Ca²⁺/Ba²⁺ complexes obtained using MD simulation were found to be in good agreement with those obtained in the DFT-based calculations.

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Graphical Abstract Conformational search on encapsulation of divalent metal cations (Ca²⁺, Zn²⁺, Ca²⁺, Ba²⁺) by the Phe-Phe dipeptide

     

The involvement of Ca<Superscript>2+</Superscript> gradients,Ca<Superscript>2+</Superscript> fluxes,and CaM kinase II in polarization and germination of<Emphasis Type="Italic"> Silvetia compressa</Emphasis> zygotes

Previous work has shown that distinct Ca²⁺ gradients precede and predict the loci of germination of the zygotes of the brown alga, Silvetia compressa (J. Agardh) E. Serrão, T.O. Cho, S.M. Boo et Brawley, that are polarized by unilateral blue light. We show here that dark-grown S. compressa zygotes also form cytosolic Ca²⁺ gradients prior to germination and then germinate from the site of elevated Ca²⁺. In no case did germination occur without a prior formation of a Ca²⁺ gradient. Using the self-referencing Ca²⁺-selective probe, we measured highly localized influx of Ca²⁺ during photopolarization, indicating that extracellular stores supply at least some of the Ca²⁺ needed to construct a gradient. Finally, we find that germination was inhibited by a bath-applied inhibitor of calcium/calmodulin-dependent kinase II (CaM kinase II), KN-93 (but not by its inactive analog, KN-92), and by an injected inhibitory peptide for the kinase. KN-93 did not interfere with the photopolarization of the zygotes, consistent with the view that calmodulin is not involved in the initial response to light. The KN-93 results indicate that the requirement for active CaM kinase II for germination ends about 2 h before overt germination. We conclude that Ca²⁺ gradients, generated in part by localized calcium entry from the seawater, are an essential part of the process of polarity development and expression in these cells, regardless of the nature of the external cue that directs the orientation of the axis. Calmodulin and CaM kinase II are involved in interpreting (but not in establishing) the calcium gradient, allowing germination to occur at the site of elevated calcium, but CaM kinase II appears not to be involved in the initiation of germination.