Stem cell recovering effect of copper‐free GHK in skin

The peptide Gly‐His‐Lys (GHK) is a naturally occurring copper(II)‐chelating motifs in human serum and cerebrospinal fluid. In industry, GHK (with or without copper) is used to make hair and skin care products. Copper‐GHK plays a physiological role in the process of wound healing and tissue repair by stimulating collagen synthesis in fibroblasts. We also reported that copper‐GHK promotes the survival of basal stem cells in the skin. However, the effects of copper‐free GHK (GHK) have not been investigated well. In this study, the effects of GHK were studied using cultured normal human keratinocytes and skin equivalent (SE) models. In monolayer cultured keratinocytes, GHK increased the proliferation of keratinocytes. When GHK was added during the culture of SE models, the basal cells became more cuboidal than control model. In addition, there was linear and intense staining of α6 and β1 integrin along the basement membrane. The number of p63 and proliferating cell nuclear antigen positive cells was also significantly increased in GHK‐treated SEs than in control SEs. Western blot and slide culture experiment showed that GHK increased the expression of integrin by keratinocytes. All these results showed that GHK increased the stemness and proliferative potential of epidermal basal cells, which is associated with increased expression of integrin. In conclusion, copper‐free GHK showed similar effects with copper‐GHK. Thus, it can be said that copper‐free GHK can be used in industry to obtain the effects of copper‐GHK in vivo. Further study is necessary to explore the relationship between copper‐free GHK and copper‐GHK. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

The role of charged residues in the transmembrane helices of monocarboxylate transporter 1 and its ancillary protein basigin in determining plasma membrane expression and catalytic activity

Monocarboxylate transporters MCT1-MCT4 require basigin (CD147) or embigin (gp70), ancillary proteins with a glutamate residue in their single transmembrane (TM) domain, for plasma membrane (PM) expression and activity. Here we use site-directed mutagenesis and expression in COS cells or Xenopus oocytes to investigate whether this glutamate (Glu₂₁₈ in basigin) may charge-pair with a positively charged TM-residue of MCT1. Such residues were predicted using a new molecular model of MCT1 based upon the published structure of the E. coli glycerol-3-phosphate transporter. No evidence was obtained for Arg₃₀₆ (TM 8) of MCT1 and Glu₂₁₈ of basigin forming a charge-pair; indeed E218Q-basigin could replace WT-basigin, although E218R-basigin was inactive. No PM expression of R306E-MCT1 or D302R-MCT1 was observed but D302R/R306D-MCT1 reached the PM, as did R306K-MCT1. However, both were catalytically inactive suggesting that Arg₃₀₆ and Asp₃₀₂ form a charge-pair in either orientation, but their precise geometry is essential for catalytic activity. Mutation of Arg₈₆ to Glu or Gln within TM3 of MCT1 had no effect on plasma membrane expression or activity of MCT1. However, unlike WT-MCT1, these mutants enabled expression of E218R-basigin at the plasma membrane of COS cells. We propose that TM3 of MCT1 lies alongside the TM of basigin with Arg₈₆ adjacent to Glu₂₁₈ of basigin. Only when both these residues are positively charged (E218R-basigin with WT-MCT1) is this interaction prevented; all other residue pairings at these positions may be accommodated by charge-pairing or stabilization of unionized residues through hydrogen bonding or local distortion of the helical structure.     

Synthesis of new vasotocin analogues: effects on renal water and ion excretion in rats

Vasopressin and nonmammalian hormone vasotocin are known to increase the water permeability of mammalian collecting ducts, frog skin and the urinary bladder. Neurohypophysial nonapeptides have also been shown to interfere with the regulation of renal ion transport. The subject of this study was a search for vasopressin and vasotocin analogues with selective effects on renal water, sodium and potassium excretion. During this study, we synthesised the following peptides: 13 vasotocin analogues modified at positions 4 (Thr or Arg), 7 (Gly or Leu) and 8 (d ‐Arg, Lys or Glu); 4 vasopressin analogues modified at positions 4 and 8; and 9 peptides shortened or extended at the C‐terminal or with substitutions for Gly‐NH₂. Most of these peptides had mercaptopropionic acid (Mpa) instead of Cys in position 1. The effects of these nonapeptides on renal water, sodium and potassium transport were evaluated in in vivo experiments using Wistar rats. Some nonapeptides possessed antidiuretic, natriuretic and kaliuretic activities ([Mpa¹]‐arginine vasotocin, [Mpa¹, homoArg⁸]‐vasotocin, [Mpa¹, Thr⁴]‐arginine vasotocin and [Mpa¹, Arg⁴]‐arginine vasopressin). Substitutions at positions 4 and 8 increased the selectivity of peptide actions. The antidiuretic [d ‐Arg⁸]‐vasotocin analogues had no effects on sodium excretion. [Mpa¹, Arg⁴]‐arginine vasotocin was antidiuretic and kaliuretic but not natriuretic. [Mpa¹, Glu⁸]‐oxytocin had weak natriuretic activity without any effects on water and potassium transport. In accordance with the data obtained, synthesised vasotocin analogues could be good candidates for pharmaceuticals selectively regulating renal sodium and potassium transport, which is of clinical importance. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Lecithin soybean phospholipid nano-transfersomes as potential carriers for transdermal delivery of the human growth hormone

Pharmaceutical molecules such as peptides and proteins are usually injected into the body. Numerous efforts have been made to find new noninvasive ways to administer these peptides. In this study, highly flexible vesicles (transfersomes [TFs]) were designed as a new modern transdermal drug delivery system for systemic drug administration through the skin, which had also been evaluated in vitro. In this study, two growth hormone-loaded TF formulations were prepared, using soybean lecithin and two different surfactants; F₁_sodium deoxycholate and F ₂_sodium lauryl sulfate. Thereafter, the amount of skin penetration by the two formulas was assessed using the Franz diffusion cell system. TF formulations were evaluated for size, zeta potential and in vitro skin penetration across the rat skin. Results indicated that vesicle formulations were stable for 4 weeks and their mean sizes were 241.33 ± 17 and 171 ± 12.12 nm in the F ₁ and F ₂ formulation, respectively. After application to rat skin, transport of the human growth hormone (hGH) released from the TF formulations was found to be higher than that of the hGH alone. Maximum amounts of transdermal hormone delivery were estimated to be 489.54 ± 8.301 and 248.46 ± 4.019 ng·cm−2 , for F ₁ and F ₂, respectively. The results demonstrate the capability of the TF-containing growth hormone in transdermal delivery and superiority of the F ₁ to F ₂ TFs.     

The effect of conjugation on antitumor activity of vindoline derivatives with octaarginine,a cell‐penetrating peptide

Some Vinca alkaloids (eg, vinblastine, vincristine) have been widely used as antitumor drugs for a long time. Unfortunately, vindoline, a main alkaloid component of Catharanthus roseus (L.) G. Don, itself, has no antitumor activity. In our novel research program, we have prepared and identified new vindoline derivatives with moderate cytostatic activity. Here, we describe the effect of conjugation of vindoline derivative with oligoarginine (tetra‐, hexa‐, or octapeptides) cell‐penetrating peptides on the cytostatic activity in vitro and in vivo. Br‐Vindoline‐(l )‐Trp‐OH attached to the N‐terminus of octaarginine was the most effective compound in vitro on HL‐60 cell line. Analysis of the in vitro activity of two isomer conjugates (Br‐vindoline‐(l )‐Trp‐Arg₈ and Br‐vindoline‐(d )‐Trp‐Arg₈ suggests the covalent attachment of the vindoline derivatives to octaarginine increased the antitumor activity significantly against P388 and C26 tumour cells in vitro. The cytostatic effect was dependent on the presence and configuration of Trp in the conjugate as well as on the cell line studied. The configuration of Trp notably influenced the activity on C26 and P388 cells: conjugate with (l )‐Trp was more active than conjugate with the (d )‐isomer. In contrast, conjugates had very similar effect on both the HL‐60 and MDA‐MB‐231 cells. In preliminary experiments, conjugate Br‐vindoline‐(l )‐Trp‐Arg₈ exhibited some inhibitory effect on the tumor growth in P388 mouse leukemia tumor‐bearing mice. Our results indicate that the conjugation of modified vindoline could result in an effective compound even with in vivo antitumor activity.     

Synthesis and properties of cyclic gomesin and analogues

Gomesin (Gm) was the first antimicrobial peptide (AMP) isolated from the hemocytes of a spider, the Brazilian mygalomorph Acanthoscurria gomesiana. We have been studying the properties of this interesting AMP, which also displays anticancer, antimalarial, anticryptococcal and anti‐Leishmania activities. In the present study, the total syntheses of backbone‐cyclized analogues of Gm (two disulfide bonds), [Cys(Acm)^2,15]‐Gm (one disulfide bond) and [Thr^2,6,11,15,d ‐Pro⁹]‐Gm (no disulfide bonds) were accomplished, and the impact of cyclization on their properties was examined. The consequence of simultaneous deletion of pGlu¹ and Arg¹⁶‐Glu‐Arg¹⁸‐NH₂ on Gm antimicrobial activity and structure was also analyzed. The results obtained showed that the synthetic route that includes peptide backbone cyclization on resin was advantageous and that a combination of 20% DMSO/NMP, EDC/HOBt, 60 °C and conventional heating appears to be particularly suitable for backbone cyclization of bioactive peptides. The biological properties of the Gm analogues clearly revealed that the N‐terminal amino acid pGlu¹ and the amidated C‐terminal tripeptide Arg¹⁶‐Glu‐Arg¹⁸‐NH₂ play a major role in the interaction of Gm with the target membranes. Moreover, backbone cyclization practically did not affect the stability of the peptides in human serum; it also did not affect or enhanced hemolytic activity, but induced selectivity and, in some cases, discrete enhancements of antimicrobial activity and salt tolerance. Because of its high therapeutic index, easy synthesis and lower cost, the [Thr^2,6,11,15,d ‐Pro⁹]‐Gm analogue remains the best active Gm‐derived AMP developed so far; nevertheless, its elevated instability in human serum may limit its therapeutic potential. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Coassemble dopamine and GHK tripeptide into fluorescent nanoparticles for pH sensing

Fluorescent nanostructures have been widely applied to biomedical researches and clinical diagnosis such as biolabeling/imaging/sensing and have even acted as therapy reagents. Peptide‐based fluorescent nanostructures attract recent interest from biomedical researchers. Inspired by the natural existence of GHK‐Cu complex with a growth factor‐like effect in human blood, here we have developed a novel approach for designing nanosensors through the co‐assembling of two kinds of biomolecules. By making best use of both π‐π stacking between carbon rings and the easy‐oxidation property of an important transmitter molecule, dopamine (DA), we successfully built up a supersensitive and robust fluorescent pH nanosensor by co‐assembling oxidized DA (DA_ox) with a tripeptide GHK. The GHK‐DA_ox nanostructures have a quantum yield of 20.82%, which might be the brightest one among all the current co‐assembling structures merely through unmodified biomolecules. We envision this approach could open a new avenue for not only hybrid nanostructure construction, but also may inspire the bioengineering of in vivo luminescent probes.     

α‐MSH tripeptide analogs activate the melanocortin 1 receptor and reduce UV‐induced DNA damage in human melanocytes

One skin cancer prevention strategy that we are developing is based on synthesizing and testing melanocortin analogs that reduce and repair DNA damage resulting from exposure to solar ultraviolet (UV) radiation, in addition to stimulating pigmentation. Previously, we reported the effects of tetrapeptide analogs of α‐melanocortin (α‐MSH) that were more potent and stable than the physiological α‐MSH, and mimicked its photoprotective effects against UV‐induced DNA damage in human melanocytes. Here, we report on a panel of tripeptide analogs consisting of a modified α‐MSH core His⁶‐d ‐Phe⁷‐Arg⁸, which contained different N‐capping groups, C‐terminal modifications, or arginine mimics. The most potent tripeptides in activating cAMP formation and tyrosinase of human melanocytes were three analogs with C‐terminal modifications. The most effective C‐terminal tripeptide mimicked α‐MSH in reducing hydrogen peroxide generation and enhancing nucleotide excision repair following UV irradiation. The effects of these three analogs required functional MC1R, as they were absent in human melanocytes that expressed non‐functional receptor. These results demonstrate activation of the MC1R by tripeptide melanocortin analogs. Designing small analogs for topical delivery should prove practical and efficacious for skin cancer prevention.     

Two amino acid changes in the R3 repeat cause functional divergence of two clustered <Emphasis Type="Italic">MYB10</Emphasis> genes in peach

R2R3-MYB genes play a pivotal role in regulating anthocyanin accumulation. Here, we report two tandemly duplicated R2R3-MYB genes in peach, PpMYB10.1 and PpMYB10.2, with the latter showing lower ability to induce anthocyanin accumulation than the former. Site-directed mutation assay revealed two amino acid changes in the R3 repeat, Arg/Lys⁶⁶ and Gly/Arg⁹³, responsible for functional divergence between these two PpMYB10 genes. Anthocyanin-promoting activity of PpMYB10.2 was significantly increased by a single amino acid replacement of Arg⁹³ with Gly⁹³. However, either the Gly⁹³ → Arg⁹³ or Arg⁶⁶ → Lys⁶⁶ substitutions alone showed little impact on anthocyanin-promoting activity of PpMYB10.1, but simultaneous substitutions caused a significant decrease. Reciprocal substitution of Arg/Gly⁹³ could significantly alter binding affinity to PpbHLH3, while the Arg⁶⁶ → Lys⁶⁶ substitution is predicted to affect the folding of the MYB DNA-binding domain, instead of PpbHLH3-binding affinity. Overall, the change of anthocyanin-promoting activity was accompanied with that of bHLH-binding affinity, suggesting that DNA-binding affinity of R2R3-MYBs depends on their bHLH partners. Our study is helpful for understanding of functional evolution of R2R3-MYBs and their interaction with DNA targets.     

12(R)-hydroxy-5,8,10,14-eicosatetraenoic acid is a chemoattractant fo human polymorphonuclear leucocytes in vitro

Increased amounts of 12-hydroxy - 5,8,10,14-eicosatetraenoic acid (12-HETE) are found in the lesional skin of patients with the skin disease psoriasis when compared to clinically normal skin. Stereochemical analysis has recently shown that the 12-HETE present in lesional psoriatic scale is the (R), and not the (S) hydroxyl enantiomer, produced by platelets. Since the chemoattractant activity of 12(R)-HETE has not previously been described, the (R) and (S) hydroxyl enantiomers of 12-HETE have now been synthesised and their chemokinetic activity compared in vitro. 12(R)-HETE, was more potent than 12(S)-HETE as a chemokinetic agent for human polymorphonuclear leucocytes but 2000 times less potent than leukotriene B₄. In contrast to results obtained with the 12-HETE enantiomers, the chemoattractant compound 5(S)-HETE was found to be more potent than the 5(R) hydroxyl enantiomer. Thus, the configuration of the hydroxyl group appears to be of importance to the chemokinetic activity of the HETEs, and the increased potency of the 12(R) enantiomer may enhance its significance as a mediator of inflammation in psoriasis.     

A yeast arginine specific tRNA is a remnant aspartate acceptor

High specificity in aminoacylation of transfer RNAs (tRNAs) with the help of their cognate aminoacyl-tRNA synthetases (aaRSs) is a guarantee for accurate genetic translation. Structural and mechanistic peculiarities between the different tRNA/aaRS couples, suggest that aminoacylation systems are unrelated. However, occurrence of tRNA mischarging by non-cognate aaRSs reflects the relationship between such systems. In Saccharomyces cerevisiae, functional links between arginylation and aspartylation systems have been reported. In particular, it was found that an in vitro transcribed tRNA^Asp is a very efficient substrate for ArgRS. In this study, the relationship of arginine and aspartate systems is further explored, based on the discovery of a fourth isoacceptor in the yeast genome, tRNA₄^Arg. This tRNA has a sequence strikingly similar to that of tRNA^Asp but distinct from those of the other three arginine isoacceptors. After transplantation of the full set of aspartate identity elements into the four arginine isoacceptors, tRNA₄^Arg gains the highest aspartylation efficiency. Moreover, it is possible to convert tRNA₄^Arg into an aspartate acceptor, as efficient as tRNA^Asp, by only two point mutations, C38 and G73, despite the absence of the major anticodon aspartate identity elements. Thus, cryptic aspartate identity elements are embedded within tRNA₄^Arg. The latent aspartate acceptor capacity in a contemporary tRNA^Arg leads to the proposal of an evolutionary link between tRNA₄^Arg and tRNA^Asp genes.     

Enantiomeric Specificity of Biologically Significant Cu(II) and Zn(II) Chromone Complexes Towards DNA

Novel chiral Schiff base ligands (R)/(S)‐2‐amino‐3‐(((1‐hydroxypropan‐2‐yl)imino)methyl)‐4H‐chromen‐4‐one (L₁ and L₂) derived from 2‐amino‐3‐formylchromone and (R/S)‐2‐amino‐1‐propanol and their Cu(II)/Zn(II) complexes ( R1 , S1 , R2 , and S2 ) were synthesized. The complexes were characterized by elemental analysis, infrared (IR), hydrogen (¹H) and carbon (¹³C) nuclear magnetic resonance (NMR), electrospray ionization‐mass spectra (ESI‐MS), and molar conductance measurements. The DNA binding studies of the complexes with calf thymus were carried out by employing different biophysical methods and molecular docking studies that revealed that complexes R1 and S1 prefers the guanine–cytosine‐rich region, whereas R2 and S2 prefers the adenine–thymine residues in the major groove of DNA. The relative trend in K_b values followed the order R1 S1 R2 S2 . This observation together with the findings of circular dichroic and fluorescence studies revealed maximal potential of (R)‐enantiomeric form of complexes to bind DNA. Furthermore, the absorption studies with mononucleotides were also monitored to examine the base‐specific interactions of the complexes that revealed a higher propensity of Cu(II) complexes for guanosine‐5′‐monophosphate disodium salt, whereas Zn(II) complexes preferentially bind to thymidine‐5′‐monophosphate disodium salt. The cleavage activity of R1 and R2 with pBR322 plasmid DNA was examined by gel electrophoresis that revealed that they are good DNA cleavage agents; nevertheless, R1 proved to show better DNA cleavage ability. Topoisomerase II inhibitory activity of complex R1 revealed that the complex inhibits topoisomerase II catalytic activity at a very low concentration (25 μM). Furthermore, in vitro antitumor activity of complexes R1 and S1 were screened against human carcinoma cell lines of different histological origin. Chirality 24:977–986, 2012. © 2012 Wiley Periodicals, Inc.     

Cooperative role of endogenous leucotrienes and platelet‐activating factor in ischaemia–reperfusion‐mediated tissue injury

Insufficient oxygen delivery to organs leads to tissue dysfunction and cell death. Reperfusion, although vital to organ survival, initiates an inflammatory response that may both aggravate local tissue injury and elicit remote organ damage. Polymorphonuclear neutrophil (PMN) trafficking to remote organs following ischaemia/reperfusion (I/R) is associated with the release of lipid mediators, including leucotriene (LT) B₄, cysteinyl‐LTs (CysLTs) and platelet‐activating factor (PAF). Yet, their potentially cooperative role in regulating I/R‐mediated inflammation has not been thoroughly assessed. The present study aimed to determine the cooperative role of lipid mediators in regulating PMN migration, tissue oedema and injury using selective receptor antagonists in selected models of I/R and dermal inflammation. Our results show that rabbits, pre‐treated orally with BIIL 284 and/or WEB 2086 and MK‐0571, were protected from remote tissue injury following I/R or dermal inflammation in an additive or synergistic manner when the animals were pre‐treated with two drugs concomitantly. The functional selectivity of the antagonists towards their respective agonists was assessed in vitro, showing that neither BIIL 284 nor WEB 2086 prevented the inflammatory response to IL‐8, C5a and zymosan‐activated plasma stimulation. However, these agonists elicited LTB₄ biosynthesis in isolated rabbit PMNs. Similarly, a cardioprotective effect of PAF and LTB₄ receptor antagonists was shown following myocardial I/R in mice. Taken together, these results underscore the intricate involvement of LTB₄ and PAF in each other's responses and provide further evidence that targeting both LTs and PAF receptors provides a much stronger anti‐inflammatory effect, regulating PMN migration and oedema formation.     

Essential roles of dopamine D4 receptors and the type 1 adenylyl cyclase in photic control of cyclic AMP in photoreceptor cells

Light and dopamine regulate many physiological functions in the vertebrate retina. Light exposure decreases cyclic AMP formation in photoreceptor cells. Dopamine D₄ receptor (D₄R) activation promotes light adaptation and suppresses the light‐sensitive pool of cyclic AMP in photoreceptor cells. The key signaling pathways involved in regulating cyclic AMP in photoreceptor cells have not been identified. In the present study, we show that the light‐ and D₄R‐signaling pathways converge on the type 1 Ca²⁺/calmodulin‐stimulated adenylyl cyclase (AC1) to regulate cyclic AMP synthesis in photoreceptor cells. In addition, we present evidence that D₄R activation tonically regulates the expression of AC1 in photoreceptors. In retinas of mice with targeted deletion of the gene (Adcy1) encoding AC1, cyclic AMP levels and Ca²⁺/calmodulin‐stimulated adenylyl cyclase activity are markedly reduced, and cyclic AMP accumulation is unaffected by either light or D₄R activation. Similarly, in mice with disruption of the gene (Drd4) encoding D₄R, cyclic AMP levels in the dark‐adapted retina are significantly lower compared to wild‐type retina and are unresponsive to light. These changes in Drd4?/? mice were accompanied by significantly lower Adcy1 mRNA levels in photoreceptor cells and lower Ca²⁺/calmodulin‐stimulated adenylyl cyclase activity in retinal membranes compared with wild‐type controls. Reduced levels of Adcy1 mRNA were also observed in retinas of wild‐type mice treated chronically with a D₄R antagonist, L‐745870. Thus, activation of D₄R is required for normal expression of AC1 and for the regulation of its catalytic activity by light. These observations illustrate a novel mechanism for cross‐talk between dopamine and photic signaling pathways regulating cyclic AMP in photoreceptor cells.     

Classification of 2-pore domain potassium channels based on rectification under quasi-physiological ionic conditions

It is generally expected that 2-pore domain K⁺ (K_2P) channels are open or outward rectifiers in asymmetric physiological K⁺ gradients, following the Goldman-Hodgkin-Katz (GHK) current equation. Although cloned K_2P channels have been extensively studied, their current-voltage (I-V) relationships are not precisely characterized and previous definitions are contradictory. Here we study all the functional channels from 6 mammalian K_2P subfamilies in transfected Chinese hamster ovary cells with patch-clamp technique, and examine whether their I-V relationships are described by the GHK current equation. K_2P channels display 2 distinct types of I-V curves in asymmetric physiological K⁺ gradients. Two K_2P isoforms in the TWIK subfamily conduct large inward K⁺ currents and have a nearly linear I-V curve. Ten isoforms from 5 other K_2P subfamilies conduct small inward K⁺ currents and exhibit open rectification, but fits with the GHK current equation cannot precisely reveal the differences in rectification among K_2P channels. The Rectification Index, a ratio of limiting I-V slopes for outward and inward currents, is used to quantitatively describe open rectification of each K_2P isoform, which is previously qualitatively defined as strong or weak open rectification. These results systematically and precisely classify K_2P channels and suggest that TWIK K⁺ channels have a unique feature in regulating cellular function.     

Vasopressin inhibits the adenylate cyclase activity of human platelet particulate fraction through V1-receptors

Arg⁸-vasopressin inhibited the adenylate cyclase activity of human platelet particulate fraction up to a maximum of 27% (IC₅₀ = 1.2 nM). This inhibition required the presence of 10 μM GTP and was optimal with 100 mM NaCl. Orn⁸-vasopressin had similar effects. 1-Deamino-Val⁴, D-Arg⁸-vasopressin did not by itself affect adenylate cyclase activity but competitively inhibited the action of Arg⁸-vasopressin (pA₂ = 7.74). Arg⁸-vasopressin did not inhibit adenylate cyclase in intact platelets but instead caused platelet aggregation, an effect that was also competitively inhibited by 1-deamino-Val⁴, D-Arg⁸-vasopressin (pA₂ = 7.82). Thus, platelets possess vasopressin receptors of the V₁ type that, under appropriate conditions, can mediate either inhibition of platelet adenylate cyclase or platelet aggregation.     

Classification of 2-pore domain potassium channels based on rectification under quasi-physiological ionic conditions

It is generally expected that 2-pore domain K⁺ (K_2P) channels are open or outward rectifiers in asymmetric physiological K⁺ gradients, following the Goldman-Hodgkin-Katz (GHK) current equation. Although cloned K_2P channels have been extensively studied, their current-voltage (I-V) relationships are not precisely characterized and previous definitions are contradictory. Here we study all the functional channels from 6 mammalian K_2P subfamilies in transfected Chinese hamster ovary cells with patch-clamp technique, and examine whether their I-V relationships are described by the GHK current equation. K_2P channels display 2 distinct types of I-V curves in asymmetric physiological K⁺ gradients. Two K_2P isoforms in the TWIK subfamily conduct large inward K⁺ currents and have a nearly linear I-V curve. Ten isoforms from 5 other K_2P subfamilies conduct small inward K⁺ currents and exhibit open rectification, but fits with the GHK current equation cannot precisely reveal the differences in rectification among K_2P channels. The Rectification Index, a ratio of limiting I-V slopes for outward and inward currents, is used to quantitatively describe open rectification of each K_2P isoform, which is previously qualitatively defined as strong or weak open rectification. These results systematically and precisely classify K_2P channels and suggest that TWIK K⁺ channels have a unique feature in regulating cellular function.     

Interactions of vasopressin and oxytocin receptors with vasopressin analogues substituted in position 2 with 3,3′‐diphenylalanine – a molecular docking study

Vasopressin and oxytocin receptors belong to the superfamily of G protein‐coupled receptors and play an important role in many physiological functions. They are also involved in a number of pathological conditions being important drug targets. In this work, four vasopressin analogues substituted at position 2 with 3,3′‐diphenylalanine have been docked into partially flexible vasopressin and oxytocin receptors. The bulky residue at position 2 acts as a structural restraint much stronger in the oxytocin receptor (OTR) than in the vasopressin V2 receptor (V2R), resulting in a different location of the analogues in these receptors. This explains the different, either agonistic or antagonistic, activities of the analogues in V2R and OTR, respectively. In all complexes, the conserved polar residues serve as anchor points for the ligand both in OTR and V2R. Strong interactions of the C‐terminus of analogue II ([Mpa¹,d ‐Dpa²,Val⁴,d ‐Arg⁸]VP) with extracellular loop 3 may be responsible for its highest activity at V2R. It also appears that V2R adapts more readily to the docking analogues by conformational changes in the aromatic side chains triggering receptor activation. A weak activity at V1a vasopressin receptor appears to be caused by weak receptor–ligand interactions. Results of this study may facilitate a rational design of new analogues with the highest activity/selectivity at vasopressin and OTRs. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Synthesis and biological activity of lipophilic analogs of the cationic antimicrobial active peptide anoplin

Anoplin is a short natural cationic antimicrobial peptide which is derived from the venom sac of the solitary wasp, Anoplius samariensis. Due to its short sequence G¹LLKR⁵IKT⁸LL‐NH₂, it is ideal for research tests. In this study, novel analogs of anoplin were prepared and examined for their antimicrobial, hemolytic activity, and proteolytic stability. Specific substitutions were introduced in amino acids Gly¹, Arg⁵, and Thr⁸ and lipophilic groups with different lengths in the N‐terminus in order to investigate how these modifications affect their antimicrobial activity. These cationic analogs exhibited higher antimicrobial activity than the native peptide; they are also nontoxic at their minimum inhibitory concentration (MIC) values and resistant to enzymatic degradation. The substituted peptide GLLKF⁵IKK⁸LL‐NH₂ exhibited high activity against Gram‐negative bacterium Zymomonas mobilis (MIC = 7 µg/ml), and the insertion of octanoic, decanoic, and dodecanoic acid residues in its N‐terminus increased the antimicrobial activity against Gram‐positive and Gram‐negative bacteria (MIC = 5 µg/ml). The conformational characteristics of the peptide analogs were studied by circular dichroism. Structure activity studies revealed that the substitution of specific amino acids and the incorporation of lipophilic groups enhanced the amphipathic α‐helical conformation inducing better antimicrobial effects. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Conserved residues are critical for Haloferax volcanii archaeosortase catalytic activity: Implications for convergent evolution of the catalytic mechanisms of non‐homologous sortases from archaea and bacteria

Proper protein anchoring is key to the biogenesis of prokaryotic cell surfaces, dynamic, resilient structures that play crucial roles in various cell processes. A novel surface protein anchoring mechanism in Haloferax volcanii depends upon the peptidase archaeosortase A (ArtA) processing C‐termini of substrates containing C‐terminal tripartite structures and anchoring mature substrates to the cell membrane via intercalation of lipid‐modified C‐terminal amino acid residues. While this membrane protein lacks clear homology to soluble sortase transpeptidases of Gram‐positive bacteria, which also process C‐termini of substrates whose C‐terminal tripartite structures resemble those of ArtA substrates, archaeosortases do contain conserved cysteine, arginine and arginine/histidine/asparagine residues, reminiscent of His‐Cys‐Arg residues of sortase catalytic sites. The study presented here shows that ArtA^WT‐GFP expressed in trans complements ΔartA growth and motility phenotypes, while alanine substitution mutants, Cys¹⁷³ (C173A), Arg²¹⁴ (R214A) or Arg²⁵³ (R253A), and the serine substitution mutant for Cys¹⁷³ (C173S), fail to complement these phenotypes. Consistent with sortase active site replacement mutants, ArtA^C173A‐GFP, ArtA^C173S‐GFP and ArtA^R214A‐GFP cannot process substrates, while replacement of the third residue, ArtA^R253A‐GFP retains some processing activity. These findings support the view that similarities between certain aspects of the structures and functions of the sortases and archaeosortases are the result of convergent evolution.