Synthesis,conformation, and membrane modifying properties of the trikoningin KB lipopeptaibols: Effect of hydrophobicity and chirality in position 1

Summary We synthesized by solution-phase methods the naturally occurring, 10-amino acid residue lipopeptaibol antibiotics trikoningins KBI and KBII, and the [l-Iva¹] KB analogue, in which the amino acid in position 1 is different, with the aim at investigating the effect of hydrophobicity and chirality in that position. A solution conformational analysis, using FT-IR absorption and CD techniques, indicated that all of the three decapeptides are predominantly helical in a membrane-mimetic environment. Permeability measurements showed an increase of the activity from the [Aib¹] peptide to the more hydrophobic [Iva¹] peptides. Conversely, the effect of a change in chirality, obtained by replacingd-Iva¹ withl-Iva, turned out to be of minor significance.     

Potassium-induced insulin release and voltage noise measurements in single mouse islets of Langerhans

Summary Insulin release and membrane potential fluctuations in response to increased extracellular potassium [K⁺] _o have been measured in single perifused islets of Langerhans from normal mice. An increase in [K⁺] _o from 5mm to 50mm induced a transient insulin release with a peak at about 1 min. The peak value was [K⁺] _o -dependent but the half-timet _1/2 for the decline was constant at nearly 1 min. 2.5mm cobalt completely inhibited the potassium-induced stimulation of insulin release. The insulin release elicited by 28 and 50mm [K⁺] _o was similar in terms of peak, total release and half-time from maximum release. Stepwise increase in [K⁺] _o from 10 to 28 to 50mm resulted in a normal response to 28mm but no peak of release after the 28 to 50mm increase. The results indicate good correlation between excess voltage noise, thought to reflect calcium channel activity, and insulin release evoked by changing extracellular potassium.     

Effect of exo-16,17-dihydro-gibberellin A5 on gibberellin A20 metabolism in seedlings of dwarf rice (Oryza sativa L. cv. Tan-ginbozu)

Several of the 16,17-dihydro gibberellins (GAs) inhibit elongation in a variety of species. In a study of their mechanism of action we have investigated the effect of exo-16,17-dihydro-Ga₅ (diHGA₅) on the metabolism of GA₂₀ in dwarf rice (Oryza sativa cv. Tan-ginbozu). A mixture of [³H]- and [³H]-GA₂₀ (100 ng per plant) was applied in microdrops to 4 d old seedlings which were harvested 72 h later. Concurrent treatment with diHGA₅ at 100 ng or 333 ng per plant reduced GA₂₀-induced elongation of the second leaf sheath by 41–66%. There was a concomitant reduction in the amount of [²H₂]GA₁ present at harvest, measured by gas chromatography-mass spectrometry-selected ion monitoring. The [²H₂]GA₂₉ content was also reduced. There was no clear effect of diHGA₅ on the total radioactivity recovered, or on conversion of the [³H]GA₂₀ to putative [³H]GA conjugates, or on the amount of [²H₂]GA₂₀ found. No free [²H₂]GA₈ was detected. In other experiments there was little effect of diHGA₅ on elongation induced by treatment with GA₁. We conclude that diHGA₅ inhibited GA₂₀-induced elongation in dwarf rice shoots at least partly by reducing the 3-hydroxylation of GA₂₀ to GA₁.Abbreviations diHGA₅ = exo- 16, 17-dihydro-gibberellin A₅ - GA = gibberellin - GC-MS-SIM = gas chromatography-mass spectrometry-selected ion monitoring     

Comparative studies on [Ca2+]i-level of fibroblasts from Alzheimer patients and control individuals

The accumulation of the -amyloid peptide (AP) in the brain, produced from the ubiquitously expressed amyloid precursor protein (APP) is a defining feature of Alzheimer's disease (AD). Consistent with studies demonstrating the importance of skin biopsy in the diagnosis of neurodegenerative disorders, we investigated whether differences in intracellular free calcium levels ([Ca²⁺]_i) of cultured cutaneous fibroblasts derived from sporadic AD patients and from age-matched control individuals might be present. [Ca²⁺]_i was measured in Fura-2AM-loaded human fibroblasts by dual wavelength spectrofluorimetry. AD cells exhibited lower [Ca²⁺]_i as compared to the control cultures. Exposure of fibroblasts to AP resulted in increased [Ca²⁺]_i of the control cells, but not of AD fibroblasts. Our test could prove useful in supporting the diagnosis of (sporadic) AD in patients suspected of suffering from the disease.     

Novel analogs of alloferon: Synthesis,conformational studies,pro-apoptotic and antiviral activity

In this study, we report the structure-activity relationships of novel derivatives of the insect peptide alloferon (H-His-Gly-Val-Ser-Gly-His-Gly-Gln-His-Gly-Val-His-Gly-OH). The peptide structure was modified by exchanging His at position 9 or 12 for natural or non-natural amino acids. Biological properties of these peptides were determined in antiviral in vitro test against Human Herpes Virus 1 McIntrie strain (HHV-1_MC) using a Vero cell line. The peptides were also evaluated for the pro-apoptotic action in vivo on hemocytes of the Tenebrio molitor beetle. Additionally, the structural properties of alloferon analogs were examined by the circular dichroism in water and methanol. It was found that most of the evaluated peptides can reduce the HHV-1 titer in Vero cells. [Ala⁹]-alloferon exhibits the strongest antiviral activity among the analyzed compounds. However, no cytotoxic activity against Vero cell line was observed for all the studied peptides. In vivo assays with hemocytes of T. molitor showed that [Lys⁹]-, [Phg⁹]-, [Lys¹²]-, and [Phe¹²]-alloferon exhibit a twofold increase in caspases activity in comparison with the native peptide. The CD conformational studies indicate that the investigated peptides seem to prefer the unordered conformation.     

H, C NMR and UV spectroscopy studies of gold(III)-tetracyanide complex with l-cysteine, glutathione, captopril, l-methionine and dl-seleno-methionine in aqueous solution

Auricyanide [Au(CN)₄]⁻ interaction with biologically important thiols, thioether and selenoether were carried out and monitored using ¹H, ¹³C NMR and UV spectroscopy. These ligands include l-cysteine, glutathione, captopril, l-methionine and dl-seleno-methionine. Thiols show very strong affinity to be oxidized into the disulfide by auricyanide, which gets reduced to aurocyanide [Au(CN)₂]⁻. l-cysteine reaction mechanism with [Au(CN)₄]⁻ was found to be dependent on reactants mole ratio. While l-methionine was completely inert toward auricyanide, dl-Se-methionine showed some reactivity with [Au(CN)₄]⁻ after raising solution pH to 12 that facilitated cyanide exchange.     

β-Adrenergic-agonist stimulated taurine release from astroglial cells is modulated by extracellular [K+] and osmolarity

Astroglial cells are known to release taurine in response to stimulation by a variety of stimuli including -adrenergic receptor agonists such as isoproterenol (IPR). The effects of changing osmolarity and extracellular [K⁺] on IPR-stimulated taurine release were studied with LRM55 cells, a continuous astroglial cell line. IPR-stimulated taurine release decreased almost 8% for each 1% increase in osmolarity, indicating that IPR-stimulated release is highly regulated by the osmolarity of the medium. IPR-stimulated taurine release was greatly enhanced when external [K⁺] was increased isosmotically by substituting KCl for NaCl but was strongly suppressed when external [K⁺] was increased hyperosmotically by adding KCl to the medium. Both IPR-stimulated and K⁺-stimulated taurine release depended on external [Cl^–]; IPR-stimulated release declined approximately in parallel to K⁺-stimulated release as [Cl^–] in the medium was reduced. The high sensitivity of IPR-stimulated release to factors that change cell volume (osmolarity, external [K⁺], external [Cl^–]) is consistent with the idea that IPR, elevated [K⁺], and reduced osmolarity all elicit taurine release via a single tension-controlled mechanism.Special issue dedicated to Dr. Claude Baxter.     

[D‐(p‐Benzoylphenylalanine)13, tyrosine19]‐melanin‐concentrating hormone,a potent analogue for MCH receptor crosslinking

A photoreactive analogue of human melanin‐concentrating hormone was designed, [d‐ Bpa¹³,Tyr¹⁹]‐MCH, containing the d‐ enantiomer of photolabile p‐benzoylphenylalanine (Bpa) in position 13 and tyrosine for radioiodination in position 19. The linear peptide was synthesized by the continuous‐flow solid‐ phase methodology using Fmoc‐strategy and PEG‐PS resins, purified to homogeneity and cyclized by iodine oxidation. Radioiodination of [d ‐Bpa¹³,Tyr¹⁹]‐MCH at its Tyr¹⁹ residue was carried out enzymatically using solid‐ phase bound glucose oxidase/lactoperoxidase, followed by purification on a reversed‐ phase mini‐column and HPLC. Saturation binding analysis of [¹²⁵I]‐[d‐ Bpa¹³,Tyr¹⁹]‐MCH with G4F‐7 mouse melanoma cells gave a K_D of 2.2±0.2×10⁻¹⁰ mol/l and a B_max of 1047±50 receptors/cell. Competition binding analysis showed that MCH and rANF(1–28) displace [¹²⁵I]‐[d‐ Bpa¹³,Tyr¹⁹]‐MCH from the MCH binding sites on G4F‐7 cells whereas α‐MSH has no effect. Receptor crosslinking by UV‐irradiation of G4F‐7 cells in the presence of [¹²⁵I]‐[d‐ Bpa¹³,Tyr¹⁹]‐MCH followed by SDS‐polyacrylamide gel electrophoresis and autoradiography yielded a band of 45–50 kDa. Identical crosslinked bands were also detected in B16‐F1 and G4F mouse melanoma cells, in RE and D10 human melanoma cells as well as in COS‐7 cells. Weak staining was found in rat PC12 phaeochromocytoma and Chinese hamster ovary cells. No crosslinking was detected in human MP fibroblasts. These data demonstrate that [¹²⁵I]‐[d‐ Bpa¹³,Tyr¹⁹]‐MCH is a versatile photocrosslinking analogue of MCH suitable to identify MCH receptors in different cells and tissues; the MCH receptor in these cells appears to have the size of a G protein‐coupled receptor, most likely with a varying degree of glycosylation. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

Excitatory action of γ-aminobutyric acid (GABA) on crustacean neurosecretory cells

Summary 1. Intracellular and voltage-clamp recordings were obtained from a selected population of neuroscretory (ns) cells in the X organ of the crayfish isolated eyestalk. Pulses of -aminobutyric acid (GABA) elicited depolarizing responses and bursts of action potentials in a dose-dependent manner. These effects were blocked by picrotoxin (50 µM) but not by bicuculline. Picrotoxin also suppressed spontaneous synaptic activity.2. The responses to GABA were abolished by severing the neurite of X organ cells, at about 150 µm from the cell body. Responses were larger when the application was made at the neuropil level.3. Topical application of Cd²⁺ (2 mM), while suppressing synaptic activity, was incapable of affecting the responses to GABA.4. Under whole-cell voltage-clamp, GABA elicited an inward current with a reversal potential dependent on the chloride equilibrium potential. The GABA effect was accompanied by an input resistance reduction up to 33% at a –50 mV holding potential. No effect of GABA was detected on potassium, calcium, and sodium currents present in X organ cells.5. The effect of GABA on steady-state currents was dependent on the intracellular calcium concentration. At 10^–6 M [Ca²⁺]_i, GABA (50 µM) increased the membrane conductance more than threefold and shifted the zero-current potential from–25 to–10 mV. At 10^–9 M [Ca²⁺]_i, GABA induced only a 1.3-fold increase in membrane conductance, without shifting the zero-current potential.6. These results support the notion that in the population of X organ cells sampled in this study, GABA acts as an excitatory neurotransmitter, opening chloride channels.     

Salt-induced hypertension in WKY rats: Prevention by α-lipoic acid supplementation

There is strong evidence that points to excess dietary salt as a major factor contributing to the development of hypertension. Salt sensitivity is associated with glucose intolerance and insulin resistance in both animal models and humans. In insulin resistance, impaired glucose metabolism leads to elevated endogenous aldehydes which bind to vascular calcium channels, increasing cytosolic [Ca²⁺]_i and blood pressure. In an insulin resistant animal model of hypertension, spontaneously hypertensive rats (SHRs), dietary supplementation with lipoic acid lowers tissue aldehydes and plasma insulin levels and normalizes blood pressure. The objective of this study is to examine the effects of a high salt diet on tissue aldehydes, cytosolic [Ca²⁺]_i and blood pressure in WKY rats and to investigate whether dietary supplementation with lipoic acid can prevent a salt induced increase in blood pressure. Starting at 7 weeks of age, WKY rats were divided into three groups of six animals each and treated for 10 weeks with diets as follows: WKY-normal salt (0.7% NaCl); WKY-high salt (8% NaCl); WKY-high salt + lipoic acid (8% NaCl diet + lipoic acid 500 mg/Kg feed). At completion, animals in the high salt group had elevated systolic blood pressure, platelet [Ca²⁺]_i, and tissue aldehyde conjugates compared with the normal salt group and showed smooth muscle cell hyperplasia in the small arteries and arterioles of the kidneys. Dietary -lipoic acid supplementation in high salt-treated WKY rats normalized systolic blood pressure and cytosolic [Ca²⁺]_i and aldehydes in liver and aorta. Kidney aldehydes and renal vascular changes were attenuated, but not normalized.     

The effect of amino acids,monoamines and polyamines on pyruvate dehydrogenase activity in mitochondria from rat adipocytes

Summary The ability of polyamines and other cationic compounds including monoamines, amino acids, poly-L-arginine, poly-D-lysine and poly-L-lysine, to alter pyruvate dehydrogenase (PDH) activity in mitochondria from rat epididymal adipocytes was determined. PDH was assayed with the substrate [1-¹⁴C] pyruvate in the presence of 0.05 mM Ca²⁺ and Mg²⁺. Nine of the fourteen compounds tested at 0.1 mM caused a significant increase (procaine, 3-(-morpholinopropionyl) benzo[b]thiophene [VII], spermine, spermidine, putrescine, lysine and tryptophan) or decrease (poly-L-arginine, 3-(-piperidinopropionyl) benzo[b]thiophene) in PDH activity. None of these compounds nonenzymatically decarboxylated [1-¹⁴C] pyruvate to release ¹⁴CO₂. NaF, a PDH phosphatase inhibitor, suppressed the stimulatory effects of those compounds tested: procaine, tryptophan, VII, spermine and spermidine. These results imply that these five compounds activate PDH activity through stimulation of the PDH phosphatase. When the Mg²⁺ concentration was increased from 0.05 to 4.5 mM, the stimulatory effect of spermine was increased, consistent with the finding by others that spermine lowers the Km of the enzyme for Mg²⁺. However, at Mg²⁺ concentrations greater than 0.3 mM, the stimulatory effect of VII was unaltered, procaine failed to alter PDH activity, lysine inhibited PDH activity, and poly-L-lysine stimulated PDH activity. Therefore, polyamines and other positively charged small molecules may be physiologic regulators of PDH activity.     

Structure‐activity relationship of indolicidin,a Trp‐rich antibacterial peptide

A series of Trp and Arg analogs of antibacterial indolicidin (Ind) was synthesized and the antimicrobial and hemolytic activities were investigated. [L⁹]Ind, [L¹¹]Ind, [K⁸,L⁹]Ind and [K^{6, 8},L⁹]Ind showed desirable characteristics, exhibiting negligible hemolytic activity while keeping strong antibacterial activity. The results indicated that the Trp residue at position 11 essentially contributes to both activities and one can not be exchanged for the other, whereas the Trp residues at positions 4 and 9 play important roles in antimicrobial and hemolytic activities, respectively. The Trp residues at positions 6 and 8 play no important roles in biological activities. We then found that the retro analog of Ind showed higher antibacterial activity than Ind against both Gram‐positive and Gram‐negative bacteria but remarkably lower hemolytic activity than that of Ind. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Oxidant-induced pHi/Ca2+ changes in rat aortic smooth muscle cells: The role of atrial natriuretic peptide

The aim of this study was to investigate the effects of oxidative stress on PLD activity, [Ca²⁺]_i and pHi levels and the possible relationship among them. Moreover, since atrial natriuretic peptide (ANP) protects against oxidant-induced injury, we investigated the potential protective role of the hormone in Rat Aortic Smooth Muscle (RASM) cells exposed to oxidative stress. Water-soluble 2,2-Azobis (2hyphen;amidinopropane) dihydrochloride (AAPH) was used as free radical generating system, since it generates peroxyl radicals with defined reaction and the half time of peroxyl radicals is longer than other ROS. A significant increase of PLD activity was related to a significant decrease in pHi, while [Ca²⁺]_i levels showed an increase followed by a decrease after cell exposure to AAPH. [Ca²⁺]_i changes and pHi fall induced by AAPH were prevented by cadmium which inhibits a plasma membrane Ca²⁺ ATPase coupled to Ca²⁺/H⁺ exchanger, that operates the efflux of Ca²⁺ coupled to H⁺ influx. The involvement of PLD in pHi and [Ca²⁺]_i changes was confirmed by calphostin-c treatment, a potent inhibitor of PLD, which abolished all AAPH-induced effects. Pretreatment of RASM cells with pharmacological concentrations of ANP attenuated the AAPH effects on PLD activity as well as [Ca²⁺]_i and pHi changes, while no effects were observed with physiological ANP concentrations, suggesting a possible role of the hormone as defensive effector against early events of the oxidative stress.     

Identification of a hexapeptide binding region in the nociceptin (ORL1) receptor by photo-affinity labelling with Ac-Arg-Bpa-Tyr-Arg-Trp-Arg-NH2

The interaction of Ac-Arg-Tyr-Tyr-Arg-Trp-Arg-NH₂ (HP1), a high-affinity partial agonist of the opioid receptor like (ORL1) receptor, has been investigated using the photo-labile analogue [p-benzoyl-l-Phe (Bpa)²]-HP1. In recombinant CHO cells expressing the human ORL1 receptor, [Bpa²]-HP1 binds the receptor with high affinity (K; ∼3 nM) and is as potent as HP1 in stimulating GTPγS binding (50-60% of nociceptin maximal effect). UV irradiation at 365 nm of the complex formed by the ORL1 receptor and radio-iodinated [Bpa²]-HP1 results in the irreversible labelling of a glycoprotein of M_r∼66 kDa, as determined by SDS-PAGE. Cyanogen bromide (CNBr) and enzymatic footprints of the photo-labelled receptor and an engineered receptor mutant (L113M), containing an additional CNBR cleavage site, allowed the photoreactive region to be identified as ORL1[107-113] at the C-terminal of TM helix II. In addition the presence of a disulphide bridge between Cysl23 and Cys200 has been confirmed biochemically.     

Cell calcium signalling induced by endogenous lectin carbohydrate interaction in the Jurkat T cell line

The effects of the -galactoside-binding lectin from human placenta (HPL14) on intracellular calcium concentration ([Ca²⁺]_i) were examined in the human Jurkat T cell line. The lectin induces a concentration dependent increase in [Ca²⁺]_i. This calcium signalling effect is clearly mediated through complementary cell surface galactoglycoconjugates because it can be blocked by -galactosides. The observed Ca²⁺-response involves both the release of calcium from intracellular stores and a calcium influx from the extracellular space. It is sustained in the presence of 1 mM extracellular calcium whereas it becomes transient when the influx of extracellular calcium was blocked by calcium chelation to EGTA. Voltage-sensitive calcium channel blockers like verapamil and prenylamine were without effect on the action of HPL14. Protection of the sugar binding activity of HPL14 in the absence of a thiol-reducing reagent by carboxamidomethylation (CM-HPL14) or by substitution Cys2 with serine (C2S) results in lectin proteins with considerably decreased calcium signalling efficiency. The recombinant lectin (Rec H) and the mutant protein obtained by substitution of highly conservative Trp68 with tyrosine (W68Y) induce lower levels of [Ca²⁺]_i compared to wild type lectin.Abbreviation [Ca²⁺]_i concentration of intracytoplasmic free calcium - CM carboxamidomethylation - CRD earbohydrate recognition domain - C2S mutant lectin protein in which Cys2 was replaced by serine - EGTA ethyleneglycol-bis(2-aminoethylether)-N,N,N - N-tetraacetic acid HEPES,N-(2-hydroxyethyl)piperazine-N-2-ethanesulfonic acid - HPL14 human -galactoside-binding placental lectin - Rec H recombinant human 14 kDa lectin - W68Y mutant lectin protein in which Trp68 was substituted to tyrosine     

Acetyl-L-Carnitine Arginine Amide Prevents β 25–35-Induced Neurotoxicity in Cerebellar Granule Cells

Cerebellar granule cells (CGC) at different stages of maturation in vitro (1 or 6 DIV), were treated with 25–35 and acetyl-L-carnitine arginine amide (ST857) in presence of 25 mM KC1 in the culture medium, and neuronal viability was assessed. Three days of treatment slightly modified the survival of 1 DIV-treated cells, which degenerate and die five days later -amyloid matching. Similarly, a significative neurotoxic effect was observed on 6 DIV treated-cells after 5 days of exposure to the peptide, while the death occurred within 8 days. ST857 coincubated with 25–35 was able to rescue neurons from 25–35-induced neurotoxicity. We also studied the changes in Ca²⁺ homeostasis following glutamate stimulation, in control and -amyloid treated single cells, either in presence or in absence of ST857. 25–35 did not affect basal [Ca²⁺]_i, while modified glutamate-induced [Ca²⁺]_i increase, causing a sustained plateau phase of [Ca²⁺]_i, that persisted after the removal of the agonist. ST857 pretreatment completely reverted this effect suggesting that, in CGC chronically treated with 25–35, ST857 could protect the cells by neurotoxic insults of the peptide likely interfering with the cellular mechanisms involved in the control of Ca²⁺ homeostasis.     

Uptake,exchange, and release of GABA by cerebellar glomeruli

Glomerular particles were isolated from the bovine cerebellar vermis and studied in vitro to further assess the possibility that -aminobutyric acid (GABA) is utilized as a neurotransmitter in this synaptic complex. Cerebellar glomeruli accumulated [³H]GABA at two different high affinity sites, with affinities (K _T) of 2.2×10^–6M and 3.0×10^–5M. These uptake sites could not be distinguished on the basis of their temperature sensitivities, sodium dependence, substrate specificities or responses to metabolic inhibitors. Although an exchange process contributed to the uptake measured in these experiments, a considerable amount of the [³H]GABA accumulated by glomerular particles was stored in an osmotically-sensitive, nonexchangeable pool. Glomerular particles preloaded with [³H]GABA exhibited a Ca²⁺-independent release of this amino acid in response to membrane depolarization. However, when preloaded glomerular particles were exposed to unlabeled GABA, which presumably displaced [³H]GABA from the exchangeable pool, a K^–-evoked and Ca²⁺-dependent release of the remaining [³H]GABA occurred. The observed net uptake, together with the depolarization-induced and Ca²⁺-dependent release, of [³H]GABA from glomerular particles supports the suggestion that functionally active GABAergic synapses are present in these structures.     

The pro-apoptotic action of new analogs of the insect gonadoinhibiting peptide Neb-colloostatin: Synthesis and structure–activity studies

Neb-colloostatin (SIVPLGLPVPIGPIVVGPR), an insect oostatic factor found in the ovaries of the flesh fly Neobellieria bullata, strongly induces apoptosis in insect haemocytes. To explain the role of Ser¹ and Pro⁴ residues of Neb-colloostatin in the pro-apoptotic activity of this peptide, the synthesis of a series of analogs was performed, such as: [Ac-Ser¹]- (1), [d-Ser¹]- (2), [Thr¹]- (3), [Asp¹]- (4), [Glu¹]- (5), [Gln¹]- (6), [Ala¹]- (7), [Val¹]- (8), [d-Pro⁴]-(9), [Hyp⁴]- (10), [Acp⁴]- (11), [Ach⁴]- (12), [Ala⁴]- (13), [Ile⁴]- (14), and [Val⁴]-colloostatin (15). All peptides were bioassayed in vivo for the pro-apoptotic action on haemocytes of Tenebrio molitor. Additionally, the structural properties of Neb-colloostatin and its analogs were examined by the circular dichroism in water and methanol. Peptides 1, 4, 5, 7, 8, 10, 12, 14, and 15 strongly induce T. molitor haemocytes to undergo apoptosis and they show about 120–230% of the Neb-colloostatin activity at a dose of 1 nM. The CD conformational studies show that the investigated peptides seem to prefer the unordered conformation.