ISOLATION, CHEMICAL AND BIOLOGICAL CHARACTERIZATION OF MARGARATENSIN, A NEUROTENSIN-RELATED PEPTIDE FROM THE SKIN OF Rana margaratae

A new neurotensin (NT)-related peptide, margaratensin, was obtained by Sep-Pak C_(18) and RP-HPLC from methanol extracts of the skin of Chinese frog Rana margaratae. The structure of the peptide has been determined to be Asp-Lys-Arg-Pro-Tyr-lle-Leu-His-Glu, which is found to be homologous to the COOH-terminal sequence of NT, but has an extra His-Glu at the COOH-terminus. The synthetic preparation was shown to be indistinguishable from the native peptide during HPLC, amino acid analysis and bioassay. Margaratensin exhibited a hypotensive effect in the rat but the response was weaker than NT. The peptide could induce a potent and reproducible contractile activity on GPI which was different from xenopsin, another NT-related peptide from amphibian skin.     

Multivalent Ligands for Inducing Receptor-Receptor Interactions

To obtain enchanced and/or unique cellular responses to peptide hormones by simultaneous activation of two different receptor systems, chimera peptide hormones in which enkephalin (EK) is connected through sarcosine oligomer (n) to the message segment of neurotensin [NT(8–13)] were synthesized. Interaction of these two receptor systems was studied by receptor-binding assay and determination of cAMP and cGMP level in cytosol of NG108-15 cells. It was found that EK-12-NT(8- 13) induced specific cell response in terms of the second messenger level. The specific cell response was explained in terms of the enhancement of receptor-receptor interaction in cytosol due to activation of nearby occurring receptors of different kinds. It was also found that EK-12- NT(8-13) has a high affinity toward neurotensin receptor but a low affinity toward enkephalin receptor. To strengthen and prolong the activity of peptide hormones by simultaneous binding with two or more receptors in the target cell, enkephalin/lipid conjugates were immobilized on the surface of polymerized liposomes. It was found that multivalent ligands with a high receptor affinity were synthesized, which was sensibly influenced by the presence of a spacer chain connecting the enkephalin unit to the lipid part. It was found that the introduction of anionic charges to the polymerized liposomes strongly affected the receptor affinity of immobilized ligands.     

Detection of testosterone,nandrolone and precursors in horse hair

Growing interest among several horse-breeder associations has initiated the development of a screening procedure to test for anabolic agents in hair, which has the advantage over blood and urine specimens of allowing long-term detection. An analytical method was established to monitor in tails or manes several anabolic substances available as veterinary medicines or as so-called nutritional supplements (clenbuterol, different esters or prohormones of nandrolone and testosterone). The analytical procedure to detect steroids in hair samples consists of the following steps: decontamination of the hair strand or segment with methanol/water (1:1), milling, extraction of the hair material in an ultrasonic bath using methanol, purification by liquid–liquid extraction (n-pentane/methanol, 25:1) and HPLC cleanup, derivatisation of the relevant LC fractions with MSTFA, and measurement using GC-MS/MS technique. The first objective of our study was the detection of exogenous nandrolone (nortestosterone, NT) in the horse hair; therefore nandrolone-associated compounds [nandrolone dodecanoate administered intramuscularly (i.m.) and a mixture of 4-estrenediol and 4-estrenedione, transdermal] were administered to four geldings. The highest concentrations of NT following i.m. treatment were measured after 10 days in a 2-cm hair segment (up to 18 pg/mg); NT was detectable for up to 120 days and in some cases up to 330 days in tail hair (limit of detection 0.3 pg/mg). Following transdermal application, nandrolone as well as the administered prohormones were identified in tail and mane until the latest sampling at 3 months. Furthermore, untreated stallions (128) were investigated to estimate the range of endogenous levels of NT and testosterone (T) in hair. Maximum values of 3 pg/mg (NT) and 1 pg/mg (T) were quantified originating from endogenous formation in the male horse. Additionally, a possible relationship between steroid concentrations in hair specimens and the age of stallions was appraised. NT and T were not detected in hair samples of control geldings. Following nandrolone treatment of geldings, highest values in hair exceeded the endogenous amount detected in untreated stallions. Therefore comparison of concentrations measured in control samples with the estimated endogenous levels could give a clue to exogenous application in cases of abnormally high amounts of NT or T. The possibility of the evaluation of threshold values is discussed as a means to verify an exogenous administration of NT and T in hair samples. Furthermore, the detection of a synthetic substance in hair, e. g. the parent steroid ester by itself, would be unequivocal proof of an exogenous origin of NT or T and the previous medication of the stallion.     

Development of an efficient transdermal delivery system of small interfering RNA using functional peptides, Tat and AT-1002

Topical use of small interfering RNA (siRNA) as a therapeutic nucleic acid is increasingly studied for the treatment of skin diseases and for the improvement of skin properties. However, naked siRNA transdermal delivery is limited by its low stability in the body and low permeability into target cells. This is due to various skin barriers such as the stratum corneum that has multiple lipid bilayers and epidermal layers that have tight junctions. In this study, we investigate non-invasive transdermal siRNA delivery using two functional peptides: AT1002, which is a tight junction modulator and 6-mer synthetic peptide belonging to a novel class of compounds that reversibly increases paracellular transport of molecules across the epithelial barrier; and Tat, which is a cell-penetrating peptide applicable as a transdermal siRNA delivery enhancer. We examined whether expression of the tight junction protein zonula occludens protein 1 (ZO-1) was detected in mouse skin applied with AT1002. Additionally, siRNA stabilities for RNaseA using Tat and AT1002 were assessed. We also determined the intradermal delivery efficiency of siRNA using functional peptides by confocal laser microscopy of fluorescently labeled siRNA in mouse skin. We found that the Tat analog and AT1002 strongly increased siRNA stability against RNaseA. In addition, ZO-1 disappeared from the skin after treatment with AT1002, yet recovered with time after washing. Finally, we also found that Tat and AT1002 peptides accelerate transdermal siRNA delivery both widely and effectively. Thus, combination of Tat and AT1002 is expected to be a transdermal delivery enhancer of siRNA.     

Investigation on the dynamics of electron transport and recombination in TiO2 nanotube/nanoparticle composite electrodes for dye-sensitized solar cells

In this work, we report on fabrication and characterization of dye-sensitized solar cells based on TiO(2) nanotube/nanoparticle (NT/NP) composite electrodes. TiO(2) nanotubes were prepared by anodization of Ti foil in an organic electrolyte. The nanotubes were chemically separated from the foil, ground and added to a TiO(2) nanoparticle paste, from which composite NT/NP electrodes were fabricated. In the composite TiO(2) films the nanotubes existed in bundles with a length of a few micrometres. By optimizing the amount of NT in the paste, dye-sensitized solar cells with an efficiency of 5.6% were obtained, a 10% improvement in comparison to solar cells with pure NP electrodes. By increasing the fraction of NT in the electrode the current density increased by 20% (from 11.1 to 13.3 mA cm(-2)), but the open circuit voltage decreased from 0.78 to 0.73 V. Electron transport, lifetime and extraction studies were performed to investigate this behavior. A higher fraction of NT in the paste led to more and deeper traps in the resulting composite electrodes. Nevertheless, faster electron transport under short-circuit conditions was found with increased NT content, but the electron lifetime was not improved. The electron diffusion length calculated for short-circuit conditions was increased 3-fold in composite electrodes with an optimized NT fraction. The charge collection efficiency was more than 90% over a wide range of light intensities, leading to improved solar cell performance.     

Lanthiopeptin,a new peptide effective against herpes simplex virus: Structural determination and comparison with Ro 09-0198, an immunopotentiating peptide

The structure of lanthiopeptin, a new lanthionine peptide effective against Herpes virus, was newly determined. It has a unique tetracyclic sequence which is very similar to but different from the proposed structure for Ro 09-0198. However, Ro 09-0198 was found to be quite the same compound as lanthiopeptin in all respects.     

Isolation and structure of ranamargarin, a new tachykinin from the skin of Chinese frog Rana margaratae

A new tetradecapeptide, ranamargarin, has been isolated by Sep-Pak C18 and HPLC from methanol extracts of the skin of the Chinese frog Rana margaratae. The sequence of the peptide is: Asp-Asp-Ala-Ser-Asp-Arg-Ala-Lys-Lys-Phe-Tyr-Gly-Leu-Met-NH2. This structure has been confirmed by synthesis. The peptide is the largest among the amphibian tachykinins and its N-terminal amino acids are quite different from those of the other tachykinins. The formation of the sulfoxide and peak-splitting of ranamargarin during purification procedures are briefly discussed.     

Photobleaching of hypocrellin B and its butylamino-substituted derivative in solutions

Peptides derived from the heavy chain of the HLA Class-I molecules have been shown to modulate immune responses both in vivo and in vitro. Using a computer-aided rational drug design approach, novel immunomodulatory peptides were designed based on peptide 2702.75-85, derived from HLA-B2702. Several peptides were identified which had increased immunomodulatory activity, including peptides RDP1258 and its D-isomer the peptide Allotrap 1258. The present study using Skh/hr hairless mouse skin model evaluated the in vivo effects of Allotrap 1258 on acute UVB-induced skin inflammation. Here we demonstrate that intraperitoneal administration of Allotrap 1258 1 h prior to UV exposure resulted in significantly diminished levels of UV-induced tumor necrosis factor (TNF)-alpha protein production in the epidermis but had no effect on other parameters of the acute UV-induced inflammatory response. By virtue of its ability to suppress TNF-alpha protein production, Allotrap 1258 could prove to be an effective modulator of inflammatory responses.     

ISOLATION AND STRUCTURE OF RANAMARGARIN,A NEW TACHYKININ FROM THE SKIN OF CHINESE FROG Rana margaratae

A new tetradecapeptide, ranamargarin, has been isolated by Sep-Pak C_(18) and HPLC from methanol extracts of the skin of the Chinese frogRana margaratae. The sequence of the peptide is: Asp-Asp-Ala-Ser-Asp-Arg-Ala-Lys-Lys-Phe-Tyr-Gly-Leu-Met-NH_2. This structure has been confirmed by synthesis. The peptide is the largest among the amphibian tachykinins and its N-terminal amino acids are quite different from those of the other tachykinins. The formation of the sulfoxide and peak-splitting of ranamargarin during purification procedures are briefly discussed.     

Multiplexed Determination of Amino‐Terminal Pro‐B‐Type Natriuretic Peptide and C‐Reactive Protein Cardiac Biomarkers in Human Serum at a Disposable Electrochemical Magnetoimmunosensor

A rapid magnetoimmunosensor for the simultaneous determination of two cardiac biomarkers, amino‐terminal pro‐B‐type natriuretic peptide (NT‐proBNP) and C‐reactive protein (CRP), in human serum is described. Specific capture antibodies were covalently immobilized onto carboxylic acid‐modified magnetic beads. The quantification of NT‐proBNP and CRP was performed by using indirect competitive and sandwich configurations, respectively, and horseradish peroxidase‐labeled tracers. The use of dual screen‐printed carbon electrodes allowed the achievement of simultaneous independent amperometric readout for each cardiac biomarker. The developed methodology showed very low limits of detection (0.47 ng mL^?1). An international standard for CRP serum spiked with NT‐proBNP was analyzed to evaluate the usefulness of the magnetoimmunosensor.     

Tandem ligation of unprotected peptides through thiaprolyl and cysteinyl bonds in water.

Tandem ligation for the synthesis and modification of proteins entails forming two or more regiospecific amide bonds of multiple free peptide segments without a protecting-group scheme. We here describe a semi-orthogonal strategy for ligating three unprotected peptide segments, two of which contain N-terminal (NT) cysteine, to form in tandem two amide bonds, an Xaa-SPro (thiaproline), and then an Xaa-Cys. This strategy exploits the strong preference of an NT-cysteinyl peptide under acidic conditions to undergo selectively an SPro-imine ligation rather than a Cys-thioester ligation. Operationally, it was performed in the N --> C direction, first by an imine ligation at pH < 3 to afford an Xaa-thiazolidine ester bond between a peptide containing a carboxyl terminal (CT)-glycoaldehyde ester and a second peptide containing both an NT-Cys and a CT-thioester. The newly created O-ester-linked segment with a CT-thioester was then ligated to another NT-cysteinyl peptide through thioester ligation at pH > 7 to form an Xaa-Cys bond. Concurrently, this basic condition also catalyzed the O,N-acyl migration of an Xaa-thiazolidine ester to the Xaa-SPro bond at the first ligation site to complete the tandem three-segment ligation. Both ligation reactions were performed in aqueous buffered solvents. The effectiveness of this three-segment ligation strategy was tested in six peptides ranging from 19 to 70 amino acids, including thiaproline --> proline analogues of somatostatins and two CC-chemokines. The thiaproline replacements in these peptides and proteins did not result in altered biological activity. By eliminating the protecting-group scheme and coupling reagents, tandem ligation of multiple free peptide segments in aqueous solutions enhances the scope of protein synthesis and may provide a useful approach for combinatorial segment synthesis.     

Matrix assisted laser desorption ionisation ion mobility separation mass spectrometry imaging of ex-vivo human skin

Matrix assisted laser desorption ionisation ion mobility separation mass spectrometry imaging (MALDI-IMS-MSI) has been employed to image the distribution of proteins in ex-vivo human skin. Using a “bottom-up” proteomics approach employing “on-tissue” digestion the distribution of abundant skin proteins; collagen, keratin, decorin and serum albumin could be mapped. Images have been recorded at 150 and 30 μm spatial resolution. Multivariate statistical analysis of the data has been employed to associate specific proteins with layers of the skin. The improved specificity given by the use of ion mobility separation in mass spectrometric imaging has been demonstrated by separation of peptide ions from phospholipids.     

Hydrolytic cleavage of pyroglutamyl-peptide bond. V. selective removal of pyroglutamic acid from biologically active pyroglutamylpeptides in high concentrations of aqueous methanesulfonic acid

Application of aqueous methanesulfonic acid (MSA) for selective chemical removal of pyroglutamic acid (pGlu) residue from five biologically active pyroglutamyl-peptides (pGlu-X-peptides, X=amino acid residue at position 2) was examined. Gonadotropin releasing hormone (Gn-RH), dog neuromedin U-8 (d-NMU-8), physalaemin (PH), a bradykinin potentiating peptide (BPP-5a) and neurotensin (NT) as pGlu-X-peptides were incubated in either 70% or 90% aqueous MSA at 25 degrees C. HPLC analysis of the incubation solutions showed that the main decomposition product was H-X-peptide derived from each pGlu-X-peptide by the removal of pGlu. The results revealed that the pGlu-X peptide bond had higher susceptibility than various internal amide bonds in the five peptides examined, including the Trp-Ser bond in Gn-RH, the C-terminal Asn-NH(2) in d-NMU-8, and the Asp-Pro bond in PH, whose acid susceptibility is well known. Thus, mild hydrolysis with high concentrations of aqueous MSA may be applicable to chemically selective removal of pGlu from pGlu-X-peptides for structural examinations.     

Temperature dependent exciton emission from herringbone aggregates of conjugated oligomers

In this work, the effect of temperature, exciton bandwidth, and size on the photoluminescence spectra of defect-free two-dimensional herringbone aggregates of pi-conjugated oligomers such as oligophenylene vinylene and oligothiophene is investigated theoretically. The model is based on exciton-phonon coupling in two-dimensional herringbone lattices with the exciton deriving from the lowest optical (1Ag-->1Bu) transition and the phonon from the most strongly coupled intramolecular vibrational mode with frequency omega0. Simple analytical expressions are obtained for the line strengths of the emission origin (0-0) and first replica (0-1) as a function of the number of molecules comprising the aggregate, N, the free exciton bandwidth, WD, and the temperature, T. At a given temperature, the 0-0 emission intensity initially scales as N/Nth, where Nth is the superradiant threshold number, but eventually converges to NT/Nth, where NT is the size independent thermal coherence number. NT is inversely proportional to temperature and proportional to the exciton band curvature (omegac) near the band bottom; NT=1+4piomegac/kbT. In striking contrast, the 0-1 line strength is relatively insensitive to temperature and size, but scales as the inverse square of WD+omega0. The insensitivity of the first replica to the exciton coherence number makes the ratio of the 0-0 to 0-1 line strengths a measure of the exciton coherence number. The ratio can be used to test for crystal purity. Comparison to experiments on thin films of quaterthiophene shows that the thermal coherence size is given by NT approximately 1+450/T (K) and that superradiance, which requires NT>Nth, can only be observed at temperatures less than 1 K.     

Distinctive Molecular Abnormalities in Benign and Malignant Skin Lesions: Studies by Raman Spectroscopy

Abstract— Near-infrared Fourier transform Raman spectroscopy is an analytical, nondestructive technique that provides information about the molecular structure of the investigated sample. The molecular structure of proteins and lipids differs between neoplastic and normal tissues and therefore Raman spectroscopy has been considered promising for the diagnosis of cancer. We aimed to compare the molecular structure of normal skin, benign and malignant skin lesions by the near-infrared Fourier transform Raman spectroscopy. Biopsies were obtained from the following skin lesions: skin tag, dermatofibroma, seborrhoeic keratosis, actinic keratosis, keratoacan-thoma, basal cell carcinoma, squamous cell carcinoma, nevus intradermal, nevus compositus, dysplastic nevus and lentigo maligna. Control skin was harvested from the vicinity of these lesions. In the Raman spectra, the secondary structure of the proteins was reflected by the amide vibrations of peptide bonds. The principal lipid vibrations were twisting and wagging (CH₂) and CH stretching vibrations. Histologically distinguishable lesions showed specific combinations of band changes indicating alterations in the protein conformation and in the molecular structure of the lipids. Histogenetically related lesions (actinic keratosis and sqamous cell carcinoma) produced similar but not identical patterns of spectral changes. Because the examined skin lesions produced reproducible and unique spectra, we suggest that Raman spectroscopy will be useful for diagnosis of skin lesions.     

PCR-based Gene Synthesis,Molecular Cloning,High Level Expression,Purification, and Characterization of Novel Antimicrobial Peptide,Brevinin-2R,in <Emphasis Type="Italic">Escherichia Coli</Emphasis>

Brevinin-2R, a member of a new family of antimicrobial peptides isolated from the skin of Rana ridibunda, displays antimicrobial activity against bacteria and fungi. In this study, we have used an assembly PCR method for the fast and extremely accurate synthesis of the brevinin-2R gene. A total of six primers were assembled in a single step PCR, and the assembly was then amplified by PCR to produce the final gene. The synthetic gene was cloned into the pET32a (+) vector to allow the expression of brevinin-2R as a Trx fusion protein in Escherichia coli. The results indicated that the expression level of the fusion protein could reach up to 25% of the total cell proteins. The expression products could be easily purified by Ni-NTA chromatography and released from the fusion protein by factor Xa protease. The peptide displayed antimicrobial activity similar to that of the purified brevinin that was reported earlier. This method allows the fast synthesis of a gene that optimized the overexpression in the E. coli system and production of sufficiently large amounts of peptide for functional and structural characterizations.     

Wavelength dependence of histological, physical, and visible changes in chronically UV-irradiated hairless mouse skin

Albino hairless mice (Skh: HR-1) exposed chronically to sub-erythemal doses of UV radiation display physical, visible and histological alterations. Using narrow bandwidth radiation covering the UV radiation spectrum from 280-380 nm, the wavelength dependence of these alterations was determined. The wavelength dependence spectra indicate that for all but one parameter measured (skin sagging), UV-B radiation is considerably more efficient than UV-A radiation in producing changes in the skin. However, in natural sunlight there is considerably more UV-A than UV-B radiation, providing the potential for UV-A to have a larger contribution to skin damage than UV-B. This argues in favor of using broad spectrum photoprotective agents to shield the skin adequately from UV-induced aging. The spectra were also used to develop potential associations among events by determining which events occur at similar wavelengths. There seems to be a correspondence between mouse visible skin wrinking (UV-B event) and two histological events: increase in glycosaminoglycans and alteration in collagen. There was no obvious correspondence among UV-A-induced events.     

Regioselective conjugation of chitosan with a laminin-related peptide, Tyr-Ile-Gly-Ser-Arg, and evaluation of its inhibitory effect on experimental cancer metastasis

A conjugate from the YIGSR peptide and chitosan has been prepared on the basis of a regioselective modification strategy of chitosan, and its antimetastatic activity has been assayed. Chitosan was converted to its organosoluble derivative, 6-O-trityl-chitosan, in 3 steps, and then coupled with the peptide portion containing a spacer amino acid, Ac-Tyr-Ile-Gly-Ser-Arg-beta Ala-OH [beta Ala; beta-alanine]. The product was treated with CHCl2CO2H to afford the desired conjugate, Ac-Tyr-Ile-Gly-Ser-Arg-beta Ala-chitosan, which proved to inhibit the experimental lung metastasis of B16BL6 melanoma cells in mice at lower doses than the parent peptide.     

Radiofrequency Irradiation Modulates TRPV1-Related Burning Sensation in Rosacea

Rosacea is a skin inflammatory condition that is accompanied by not only redness and flushing but also unseen symptoms, such as burning, stinging, and itching. TRPV1 expression in UVB-exposed skin can lead to a painful burning sensation. Upregulated TRPV1 expression helps release neuropeptides, including calcitonin gene-related peptide, pituitary adenylate cyclase-activating polypeptide, and vasoactive intestinal peptide, which can activate macrophage and inflammatory molecules. In this study, we found that radiofrequency (RF) irradiation reduced TRPV1 activation and neuropeptide expression in a UVB-exposed in vivo model and UVB- or heat-treated in an in vitro model. RF irradiation attenuated neuropeptide-induced macrophage activation and inflammatory molecule expression. Interestingly, the burning sensation in the skin of UVB-exposed mice and patients with rosacea was significantly decreased by RF irradiation. These results can provide experimental and molecular evidence on the effective use of RF irradiation for the burning sensation in patients with rosacea.     

An unusual kynurenine-containing opioid tetrapeptide from the skin gland secretion of the Australian red tree frog Litoria rubella. Sequence determination by electrospray mass spectrometry

The Kyn-containing peptide FP-Kyn-L(NH(2)) is an unusual minor component of the skin peptide profile of the Australian red tree frog Litoria rubella collected from an area within a 20 kilometre radius of Alice Springs in central Australia. The structure was determined by electrospray mass spectrometry and synthesis. The major component of the skin secretion is the analogous tryptophyllin peptide FPWL(NH(2)). Both peptides show opioid activity at 10(-7) M, and are likely to act via the μ opioid receptor.