De novo sequencing of peptides secreted by the skin glands of the Caucasian Green Frog Rana ridibunda

Amphibian skin glands are known to secrete various types of bioactive peptides. The array of these peptides is specific for every frog species. The present research deals with the identification of peptides isolated from the skin secretion of the Marsh frog R. ridibunda inhabiting the Kolkhida Canyon of the Caucasian region. The research is based on comprehensive high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) analysis of intact and chemically modified peptides. In particular, an oxidation procedure was applied directly to the crude skin secretion to open S--S loops whereas N-terminal acetylation was additionally carried out for one individual peptide. Sequences were determined by manual interpretation of electron capture dissociation (ECD) and collisionally induced dissociation (CID) tandem mass spectra. A total of 29 peptides were identified in the skin secretion of the Caucasian Marsh frog. The peptide profile is represented with disulfide-containing peptides belonging to the brevinin, esculentin and ranatuerin families, neuropeptides of the bradykinin and bombesin families. Two identified peptides belonging to the ranatuerins are the first peptides of this family discovered in the skin secretions of European frogs. Ten of the identified peptides coincide with those reported earlier for the European Edible frog. Another ten are identical to those found in R. ridubunda from the Moscow region. This fact verifies the described method as being an efficient analytical tool to compare intra- and interspecific variabilities.     

Analysis of the nerve growth activities of protein fractions from the venoms of Agkistrodon halys halys and Echis multisquamatus

The possibility has been shown of obtaining NGFs in the process of isolating coagulant proteinases and other biologically active substances from the venoms of theAgkistrodon halys halys and theEchis multisquamata. Results characterizing the NGFs of both species as basic (weakly basic) proteins with molecular masses of 30.0–40.0 kDa are presented.Institute of Biochemistry, Uzbekistan Academy of Sciences, Tashkent. Translated from Khimiya Prirodnykh Soedinenii, No. 2, pp. 266–270, March-April, 1992.     

A new iridoid glycoside with nerve growth factor-potentiating activity,gelsemiol 6'-trans-caffeoyl-1-glucoside,from Verbena littoralis

A new iridoid glycoside, gelsemiol 6'-trans-caffeoyl-1-glucoside (1), was isolated from Verbena littoralis, together with four known phenylethanoid glycosides, acteoside (2), 2'-acetylacteoside (3), jionoside (4), and isoverbascoside (5). Their structures were elucidated by spectral data. Compound 1 showed weak enhancement of nerve growth factor (NGF)-mediated neurite outgrowth from PC12D cells.     

Three-dimensional model of biomatrix as a method of studying blood vessels and nerve growth in tissue engineering structures

The method involves isolating and culturing ex vivo cultures of mouse dorsal root ganglia and abdominal aorta in a three-dimensional gel (Matrigel). Unlike other explant cultures, this technique allows to study the physiological and biochemical processes in the tissue explants of dorsal root ganglia and abdominal aorta in three-dimensional space but in contrast to a two-dimensional adhesive culture. Administration of the tested substances to the Matrigel allows analyzing their effects on the growth of blood vessels and neurites from the explants for 21 days. The developed method can be applied in modern cardiological and neurobiological research and explores how new therapeutic agents can accelerate the regeneration of blood vessels and nerves.     

A hevein-like protein and a class I chitinase with antifungal activity from leaves of the paper mulberry

Paper mulberry (Broussonetia papyrifera, syn. Morus papyrifera L.) is a Chinese traditional medicine and its low‐molecular‐weight extracts are reported to have antifungal activity. In this study, two proteins (PMAPI and PMAPII) with activity against Trichoderma viride were obtained from paper mulberry leaves with a fast protein liquid chromatography (FPLC) unit. The purification protocol employed (NH₄)₂SO₄ precipitation, ion‐exchange chromatography and hydrophobic‐interaction chromatography on FPLC. Molecular masses were 18,798 Da for PMAPI, and 31,178 Da for PMAPII determined by Matrix‐assisted laser desorption ionization time‐of‐flight mass spectrometry. Peptide mapping fingerprint analysis showed that PMAPI has no peptides similar to PMAPII. N‐terminal amino acid sequencing revealed that PMAPI is a hevein‐like protein, and PMAPII is a class I chitinase. They both had a half‐maximal inhibitory concentration (IC50) of 0.1 µg/µL against T. viride. This is the first report of high‐molecular‐weight extracts with antifungal activity from paper mulberry. Copyright © 2011 John Wiley & Sons, Ltd.     

Physicochemical analysis of a kerogen rock (oil shale)

A composition of a kerogen was studied with the use of chemical, physical, and spectral (IR, NMR, XRD, XPS, and ESR) methods. A yield and main characteristics of kerogen were found.     

Isolation of a protein with diaphorase activity from cotton seeds and a study of some of its properties

The diaphorase activities of the proteins of different varieties of two species of cotton plant —Gossypium hirsutum L. andG. barbadense L. — have been studied. It has been shown that the proteins of the seeds of the cotton plantG. barbadense possess a low diaphorase activity in comparison with the proteins of the seeds of aG. hirsutum plant. On an electrophoretogram of the proteins, diaphorase activity was localized in two zones, with R_f 0.45 and 0.70. The diaphorase with R_f 0.45 has been isolated by electrophoresis in polyacrylamide gel (PAAG) and some of its properties have been studied. The diaphorase isolated oxidizes NADH and NADPH in the presence of various artificial electron Acceptors, and has two pH optima (at 7.20 and 8.70) and is characterized by relative thermal stability (at 80°C). In the case of the total extract, brief boiling does not lead to inactivation of the enzyme, which shows the presence in cotton seeds of a factor stabilizing this diaphorase. The molecular weight of the proteins isolated, according to gel filtration on Sephadex G-150, is 59,000, and from the results of SDS-PAAG electrophoresis it is 13,600, which shows a tetrameric structure of the enzyme.Institute of Experimental Plant Biology, Academy of Sciences of the Uzbek SSR, Tashkent. Translated from Khimiya Prirodnykh Soedinenii, No. 3, pp. 416–421, May–June, 1987.     

Biological activity of the protein complex from soy meal

The amino-acid composition of the active fraction of a protein complex was determined. An analysis of the N-terminal amino acids found threonine, glycine, serine, proline, and valine at N-terminuses. The protein complex at a dose of 100 μg/mL suppressed incorporation of ¹⁴C-thymidine marker by 64.0 ± 1.9% in a radiometric cytotoxicology test for KML murine melanoma cell proliferation. The protein complex contained protease inhibitors, inhibited the growth of tumor strain AKATON by greater than 60%, and did not inhibit bone marrow cell proliferation.     

Purification of nerve growth factor from the venom of the Central Asian cobraNaja oxiana

A highly purified electrophoreticaly homogeneous protein with a NGF activity of 10·10⁵ BU/mg of protein have been isolated from the venom of the Central Asian cobra by gel-filtration and ion-exchange chromatography followed by preparative isolectric focusing in a thin layer of Sephadex. It has been shown that the NGF isolated is characterized by a molecular weight in the range of 20–30 kD and a pI value of about 7.0.     

Purification of decorin core protein from human lung tissue

A chromatographic method to purify decorin core protein from human lung tissue is described. The method is simple and rapid, using a combination of two-anion exchange and one reversed phase chromatography steps and the enzymatic digestion with chondroitinase ABC. Approximately 170 microg decorin core protein were purified from 25 g of lung tissue with an enrichment factor of 1800-fold relative to the initial protein content. SDS-PAGE analysis of the final product revealed a single 42 kDa protein band, which was recognized by anti-decorin antibodies upon Western blotting and identified by mass spectrometry. Further digestion with PNGase F evidenced the presence of three N-linked oligosaccharides on the core protein. This method forms the basis for studying structural alterations of decorin related to the pathology of diseases where tissue destruction plays a role.     

Biosensor analysis of beta-lactams in milk using the carboxypeptidase activity of a bacterial penicillin binding protein

The applicability of a beta-lactam receptor protein for detection of beta-lactam antibiotics in milk using surface plasmon resonance (SPR) biosensor technology was investigated. The advantage of using a receptor protein instead of antibodies for detection of beta-lactams is that a generic assay, specific for the active form of the beta-lactam structure, is obtained. Two assays based on the enzymatic activity of the DD-carboxypeptidase from Actinomadura R39 were developed, using a Biacore SPR biosensor. The carboxypeptidase converts a tri-peptide into a di-peptide, a reaction which is inhibited in the presence of beta-lactams. Polyclonal antibodies against the 2 peptides were developed and used to measure the amount of enzymatic product formed (di-peptide assay) or the amount of remaining enzymatic substrate (tri-peptide assay), respectively. The 2 assays showed similar performances with respect to detection limits (1.2 and 1.5 microg/kg, respectively) and precision (coefficient of variation <5%) for penicillin G in milk. Several other beta-lactams were detected at or near their respective maximum residue limit. Furthermore, the 2 peptide assays were evaluated against 5 commercial kit tests in the screening of 195 producer milk samples. The biosensor assays showed 0% false-negative and 27% false-positive results, whereas the figures were 0% false-negative and 27-53% false-positive results for other screening tests investigated.     

Identification of a light-regulated protein kinase activity from seedlings of Arabidopsis thaliana

Protein kinase transduction pathways are thought to be involved in light signaling in plants, but other than the photoreceptors, no protein kinase activity has been shown to be light-regulated in vivo. Using an in-gel protein kinase assay technique with histone H III SS as an exogenous substrate, we identified a light-regulated protein kinase activity with an apparent molecular weight ca 50 kDa. The kinase activity increased transiently after irradiation of dark-grown seedlings with continuous far red light (FR) and blue light (B) and decreased after irradiation with red light (R). The maximal activation was achieved after 30 min to 1 h with FR or B. After irradiation times longer than 2 h, the kinase activity decreased to below the sensitivity level of the assay. In Arabidopsis mutants lacking either the photoreceptors phytochrome A, phytochrome B or the blue-light receptor cryptochrome 1, kinase activity was undetectable, whereas in the photomorphogenic mutants cop1 and det1 the kinase activity was also observed in the absence of light signals, though still stimulated by B and FR. Interestingly, the R inhibition of the kinase activity was lost in the mutant hy5. Pretreatment with cycloheximide blocked the kinase activity.     

Physicochemical features and antioxidant activity of polysaccharides from Herba Patriniae by gradient ethanol precipitation

Four polysaccharides BJP50, BJP60, BJP70 and BJP80 (total named BJPs) were separated from Herba Patriniae via gradient ethanol precipitation with 50%, 60%, 70% and 80%, respectively. After decolorization and deproteinization, their physicochemical features and antioxidant activities were investigated. The results showed that the total sugar content of BJPs accounted above 50% but no protein contained, while BJP50 and BJP60 contained a small amount of uronic acid. GC analysis indicated that BJPs were mainly composed of galactose, arabinose, glucose, mannose, xylose and rhamnose. From BJP50 to BJP80, the types of monosaccharides and the content of arabinose, glucose and mannose increased but that of galactose and rhamnose decreased, and their molecular weights gradually reduced from 2.3 × 10⁶ to 4.5 × 10³. BJPs had a good thermal stability with the order of BJP80 > BJP70 > BJP60 > BJP50. The vitro bioactivity assay showed that BJP80 and BJP70 exhibited stronger scavenging capacities for DPPH, ABTS and hydroxyl free radicals than that of BJP60 and BJP50. As the concentration reached 4 mg/mL, the scavenging capacities of BJP80 and BJP70 on DPPH and hydroxyl free radical were up to 92% and 95%, respectively, and their antioxidant activities gradually approached to the positive control.     

Purification and initial characterization of a novel protein with factor Xa activity from Lonomia obliqua caterpillar spicules

A novel protein with factor Xa-like activity was isolated from Lonomia obliqua caterpillar spicules by gel filtration chromatography and reversed-phase high-performance liquid chromatography. The protein had a mass of 20745.7 Da, as determined by mass spectrometry, and contained four Cys residues. Enzymatic hydrolysis followed by de novo sequencing by tandem mass spectrometry was used to determine the primary structure of the protein and the cysteine residues linked by disulfide bridges. The positions of 24 sequenced tryptic peptides, including the N-terminal, were deduced by comparison with a homologous protein from the superfamily Bombycoidea. Approximately 90% of the primary structure of the active protein was determined.