Cloning from tissue surrogates: antimicrobial peptide (esculentin) cDNAs from the defensive skin secretions of Chinese ranid frogs

The defensive skin secretions of amphibians are a rich source of bioactive peptides. Here we describe a rapid technique for skin granular gland transcriptome cloning from a surrogate tissue-the secretion itself. cDNA libraries were constructed from lyophilized skin secretion from each of the Chinese frogs (Rana schmackeri, Rana versabilis, and Rana plancyi fukienensis) using magnetic oligo(dT) bead-captured polyadenylated mRNA as templates. Specific esculentin cDNAs were amplified by 3'-RACE using a degenerate primer designed for a consensus nucleotide sequence in the 5' untranslated region of previously characterized ranid frog peptide cDNAs. The cloned cDNAs were found to encode the antimicrobial peptides esculentins 1 and 2 from each of the species examined. The presence of predicted peptide structures in skin secretions was confirmed by MALDI-TOF mass spectrometry and automated Edman degradation. This experimental approach can thus rapidly expedite parallel transcriptome and peptidome analysis of amphibian granular gland secretions without harming or sacrificing donor animals.

Kinestatin: a novel bradykinin B2 receptor antagonist peptide from the skin secretion of the Chinese toad, Bombina maxima

We have isolated a novel bradykinin B2-receptor antagonist peptide, kinestatin, from toad (Bombina maxima) defensive skin secretion. Mass spectroscopy established a molecular mass of 931.56 Da and a provisional structure: pGlu-Leu/Ile-Pro-Gly-Leu/Ile-Gly-Pro-Leu/Ile-Arg.amide. The unmodified sequence, -QIPGLGPLRG-, was located at the C-terminus of a 116-amino-acid residue open-reading frame following interrogation of a sequenced B. maxima skin cDNA library database. This confirmed the presence of appropriate primary structural attributes for the observed post-translational modifications present on the mature peptide and established residue 2 as Ile and residues 5/8 as Leu. Kinestatin represents a prototype novel peptide from amphibian skin.

The preparation, solubilisation and binding characteristics of a beta 2-adrenoceptor isolated from transfected Chinese hamster cells

beta2-Adrenoceptor agonists (beta -agonists) are well known for their growth promoting and repartitioning effects in many species. Although the use of these compounds to increase muscle mass in stockfarming is prohibited within the EU, under directive 96/22/EC, significant illegal use still occurs. With legal and illegal synthesis of new structurally related compounds, the detection of traditional beta -agonists and new derivatives becomes increasingly problematical. This method describes the isolation and solubilisation of a beta2-adrenoceptor from a transfected Chinese hamster ovary cell line, using the detergent digitonin. The solubilised receptor retained its activity and was isolated from the cell membrane at a concentration of 550 +/- 100 fmol mg(-1) of solubilised protein. Competition analysis using the tritiated antagonist dihydroalprenolol revealed receptor affinity for five structurally different beta -agonists, with IC50 values ranging from 2.1 +/- 0.76 X 10(-7) M for salmeterol to 1.1 +/- 0.62 x 10(-5) M for ractopamine. This study has demonstrated that transfected cell lines with a high expression of beta2-adrenoceptors are a convenient source of active receptor material. Solubilised beta (2)-adrenoceptors could form the basis of a multi-analyte screening assay for use in routine screening.

The preparation, solubilisation and binding characteristics of a beta 2-adrenoceptor isolated from transfected Chinese hamster cells

beta2-Adrenoceptor agonists (beta -agonists) are well known for their growth promoting and repartitioning effects in many species. Although the use of these compounds to increase muscle mass in stockfarming is prohibited within the EU, under directive 96/22/EC, significant illegal use still occurs. With legal and illegal synthesis of new structurally related compounds, the detection of traditional beta -agonists and new derivatives becomes increasingly problematical. This method describes the isolation and solubilisation of a beta2-adrenoceptor from a transfected Chinese hamster ovary cell line, using the detergent digitonin. The solubilised receptor retained its activity and was isolated from the cell membrane at a concentration of 550 +/- 100 fmol mg(-1) of solubilised protein. Competition analysis using the tritiated antagonist dihydroalprenolol revealed receptor affinity for five structurally different beta -agonists, with IC50 values ranging from 2.1 +/- 0.76 X 10(-7) M for salmeterol to 1.1 +/- 0.62 x 10(-5) M for ractopamine. This study has demonstrated that transfected cell lines with a high expression of beta2-adrenoceptors are a convenient source of active receptor material. Solubilised beta (2)-adrenoceptors could form the basis of a multi-analyte screening assay for use in routine screening.

Hydrophilic Interaction Ultra-High Performance Liquid Chromatography Coupled with Triple-Quadrupole Mass Spectrometry for Determination of Nucleosides and Nucleobases in Animal Horns

Traditional Chinese Medicines (TCMs) derived from animal horns are one of the most important types of Chinese medicine. In the present study, a fast and sensitive analytical method was established for qualitative and quantitative determination of 14 nucleosides and nucleobases in animal horns using hydrophilic interaction ultra-high performance liquid chromatography coupled with triple-quadruple tandem mass spectrometry (HILIC-UPLC-QQQ-MS/MS) in selective reaction monitoring (SRM) mode. The method was optimized and validated, and showed good linearity, precision, repeatability, and accuracy. The method was successfully used to determine contents of the 14 nucleosides and nucleobases in 25 animal horn samples. Hierarchical clustering analysis (HCA) and principal component analysis (PCA) were performed and the 25 samples were thereby divided into two groups, which agreed with taxonomy. The method may enable quick and effective search of substitutes for precious horns.

De novo sequencing of two novel peptides homologous to calcitonin-like peptides, from skin secretion of the Chinese Frog, Odorrana schmackeri

An MS/MS based analytical strategy was followed to solve the complete sequence of two new peptides from frog (Odorrana schmackeri) skin secretion. This involved reduction and alkylation with two different alkylating agents followed by high resolution tandem mass spectrometry. De novo sequencing was achieved by complementary CID and ETD fragmentations of full-length peptides and of selected tryptic fragments. Heavy and light isotope dimethyl labeling assisted with annotation of sequence ion series. The identified primary structures are GCD[I/L]STCATHN[I/L]VNE[I/L]NKFDKSKPSSGGVGPESP-NH2 and SCNLSTCATHNLVNELNKFDKSKPSSGGVGPESF-NH2, i.e. two carboxyamidated 34 residue peptides with an aminoterminal intramolecular ring structure formed by a disulfide bridge between Cys2 and Cys7. Edman degradation analysis of the second peptide positively confirmed the exact sequence, resolving I/L discriminations. Both peptide sequences are novel and share homology with calcitonin, calcitonin gene related peptide (CGRP) and adrenomedullin from other vertebrates. Detailed sequence analysis as well as the 34 residue length of both O. schmackeri peptides, suggest they do not fully qualify as either calcitonins (32 residues) or CGRPs (37 amino acids) and may justify their classification in a novel peptide family within the calcitonin gene related peptide superfamily. Smooth muscle contractility assays with synthetic replicas of the S–S linked peptides on rat tail artery, uterus, bladder and ileum did not reveal myotropic activity.

Intraocular pressure, central corneal thickness, and glaucoma in chinese adults: the liwan eye study.

PURPOSE: To describe the distribution of central corneal thickness (CCT), intraocular pressure (IOP), and their determinants and association with glaucoma in Chinese adults.DESIGN: Population-based cross-sectional study.METHODS: Chinese adults aged 50 years and older were identified using cluster random sampling in Liwan District, Guangzhou. CCT (both optical [OCCT] and ultrasound [UCCT]), intraocular pressure (by Tonopen, IOP), refractive error (by autorefractor, RE), radius of corneal curvature (RCC), axial length (AL), and body mass index (BMI) were measured, and history of hypertension and diabetes (DM) was collected by questionnaire. Right eye data were analyzed.RESULTS: The mean values of OCCT, UCCT, and IOP were 512 ± 29.0 μm, 542 ± 31.4 μm, and 15.2 ± 3.1 mm Hg, respectively. In multiple regression models, CCT declined with age (P < .001) and increased with greater RCC (P < .001) and DM (P = .037). IOP was positively associated with greater CCT (P < .001), BMI (P < .001), and hypertension (P < .001). All 25 persons with open-angle glaucoma had IOP <21 mm Hg. CCT did not differ significantly between persons with and without open- or closed-angle glaucoma. Among 65 persons with ocular hypertension (IOP >97.5th percentile), CCT (555 ± 29 μm) was significantly (P = .01) higher than for normal persons.CONCLUSIONS: The distributions of CCT and IOP in this study are similar to that for other Chinese populations, though IOP was lower than for European populations, possibly due to lower BMI and blood pressure. Glaucoma with IOP <21 mm Hg is common in this population. We found no association between glaucoma and CCT, though power (0.3) for this analysis was low.