Synthesis of salmon calcitonin, a high activity hypocalcemic hormone

A total synthesis of salmon calcitonin is described. The C-terminal nonapeptide amide 24–32 was coupled with the heptapeptide 17–23. The resulting hexadecapeptide amide 17–32 was reacted with the heptapeptide 10–16 to give the tricosapeptide 10–32. This was condensed with the N-terminal nonapeptide 1–9, yielding the protected dotriacontapeptide amide 1–32. After removal of the protective groups and purification by gel-filtration the free peptide obtained exhibited the physical, chemical and biological properties of the natural hormone. Its hypocalcaemic activity (ca. 3500 MRC U/mg) is 20–30 times higher than that of porcine or human calcitonin.     

Synthesis and study of the biological activity of fragments of salmon calcitonin II

A number of fragments of salmon calcitonin II possessing analgesic activity of the nonopioid type have been synthesized.Institute of High-Molecular-Mass Compounds, Russian Academy of Sciences. Institute of Experimental Medicine, Russian Academy of Medical Sciences, St. Petersburg. Translated from Khimiya Prirodnykh Soedinenii, No. 1, pp. 126–132, January–February, 1993.     

Existence of a common precursor to ACTH and endorphin in the anterior and intermediate lobes of the rat pituitary.

Extracts of rat anterior and intermediate-posterior pituitary were fractionated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and assayed for immunoactive ACTH and endorphin. In both lobes the major forms of immunoactive ACTH have apparent molecular weights of 31,000 (31K), 20--21K, 14K, and 4.5K, and the major forms of immunoactive endorphin have apparent molecular weights of 31K (coincident with the peak of immunoactive ACTH), 13K (a betaLPH-like peptide), and 3.5K (a beta-endorphin-like peptide). However, the quantitative distribution of immunoactivity among the various forms differs greatly between the lobes. Assays using an extreme COOH-terminal ACTH antiserum indicate that the 31K ACTH/endorphin molecule in rat anterior and intermediate pituitary is similar to the pro-ACTH/endorphin molecule from mouse pituitary tumor cells. A radioimmunoassay that is specific for the NH2-terminal non-ACTH, nonendorphin segment (referred to as 16K fragment) of the mouse pro-ACTH/endorphin molecule was used to assay extracts of rat pituitary. In addition to detecting material at 31K and 20--21K, the 16K fragment radioimmunoassay detects significant amounts of cross-reactive material with an apparent molecular weight of 16K in extracts of both lobes. This result also suggests that the structure and processing of the rat 31K ACTH/endorphin molecule is similar to that of mouse tumor cell pro-ACTH/endorphin. Cell suspensions were prepared from the anterior and intermediate lobes of the rat pituitary and maintained in culture for a 24-h period. The isolated cells from both lobes incorporate [3H] phenylalanine into immunoprecipitable ACTH- and endorphin-containing molecules. By sequential immunoprecipitation with ACTH and endorphin antisera, it is possible to demonstrate directly that a single molecule (31K ACTH/endorphin) has antigenic determinants for both ACTH and endorphin. Significant amounts of 31K ACTH/endorphin are released into the culture medium by isolated anterior lobe and intermediate lobe cells. The isolated intermediate lobe cells synthesize and secrete relatively large amounts of a beta-endorphin-like molecule; the isolated anterior lobe cells secrete significant amounts of both a betaLPH-like molecule and a beta-endorphin-like molecule. These same quantitative differences between anterior and intermediate lobe tissue were observed in immunoassays of extracts of the separated lobes and probably reflect differences in the processing of the common precursor. The isolated anterior lobe cells can be stimulated to release increased amounts of immunoprecipitable ACTH and endorphin by incubation with a cyclic AMP analog and a phosphodiesterase inhibitor.     

Pharmacokinetic disposition of polyethylene glycol-modified salmon calcitonins in rats

This study first reports the pharmacokinetic disposition of polyethylene glycol (PEG)-modified salmon calcitonin (sCT) based on the number of attached PEG molecules. PEG-modified sCT was prepared by covalent linkage with succinimidyl carbonate monomethoxy polyethylene glycol. Mono- and di-PEG-sCTs were separated by size exclusion and reverse phase HPLC, and radioiodinated by the chloramine-T method with Na125I. 125I-mono-PEG sCT, 125I-di-PEG-sCT and unmodified 125I-sCT were administered to rats by i.v. injection. Serial blood samples, urine and various tissue samples were taken for the determination of radioactivity. Di-PEG-sCT exhibited significantly reduced systemic clearance (2.3 vs. 11.1 ml/min/kg) and steady-state volume of distribution (229.9 vs. 603.1 ml/kg), while mono-PEG-sCT showed a prolonged elimination half-life (189.1 min vs. 59.8 min) compared with unmodified sCT. The extent of urinary excretion of the PEG-modified sCTs was higher than for the unmodified sCT, but all these chemicals were excreted in urine in small quantities (< or = 0.6%). There was a tendency toward reduced accumulation of PEGylated sCTs in tissues, with its reduction being inversely proportional to the molecular size. Accumulation of the total radioactivity of the unmodified and PEG-modified sCTs was highest in the liver, followed by kidneys, lungs, spleen, heart and thyroid. When expressed per tissue gram weight, however, the highest radioactivity was found in the kidneys. PEGylated sCTs may have greater therapeutic potential via reduced systemic clearance and prolonged elimination half-life over unmodified sCT.     

Production of C-terminal amidated recombinant salmon calcitonin in Streptomyces lividans

Salmon calcitonin (sCT) is one of the many bioactive peptides that require C-terminal amidation for full biologic activity. To produce fully bioactive sCT in large scale, we constructed Streptomyces lividans [pMSA], an engineering Streptomyces strain. In the expression vector, glycine-extended sCT, the substrate for amidation, and rat α-amidating enzyme cDNA were cloned under the control of the strong constitutive promoter from the Streptomyces fradiae aph gene in pIJ680. Both were expressed in a secretory manner by the recombinant strain using the expression and secretion signals of melC1. Extracellularly expressed recombinant sCT was purified to near homogeneity and characterized by enzyme immunoassay, followed by direct amino-terminal sequencing. High-performance liquid chromatography, matrixassisted laser desorption ionization-time-of-flight mass spectrometry, and bioassay in vivo demonstrated purified product to be equivalent to synthetic standard. Thus, the engineered Streptomyces strain can produce bioactive, C-terminal amidated recombinant sCT in the culture supernatant directly. The ease of the recombinant process, as well as its potential for scale-up, makes it adaptable to production demands for sCT, and it may be applied to other bioactive peptides that need C-terminal amidation.     

Metal ions as cofactors for aggregation of therapeutic peptide salmon calcitonin

The effects of multivalent metal ions (Cu(2+)/Zn(2+)/Al(3+)) on the aggregation of salmon calcitonin (sCT)--a therapeutic peptide used worldwide in the treatment of osteoporosis and Paget's disease--have been studied in vitro using NMR (both solution state and solid state), TEM, ThT-fluorescence, and FT-IR spectroscopy. Overall, the various results indicated that the metal-ions-induced conformational transitions in the peptide--mostly toward the β-sheet--facilitate the aggregation of sCT in solution. First, the solution NMR has been used to check the interaction between the peptide and the metal ions. Following this, the formation and characterization of calcitonin aggregates has been performed using TEM, solid state NMR, and FT-IR spectroscopy. The TEM and ThT-fluorescence results revealed that the sCT peptide incubated with Cu(2+) and Zn(2+) metal ions (in aqueous environment) forms globular aggregates, while that with Al(3+) ions forms fibrils. The solid state NMR and FT-IR studies revealed the presence of a substantial amount of β-sheet content in sCT aggregates (formed in the presence of these metal ions) compared to the monomeric sCT, indicating that the metal binding is concomitant with conformational changes. The present study becomes crucial while prescribing this drug peptide under physio-pathological conditions associated with an abnormal accumulation of metal ions (Cu(2+)/Zn(2+)/Al(3+)) in the body (i.e., abnormal metal ion homeostasis).     

利用鲑鱼精DNA为模板构建CdS纳米线

以鲑鱼精DNA为模板合成了CdS纳米粒子，透射电子显微观察表明所生成的CdS是一种直径约为3nm的线形结构，紫外-可见吸收光谱，拉曼光谱和X-射线光电子能谱结果表明，CdS纳米粒子的生长点为DNA中的磷酸根，并且在这一体系中可能存在从CdS到DNA中碱基的电子转移。     

A DSC study of the liquid crystalline phases of salmon sperm DNA

Thermodynamic stabilities of the liquid crystalline (LC) and gel (G) phases of salmon sperm DNA were investigated by differential scanning calorimetry (DSC). The enthalpic and the entropic components of the thermal denaturation free energy are briefly discussed. In order to evaluate the thermodynamic data for the denaturation of the gel (G) phase of DNA and compare it with those for the LC DNA phase, a hypothesis for the thermal denaturation mechanism of G DNA is proposed. A comparison between these two sets of data has shown that DNA in the LC phase is thermally and thermodynamically more stable than in the G phase over the temperature range which was investigated.     

Determination of oxytetracycline in salmon by liquid chromatography with metal-chelate fluorescence detection

A liquid chromatography (LC) method is described for the determination of oxytetracycline (OTC) in farmed Atlantic salmon muscle tissue. The method involves homogenization of salmon tissue, extraction of OTC into Mcllvaine-EDTA buffer, acid precipitation of proteins, cleanup through tandem solid-phase extraction cartridges (Strata-X and aminopropyl), elution with mobile phase containing slightly alkaline buffer and Mg2+, and LC separation with metal-chelate induced fluorescence detection. Salmon tissue was fortified with 0.10, 0.25, 0.50, 0.75, and 1.0 microg/g (ppm) oxytetracycline. Average absolute recoveries were 84, 76, 70, 76, and 85%, respectively, with relative standard deviation (RSD) values all less than 9%. The interassay average recovery was 78%, with a 4.2% RSD. Determination was based on a standard graph using peak areas with standard solutions equivalent to 0.0625, 0.125, 0.25, 0.50, and 1.0 ppm in tissue. A set of 5 matrix controls (unfortified salmon tissue) were also analyzed, in which no OTC was detected. The lowest standard was used as the limit of quantitation.     

Detection of transgenerational barium dual-isotope marks in salmon otoliths by means of LA-ICP-MS

The present study evaluates the use of an individual-specific transgenerational barium dual-isotope procedure and its application to salmon specimens from the Sella River (Asturias, Spain). For such a purpose, the use of laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) in combination with multiple linear regression for the determination of the isotopic mark in the otoliths of the specimens is presented. In this sense, a solution in which two barium-enriched isotopes (¹³⁷Ba and ¹³⁵Ba) were mixed at a molar ratio of ca. 1:3 (N _Ba137/N _Ba135) was administered to eight returning females caught during the spawning period. After injection, these females, as well as their offspring, were reared in a governmental hatchery located in the council of Cangas de Onís (Asturias, Spain). For comparison purposes, as well as for a time-monitoring control, egg and larva data obtained by solution analysis ICP-MS are also given. Otoliths (9-month-old juveniles) of marked offspring were analysed by LA-ICP-MS demonstrating a 100 % marking efficacy of this methodology. The capabilities of the molar fraction approach for 2D imaging of fish otoliths are also addressed.     

Light and host-guest inclusion mediated salmon sperm DNA/surfactant interactions

DNA/cationic surfactant interaction is relevant from the viewpoint of gene therapy, where the complexation and resulting compaction are essential to protect DNA from nuclease, and to allow entry of DNA into cells. In this work, light input and host-guest inclusion controlled DNA complexation by a novel cationic surfactant 1-[6-(4-phenylazo-phenoxy)-hexyl]-3-methylimidazolium bromide (AzoC6Mim) is reported. The surfactant is covalently attached with an azobenzene group, which undergoes reversible photoisomerizations by changing light input. Under visible light, trans-AzoC6Mim can bind to salmon sperm DNA through electrostatic attraction and hydrophobic interaction, resulting into DNA compaction. Under UV light, although cis-AzoC6Mim still binds to DNA chain, DNA/surfactant complex is decompacted owing to the decrease of surfactant hydrophobicity. On the other hand, azobenzene group can form an inclusion complex with α-CD through host-guest interaction, which removes AzoC6Mim from DNA chain and decompacts the DNA/surfactant complex.     

Influence of the mobile phase on salmon calcitonin analysis by reversed-phase liquid chromatography

The retention properties of calcitonins on a reversed-phase column are examined using salmon calcitonin as the model compound. The effect of the concentration of organic modifier, buffer strength, pH of the mobile phase, and ion-pair reagent are studied. In the absence of an ionic modifier in the eluent the calcitonin peak shapes are not symmetrical. The addition of 0.1% trifluoroacetic acid (TFA), however, results in good peak characteristics without the need to add nonvolatile salts. The retention of the calcitonins was found to be very sensitive to the concentration of the organic modifier (acetonitrile) present in the mobile phase. A change of pH between 2 and 5 has only a slight effect of the k' of salmon calcitonin, but the k' increases significantly at higher pH values. The addition of a phosphate buffer to the mobile phase and an increase in the buffer concentration (0-0.2 M) causes a decrease in the retention of salmon calcitonin. Evidence shows that reproducible, quantitatively measurable data can be obtained using reversed-phase chromatography if the ion-pairing reagent and organic modifier concentrations are carefully controlled. The system also shows a good selectivity for the calcitonin series. Therefore, both highly selective methods (qualitative) as well as quantitative methods for analytical, pharmaceutical, and manufacturing use can be developed by adjusting the high-performance liquid chromatography (HPLC) conditions as discussed.     

Speciation of mercury in salmon egg cell cytoplasm in relation with metallomics research

Speciation of mercury in salmon egg cell cytoplasm was investigated by surfactant-mediated high-performance liquid chromatography/inductively coupled plasma mass spectrometry (HPLC/ICP-MS), where an ODS (octadecylsilica) column coated with a bile acid derivative, CHAPS (3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate), was used for species separation. Prior to the speciation analysis, total Hg in the cell cytoplasm was determined by ICP-MS at m/z 202 in a flow injection mode. For the precise measurement, salmon egg cell cytoplasm was diluted five-fold with 0.1 M Tris (Tris(hydroxymethyl)aminomethane)-HNO₃ buffer solution, and the standard addition method was employed. Thus, the total concentration of Hg in cell cytoplasm was estimated to be 12.4 ng g⁻¹ on the wet weight basis. Next, the cell cytoplasm diluted five-fold with 0.1 M Tris-HNO₃ buffer solution was analyzed by surfactant-mediated HPLC with the dual detection system of a UV absorption detector and an ICP-MS instrument. Two peaks corresponding to some proteins and small molecules were mainly observed in those chromatograms. When salmon egg cell cytoplasm was diluted five-fold with 0.01 M Tris buffer solution or pure water, some precipitates appeared probably because of precipitation of hydrophobic proteins in cytoplasm. After the precipitates were eliminated with a membrane filter, the filtrate was subjected to the analysis by surfactant-mediated HPLC/UV/ICP-MS. As a result, the peaks for small molecular species of Hg were clearly observed at the retention time near 4.0 min (corresponding to low-molecular weight zone) in the chromatograms with UV absorption detection as well as with Hg- and S-specific ICP-MS detections. The small molecule bound with Hg was identified as cysteine through the cysteine-spiked experiment. In addition, the protein fraction on the chromatogram obtained by using the CHAPS-coated ODS column was further analyzed by SEC (size exclusion chromatography). Consequently, several protein peaks with molecular weight of 300, 50 and 12 kDa were observed in all the detections of UV absorption, Hg and Se, although two peaks among them were coincident in the case of S. These results indicate that Hg in salmon egg cell cytoplasm binds with proteins containing selenocysteine and/or cysteine residues in proteins.     

The potential of Raman spectroscopy for characterisation of the fatty acid unsaturation of salmon

Raman spectroscopy has been evaluated for characterisation of the degree of fatty acid unsaturation (iodine value) of salmon (Salmo salar). The Norwegian Quality Cuts from 50 salmon samples were obtained, and the samples provided an iodine value range of 147.8-170.0 g I₂/100 g fat, reflecting a normal variation of farmed salmon. Raman measurements were performed both on different spots of the intact salmon muscle, on ground salmon samples as well as on oil extracts, and partial least squares regression (PLSR) was utilised for calibration. The oil spectra provided better iodine value predictions than the other data sets, and a correlation coefficient of 0.87 with a root mean square error of cross-validation of 2.5 g I₂/100 g fat was achieved using only one PLSR component. The ground samples provided comparable results, but at least two PLSR components were needed. Higher prediction errors were obtained from Raman spectra of intact salmon muscle, and this may partly be explained by sampling uncertainties in the relation between Raman measurements and reference analysis. All PLSR models obtained were based on chemically sound regression coefficients, and thus information regarding fatty acid unsaturation is readily available from Raman spectra even in systems with high contents of protein and water. The accuracy, the robustness and the low complexity of the PLSR models obtained suggest Raman spectroscopy as a promising method for rapid in-process control of the degree of unsaturation in salmon samples.     

Genomic salmon testes DNA as a catalyst for Michael reactions in water

The DNA molecule, recognized as the carrier of genetic information in vivo, can function as an efficient organocatalyst for Michael additions of 1,3-dicarbonyl compounds to activated alkenes in aqueous media. The procedure described here is environmentally benign and offers several advantages in terms of simplicity, generality and efficiency. We have used fluorescence spectroscopy to evaluate the catalytic activity of a molecule of DNA.