Purification, characterization and cDNA cloning of two natterin-like toxins from the skin secretion of oriental catfish Plotosus lineatus

The oriental catfish Plotosus lineatus is known to contain proteinaceous toxins in the skin secretion as well as in the venom gland. However, detailed properties and primary structures of the skin toxins have not been clarified. In this study, two proteinaceous toxins (toxins I and II) were purified from the skin secretion of oriental catfish by a combination of gel filtration, anion-exchange HPLC and hydroxyapatite HPLC. Toxins I and II are monomeric simple proteins with almost the same molecular mass (35 kDa for toxin I and 37 kDa for toxin II) and are distinguishable from each other in isoelectric point (6.5 for toxin I and 5.1 for toxin II). Both toxins display lethal, edema-forming and nociceptive activities, although toxin I is significantly more potent than toxin II. The primary structures of toxins I and II were elucidated by cloning experiments based on the determined partial amino acid sequences. Toxins I (317 amino acid residues) and II (315 amino acid residues) share as high as 86% sequence identity with each other and are also highly homologous (56–75% identities) with the known fish natterin-like proteins.     

Characteristics of chitosanases from<Emphasis Type="Italic">Aspergillus fumigatus</Emphasis> KB-1

Two chitosanases produced by Aspergillus fumigatus KB-1 were purified by ion exchange and size exclusion chromatographies. Molecular weights of chitosanases were 111.23 kDa (chitosanase I) and 23.38 kDa (chitosanase II). The N-terminal amino acid sequence of chitosanase II was determined as follows: YNLPNNLKQIYDKHKGKXSXVLAKGFTN. The optimum pH of the chitosanase I and II was 6.5 and 5.5, respectively. The optimum temperatures were 60 degrees C for chitosanase land 70 degrees C for chitosanase II. Hydrolysis products of two chitosanases were analyzed by HPLC and GPC. Chitosanase I hydrolyzed substrate to glucosamine. Chitosanase II produced chitooligosaccharides.     

In-vitro evaluation of 5-lipoxygenase and cyclo-oxygenase inhibitors using bovine neutrophils and platelets and HPLC.

Inhibition of 5-lipoxygenase has been determined by monitoring the formation of leukotriene B4 and 5-hydroxyeicosatetraenoic acid in bovine polymorphonuclear leucocytes. For evaluating the inhibition of cyclo-oxygenase two different test systems are presented: the first uses 12-hydroxyheptadecatrienoic acid produced by bovine platelets as an indicator of the cyclo-oxygenase activity; the second test system monitors the prostaglandin E2 formation by bovine platelets. All arachidonic acid metabolites are quantified by reverse-phase HPLC with UV-detection.     

Novel peptide toxins from acrorhagi, aggressive organs of the sea anemone Actinia equina.

Two peptide toxins, acrorhagin I (50 residues) and II (44 residues), were isolated from special aggressive organs (acrorhagi) of the sea anemone Actinia equina by gel filtration on Sephadex G-50 and reverse-phase HPLC on TSKgel ODS-120T. The LD50 against crabs of acrorhagin I and II were estimated to be 520 and 80 microg/kg, respectively. 3'- and 5'-RACE established the amino acid sequences of the acrorhagin precursors. The precursor of acrorhagin I is composed of both signal and mature peptides and that of acrorhagin II has an additional sequence (propart) between signal and mature peptides. Acrorhagin I has no sequence homologies with any toxins, while acrorhagin II is somewhat similar to spider neurotoxins (hainantoxin-I from Selenocosmia hainana and Tx 3-2 from Phoneutria nigriventer) and cone snail neurotoxin (omega-conotoxin MVIIB from Conus magus). In addition, analogous peptides (acrorhagin Ia and IIa) were also cloned during RT-PCR experiments performed to confirm the nucleotide sequences of acrorhagins. This is the first to demonstrate the existence of novel peptide toxins in the sea anemone acrorhagi.     

Relationship between the cytolysins tenebrosin-C from Actinia tenebrosa and equinatoxin II from Actinia equina.

The chemical, physical and biological properties of the cytolysin tenebrosin-C from Actinia tenebrosa have been compared with those of equinatoxin II from Actinia equina. The two proteins are indistinguishable by reverse-phase and cation-exchange HPLC and capillary zone electrophoresis, and give similar peptide fragments upon cyanogen bromide cleavage (as judged by the chromatographic behaviour, ultraviolet absorption spectra, amino acid composition and N-terminal amino acid sequences of the peptides). Their cardiac stimulatory activities are identical, and their haemolytic activities are similar, with equinatoxin II having slightly greater activity. These data indicate that the two molecules are either identical in all 179 amino acid positions, or differ by no more than one or two residues. These findings are discussed in the context of the taxonomic relationship between the two species of sea anemone.     

Detection and quantitative determination of 3-nitropropionic acid in bovine urine

The separation and determination of 3-nitropropionic acid (NPA) in bovine urine by reverse-phase HPLC is described. The method utilizes a rapid ethyl acetate extraction and this clean-up step also improves the Chromatographic resolution of NPA in bovine plasma. A simple colorimetric method is also developed for the rapid detection and estimation of NPA in urine and the data indicate that it is in good agreement with the HPLC procedure.     

Isolation and molecular cloning of novel peptide toxins from the sea anemone Antheopsis maculata.

Three peptide toxins (Am I-III) with crab toxicity were isolated from the sea anemone Anthopleura maculata by gel filtration and reverse-phase HPLC. Am I was weakly lethal to crabs (LD50 830 microg/kg) and Am III was potently lethal (LD50 70 microg/kg), while Am II was only paralytic (ED50 420 microg/kg). The complete amino acid sequences of the three toxins were determined by cDNA cloning based on 3'-Race and 5'-Race. Although Am III (47 residues) is an analogue of the well-known type 1 sea anemone sodium channel toxins, both Am I (27 residues) and II (46 residues) are structurally novel peptide toxins. Am I is a new toxin having no sequence homologies with any toxins. Am II shares 28-39% identity with the recently characterized sea anemone toxins inhibiting specialized ion channels, BDS-I and II from Anemonia sulcata and APETx1 and 2 from Anthopleura elegantissima. The precursor proteins of the three toxins are commonly composed of a signal peptide, a propart with a pair of basic residues (Lys-Arg) at the end and the remaining portion. Very interestingly, the Am I precursor protein contains as many as six copies of Am I.     

Purification and characterization of gonadotropin I and II from pituitary glands of tuna (Thunnus obesus)

The duality of teleost pituitary gonadotropins was established in an advanced marine fish, the tuna (Thunnus obesus). Two different molecular forms of gonadotropins, designated tGTH I and tGTH II, were isolated from an alcoholic extract of pituitary glands following ion-exchange chromatography and reversed-phase HPLC. Both tGTH I and tGTH II stimulated estradiol-17β and testosterone production in tuna ovarian follicles in vitro, although responses to tGTH II were significantly greater than those to tGTH I. Each gonadotropin consisted of α- and β-subunits. tGTH I was stable in acidic conditions, whereas tGTH II dissociated into two subunits after acid treatment. Alpha subunits of tGTH I and tGTH II had identical amino acid sequences of 94 amino acid residues. The tGTH Iβ and tGTH IIβ consisted of 102 and 115 amino acid residues, respectively, and showed 35% sequence identity. tGTH Iβ is structurally more similar to salmon GTH Iβ than to salmon GTH IIβ, whereas tGTH IIβ is more similar to salmon GTH IIβ. Thus it is evident that the tuna pituitary gland produces two chemically distinct gonadotropins.     

Chemical synthesis of urotensin II,a somatostatin-like peptide in the caudal neurosecretory system of fishes

In the goby, Gillichthys mirabilis, urotensin II (a bioactive neuropeptide present in the urophysis of teleost fish) has the dodecapeptide sequence, H₂N-AGTADC-FWKYCV-OH, which is homologous with mammalian somatostatin at positions 1, 2 and 7–9. The Merrifield solid phase synthesis of Gillichthys urotensin II (UII) was accomplished by stepwise assembly from the carboxy terminus using N^α-tert.-butyloxycarbonyl (Boc) amino acids containing benzyl-derived groups for protection of side-chain functionalities. Coupling of amino acids to the growing peptide was mediated by diisopropylcarbodiimide (DIC) in the presence of 1-hydroxybenzotriazole (HOBt). Residual α-amino groups remaining after coupling were blocked by acetylation with 1-acetylimidazole. Crude, synthetic UII was extracted from the HF-treated, protected peptide-resin product, reduced with dithiothreitol (DTT), reoxidized at high dilution with O₂, and separated into its components using a single, preparative, reverse-phase HPLC step. The pure, synthetic UII, obtained in 7.6% yield from oxidized crude UII, was indistinguishable from pure, native UII in specific bioactivity, amino acid sequence, and retention time in each of two different HPLC systems.     

Total synthesis of S-carbamoylmethyl bovine apocytochrome c by segment coupling

Three peptide segments corresponding to the complete sequence of the 104 amino acid protein bovine apocytochrome c were synthesized by the solid-phase method. The peptides Ac-[Cys(Cam)^14,17, GlyS²³]-apocytochrome c-(1–23) (I), CF₃CO-[GlyS⁶⁰]-apocytochrome c-(24–60) (II), and CF₃CO-apocytochrome c-(61–104) (III) were purified by chromatography on CM-cellulose, partition chromatography and/or HPLC. Each of the peptides was reacted with citraconic anhydride to block all of the lysine side chains, and the 61–104 peptide was treated with 10% hydrazine to remove the trifluoroacetyl group, to give the corresponding peptides Ia, IIa, and IIIa. Peptides IIa and IIIa were coupled together by reaction with silver nitrate/N-hydroxysuccinimide to give the 24–104 sequence. After removal of the trifluoroacetyl group from the amino terminus, peptide Ia was also coupled. Treatment of the peptide mixture with aqueous acetic acid removed the citraconyl groups, and purification by chromatography on CM-cellulose and HPLC gave a 0.6% yield of [Cys-(Cam)^14,17]-apocytochrome c. The synthetic product was shown to be identical to a sample derived from native bovine cytochrome c by paper or gel electrophoresis, HPLC and by chymotryptic or tryptic map.     

An epidermal growth factor-like toxin and two sodium channel toxins from the sea anemone Stichodactyla gigantea.

Three peptide toxins (gigantoxins I-III) with crab toxicity were isolated from the sea anemone Stichodactyla gigantea by gel filtration on Sephadex G-50 and reverse-phase HPLC on TSKgel ODS-120T and their complete amino acid sequences were determined. Gigantoxins II (44 residues) and III (48 residues) have LD(50) (against crabs) of 70 and 120 microg/kg, respectively, and are analogous to the known type 1 and 2 sea anemone sodium channel toxins, respectively. On the other hand, gigantoxin I (48 residues) is potently paralytic to crabs (ED(50) 215 microg/kg), although its lethality is very weak (LD(50)>1000 microg/kg). Interestingly, gigantoxin I has 31-33% homologies with mammalian epidermal growth factors (EGFs), with the same location of six cysteine residues. In accordance with the sequence similarity, gigantoxin I exhibits EGF activity as evidenced by rounding of A431 cells and tyrosine phosphorylation of the EGF receptor in the cells, although much less potently than human EGF. Gigantoxin I is the first example of EGF-like toxins of natural origin.     

Amino acid sequence of a thrombin like enzyme, elegaxobin II, from the venom of Trimeresurus elegans (Sakishima-Habu).

The amino acid sequence of a thrombin like enzyme , named elegaxobin II, isolated from the venom of Trimeresurus elegans (Sakishima-habu) was determined by Edman sequencing of the peptides which was derived from digests with cyanogen bromide, achromobacter protease I, trypsin, endoproteinase Asp-N, and chymotrypsin. Elegaxobin II consisted of 233 amino acids and showed conservation of the catalytic amino acid residues (His(57), Asp(102), and Ser(195)) of chymotrypsin family serine protease in its amino acid sequence. The carboxyterminal amino acid, Leu, was determined using carboxypeptidase Y. This enzyme contains glucosamine and an N-linked glycosylation site. Elegaxobin II was 91% homologous in sequence to elegaxobin and protease I from the same snake venom, and it was 67, 75, 31 and 26% homologous in sequences to flavoxobin, KN-BJ 2, human kallikrein and bovine thrombin, respectively. Elegaxobin II lacked thrombin's ETW (146-148) loop, as well as its functionally important YPPW (60-insertion loop).     

Development of highly sensitive determination of biogenic compounds by high-performance liquid chromatography with pre-column fluorescence derivatization

A sensitive fluorescent labeling reagent, 4-(5,6-dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride (DMS-Cl), for the determination of amino compounds in HPLC was developed. DMS-Cl reacted with amino compounds in the basic medium to produce the corresponding fluorescent sulfonamides (excition 318 nm, emission 406 nm in aqueous acetonitrile). When amino acids were analyzed using reverse-phase HPLC, the detection limits (signal-to-noise ratio = 3) of almost all amino acids labeled with DMS-Cl were less than 5 fmol/injection. DMS-Cl was utilized for highly sensitive determination of amino compounds in biological samples and HPLC methods for determination of prolyl dipeptides, Pro and Hyp, in serum and urine, pipecolic acid in serum, taurine in plasma, and free and N-acetylated polyamine in urine were established. As these proposed methods are highly sensitive and reproducible and require only a small amount of biological sample, they may be useful for clinical and biochemical research.     

Identification, isolation and characterization of impurities of clindamycin palmitate hydrochloride

Clindamycin palmitate hydrochloride is a water soluble hydrochloride salt of the ester of clindamycin and palmitic acid. It is inactive in vitro, rapid in vivo hydrolysis converts this compound to the antibacterially active clindamycin. Total 12 impurities at levels ranging from 0.05% to 0.5% were detected by isocratic reverse-phase high performance liquid chromatography (HPLC) using RI detector. The molecular weights of impurities were determined by LC-MS analysis. Two impurities were starting materials and the remaining impurities were isolated from crude samples/enriched mother liquors using reverse-phase preparative HPLC. Based on the spectral data the structures of these impurities were characterized as, clindamycin palmitate sulphoxides alpha-/beta-isomers (impurity I); clindamycin laurate (impurity II); lincomycin palmitate (impurity III); clindamycin myristate (impurity IV); epiclindamycin palmitate (impurity V); clindamycin palmitate 3-isomer (impurity VI); clindamycin pentadecanoate (impurity VII); clindamycin B-palmitate (impurity VIII); clindamycin heptadecanoate (impurity IX) and clindamycin stearate (impurity X). Structural elucidation of all impurities by spectral data ((1)H NMR, (13)C NMR, MS and IR) and formation of these impurities have been discussed in detail.     

Partial amino acid sequence of porcine elastase II

The partial amino acid sequence of porcine elastase II, isolated from crude trypsin Type II, was determined. The enzyme consists of two polypeptide chains, a light chain composed of 11 residues, and a heavy chain (M_r = 23 500) with four intrachain disulfide bridges; the two chains are held together by one interchain disulfide bond. Elastase II was fragmented into several peptides by chemical cleavages with CNBr at the two methionine residues, 99 and 180 (chymotrypsinogen numbering), and with hydroxylamine at the peptide bond following DIP-Ser¹⁹⁵. About 50% of the sequence was determined and the positions of 120 amino acids were located, including the light chain residues and most of the active site residues. The partial sequence shows 64% difference between porcine elastase II and elastase I and only 26% difference between porcine elastase II and bovine chymotrypsin B.     

Primary structure of fox pituitary growth hormone

Tryptic digests of fox growth hormone (fGH) were separated by reverse phase high performance liquid chromatography (HPLC) and by paper electrophoresis. From the amino acid composition of these tryptic peptides and from their alignment with the expected tryptic peptides from bovine growth hormone (bGH), the primary structure of fGH is proposed. There are only 17 amino acid residues which are different in these two growth hormone molecules.     

A high-performance liquid chromatographic method for the simultaneous determination of nicardipine and its pyridine metabolite II in plasma

A rapid and specific method in which reverse-phase high-performance liquid chromatography (HPLC) with UV detection was used for the simultaneous determination of nicardipine and its pyridine metabolite II in human plasma is described. Nicardipine, its pyridine metabolite II, and the internal standard were extracted from plasma and partially purified by acid-base partitioning. Final purification and quantitation were achieved by HPLC by using a reverse-phase column and a UV detector (254 nm). The extraction efficiencies for nicardipine and its pyridine metabolite II from 1 mL of plasma were 77.4 and 81.1%, respectively. The sensitivity of the assay was 5 ng/mL for both nicardipine and its pyridine metabolite II, and the linear concentration range of the assay was 5-150 ng/mL for both compounds. The low coefficients of variation (less than or equal to 5%) for samples spiked with nicardipine and its pyridine metabolite II in this concentration range demonstrate good reliability and reproducibility of the assay. The HPLC procedure has been validated by comparison with a GC-electron-capture detection (ECD) procedure, which gives the combined concentration of nicardipine-its pyridine metabolite II (total) and with an HPLC/GC-ECD procedure, which gives the concentration of its pyridine metabolite II. All three methods, which were developed in our laboratory, were used to analyze nicardipine and its pyridine metabolite II in specimens of plasma from subjects treated with nicardipine hydrochloride. Good correlations were found for concentrations of nicardipine, its pyridine metabolite II, and nicardipine plus the metabolite determined by these three procedures. The HPLC procedure is suitable for use in pharmacokinetic studies following administration of nicardipine hydrochloride to humans.     

用HPLC法和氨基酸分析仪测定多维氨基酸片中18种氨基酸

目的：RP-HPLC法和氨基酸分析仪（amino acid analyzer，AAA）法测定氨基酸含量的比较。方法：采用Agilent高效液相色谱系统和日立835-50氨基酸分析仪测定多维氨基酸片中18种氨基酸的含量。RP-HPLC法采用C18柱和邻苯二甲醛柱前衍生化进行，AAA法采用离子交换色谱柱和茚三酮柱后衍生化进行。结果：HPLC法和AAA法的变异系数分别〈1．5％和3％，最小检测量分别为3pmol和30pmol。HPLC法的测定值与AAA的测定值有相关性。两种测定方法的平均相对偏差为5．74％（0．24％～9．60％）。结论：两种方法都可用于测定氨基酸，但测定结果存在差异。     

Substrate recognition by P-glycoprotein and the multidrug resistance-associated protein MRP1: a comparison

OBJECTIVES: It has recently been suggested that substrate recognition patterns for human P-glycoprotein encoded by mdr1 consist of two electron donor groups with a spatial separation of 2.5 +/- 0.3 A (type I units) or three electron donor groups with a spatial separation of the two outer groups of 4.6 +/- 0.6 A (type II units) [Seelig 1998]. Since P-gp and the multidrug resistance-associated protein (MRP1) have overlapping substrate specificity, we screened the chemical structures of 21 compounds, previously tested as MRP1 substrates, for electron donor units. In addition, we searched the putative transmembrane domains (TMD 1-12) of P-gp and (TMD 6-17) of MRP1 for amino acid side chains having the potential to interact with the respective substrates. METHODS: The three-dimensional structures of potential MRP1 substrates were modeled with a force-field approach and were then screened for electron donor units. Helical wheel projections of the 12 putative transmembrane domains of P-gp (1-12) and MRP (6-17) were analyzed for their content of amino acid residues with hydrogen bonding side chains, charged amino acid residues, and amino acid residues with pi-electron systems. RESULTS: MRP1 recognizes compounds with type I and type II units. At least one electrically neutral together with either one negatively charged type I unit or two electrically neutral type I units are required for the compound to be bound and transported. Transport increases with increasing number of electron donor units. Compounds which carry exclusively electrically neutral type I units (P-gp substrates) are transported only weakly by MRP1, and compounds with cationic type I units (P-gp substrates) are not transported at all. An analysis of the putative transmembrane alpha-helices of MRP1 and P-gp reveals that the amino acid residues with hydrogen-bond donor side chains are arranged preferentially on one side of the helix and amino acid residues with inert (non-hydrogen-bonding) side chains on the other side. In the case of MRP1, the hydrogen-bonding face also contains several cationic residues whereas, in the case of P-gp, it contains clusters of amino acid residues with beta-electron systems. CONCLUSIONS: We propose that P-gp and MRP1 recognize type I or type II units in chemical compounds having diverse structures, and that these transporters bind their substrates via hydrogen bond formation. Furthermore, we propose that transport of anionic substrates by MRP1 is facilitated by cationic amino acid residues present in the transmembrane helices of MRP1, whereas the transport of cationic substrates by P-gp is facilitated by a beta-electron slide guide.