Darier''s disease: severe eczematization successfully treated with cyclosporin

A novel calcitonin (CT) was isolated from the spotlined sardine, Sardinops melanostictus. The primary structure of sardine CT was determined as follows: H-Cys-Ser-Asn-Leu-Ser-Thr- Cys-Ala-Leu-Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys-Leu-Gln-Ser-Tyr-Pro-Arg- Thr- Asn-Val-Gly-Ala-Gly-Thr-Pro-NH2. This amino acid sequence was different from that of salmon CT in 4 amino acid residues at positions 8th, 21th, 27th and 29th. As judged by the international method of CT bioassay, hypocalcemic activity of sardine CT was calculated as 4156 IU/mg. When compared for durability of CTs, it was found that sardine CT was significantly more potent than that of salmon CT. This is the first report of CT from a marine species of teleost.     

Expression of CYP71B7, a cytochrome P450 expressed sequence Tag from Arabidopsis thaliana

c-Yes was purified 322-fold from a rat liver plasma membrane fraction to a single 60-kDa band on SDS-PAGE. The purified protein contained essentially no phosphotyrosine residues and was autophosphorylated with Mg2+. ATP exclusively at tyrosine residues with a concomitant increase in the protein-tyrosine kinase activity. The autophosphorylated c-Yes was extensively digested by trypsin and the resultant two major phosphopeptides, peptides I and II, were purified by HPLC on a reversed-phase C-18 column. The amino acid sequence of peptide I was determined to be LIEDNEYTAR, which is identical with the sequence from Leu-418 through Arg-427 of mouse c-Yes, indicating that one of the autophosphorylation sites corresponds to Tyr-424 of the mouse c-Yes. After partial determination of the N-terminal sequence of 10 amino acid residues of peptide II, the 230 bp sequence of rat cDNA that encodes the N-terminal 76 amino acid residues of c-Yes covering peptide II, was determined. From the predicted amino acid sequence, the sequence of peptide II was assumed to be from Tyr-16 through Lys-46, YTPENPTEPVNTSAGHYGVEHATAATTSSTK. The purified c-Yes phosphorylated the tyrosine residue of synthetic peptides covering Tyr-32 and its surrounding sequence but did not phosphorylate peptides covering Tyr-16 and its surrounding sequence, suggesting that the other autophosphorylation site is Tyr-32.     

A critical appraisal of the role of triangulation in nursing research

Proopiomelanocortin (POMC) is the precursor for a number of biologically active peptides such as adrenocorticotropic hormone (ACTH), alpha-melanocyte-stimulating hormone (alpha-MSH) and beta-endorphin. It is well known that these peptides are involved in the stress response in fish as well as in mammals. We have cloned two different carp POMC cDNAs called, POMC-I and POMC-II. The nucleotide sequences of 955 bp for POMC-I and 959 bp for POMC-II share 93.5% identity in their cDNAs, and the deduced amino acid sequences (both 222 amino acids) are 91.4% identical. In the ACTH and beta-MSH domain, two amino acid substitutions are found, whereas alpha-MSH and beta-endorphin are identical. For beta-MSH, the serine replacement (in POMC-I) by a glycine (in POMC-II) results in a putative amidation site Pro-X-Gly for POMC-II. We used RT-PCR to show that both POMC mRNAs are expressed in the hypophysis, hypothalamus and other parts of the brain of a single fish. Furthermore, in a phylogenetic tree based on POMC sequences the divergence of carp POMC-I and -II from tetraploid animals (salmon, trout and xenopus) is demonstrated.     

Isolation and characterization of an endogenous peptide from rat brain interacting specifically with the serotonergic 1B receptor subtypes

The existence of endogenous compounds interacting with the serotonergic system was previously postulated. In the present work, rat brain tissues were extracted by acidic and organic procedures. The resulting extract was tested for its capacity to interact with the binding of [3H]5-hydroxytryptamine ([3H]5-HT) to 5-HT1 receptors. Compounds responsible for the observed inhibitory activities were isolated and purified by high pressure liquid chromatography. A tetrapeptide corresponding to a novel amino acid sequence Leu-Ser-Ala-Leu (LSAL) was identified. It reduces the binding of [3H]5-HT to 5-HT1 receptors at low concentration (IC50 = 10(-10) M). This effect corresponds to a specific interaction at 5-HT1B receptors since LSAL does not significantly affect other neurotransmitter bindings. LSAL appears heterogeneously distributed throughout the brain (hippocampus > cerebellum > striatum > brain stem) and in peripheral tissues (kidney > lung > stomach > blood > liver > spleen). Two other peptides, Leu-Ser (LS) and Ala-Leu (AL), were also purified. They hardly affected [3H]5-HT binding compared with LSAL. They presumably represent degradation products of the functional peptide LSAL. The fact that LSAL interacts specifically with 5-HT1B receptors that inhibit the release of neurotransmitters and particularly that of 5-HT itself suggests that this peptide may be involved in mechanisms controlling 5-HT neurotransmission and, accordingly, may play an important role in pathophysiological functions related to 5-HT activity.     

RK-2: a novel rabbit kidney defensin and its implications for renal host defense

Defensins are endogenous antimicrobial peptides. We demonstrated recently the existence of RK-1, a defensin-like peptide in rabbit kidneys. Defining the renal-defensin system is an essential step toward understanding how the kidney responds to infection. Here, using a combination of high-performance liquid chromatography, matrix-assisted laser desorption time-of-flight mass spectrometry, amino acid compositional analysis, and microsequence analysis, we show that the rabbit kidney contains a novel alpha-defensin-like peptide, RK-2. This work establishes the existence of a new subfamily of alpha-defensins in the kidney.     

Hypophysectomy, high tumor necrosis factor levels, and hemoglobinemia in lethal endotoxemic shock

Insulin was isolated from the pancreas of Chondrostean fish, the Russian sturgeon, Acipenser guldenstaedti, by acid-ethanol extraction followed by ion-exchange and reverse-phase high-performance liquid chromatographies. The amino acid sequence determined by automated Edman degradation is as follows: A-chain (21-amino-acid peptide), H-Gly-Ile-Val-Glu-Gln-Cys-Cys-His-Ser-Pro-Cys-Ser-Leu-Tyr-Asp-Leu-Glu-As n-Tyr-Cys-Asn-OH; and B-chain (31-amino-acid peptide), H-Ala-Ala-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Va l-Cys-Gly-Glu-Arg-Gly-Phe-Phe-Tyr-Thr-Pro-Asn-Lys-Val-OH. The sturgeon insulin appears to be identical with one of two forms of paddlefish insulin and differs from the other form by a single substitution in the A-chain, Asp15: His15. The amino acid sequence of sturgeon insulin is more similar to the amino acid sequence of mammalian insulins than of other fish insulins. Sturgeon insulin showed parallel but weaker displacement than porcine insulin and pink salmon insulin in their respective radioimmunoassays and was less potent than porcine insulin in displacing radiolabeled porcine insulin bound to partially purified rat liver plasma membranes.     

Studies on the pituitary "fettstoffwechselhormon". VII. Analytical studies on two lipolytically high active peptides from hog pituitary glands

First results of analytical studies on the highly purified lipolytic active hog pituitary fractions P-LF II C and P-LF II D are reported. Electrophoresis and end-group determinations indicate homogeneity for both peptides. The molecular weights, amino acid compositions, and the N- and C-terminal sequences of these peptides have been established and are compared with the corresponding results of other laboratories. The relation between biological activity and the structure of lipolytic active peptides is discussed.     

Purification of a meningeal cell-derived chondroitin sulphate proteoglycan with neurotrophic activity for brain neurons and its identification as biglycan

Serum-free cultures of meningeal fibroblasts synthesize and release a chondroitin sulphate proteoglycan (CSPG) that markedly enhances survival but not adhesion of embryonic rat (embryonic day 15) neocortical neurons in vitro. The active molecule was purified from conditioned medium (meningeal cell-conditioned medium, MCM) in three steps by means of fast-performance liquid chromatography fractionation combined with a quantitative microphotometric bioassay: (i) preparative Q-Sepharose anion exchange chromatography under native conditions; (ii) rechromatography of biologically active Q-Sepharose fractions on a MonoQ column in the presence of 8 M urea; and (iii) final gel filtration of active MonoQ fractions on Superose 6 in the presence of 4 M guanidinium hydrochloride. Analytical sodium dodecyl sulphate-polyacrylamide gradient gel electrophoresis of active Superose 6 fractions revealed a single broad glycoprotein band with a molecular mass in the range of 220-340 kDa. Further characterization of the purified molecule with glycosaminoglycan:lyases revealed a core protein of 50 kDa and the nearly complete loss of neurotrophic activity after chondroitinase digestion, whereas heparitinase treatment changed neither electrophoretic mobility nor biological activity. Amino-terminal sequencing of the purified CSPG core protein revealed identity with the amino acid sequence of rat biglycan. Biglycan purified from bovine cartilage supported neuron survival with virtually the same activity as the CSPG purified from MCM (half-maximal activity approximate to 10(-8) M). In conclusion, we isolated a neurotrophic CSPG from meningeal cells with strong survival-enhancing activity for brain neurons that was identified as biglycan, a molecule not previously related to neural functions.     

Direct sequencing of neuropeptides in biological tissue by MALDI-PSD mass spectrometry

Dissected tissue pieces of the pituitary pars intermedia from the amphibian Xenopus laevis was directly subjected to matrix-assisted laser desorption/ionization (MALDI) mass analysis. The obtained MALDI peptide profile revealed both previously known and unexpected processing products of the proopiomelanocortin gene. Mass spectrometric peptide sequencing of a few of these neuropeptides was performed by employing MALDI combined with postsource decay (PSD) fragment ion mass analysis. The potential of MALDI-PSD for sequence analysis of peptides directly from unfractionated tissue samples was examined for the first time for the known desacetyl-alpha-MSH-NH2 and the presumed vasotocin neuropeptide. In addition, the sequence of an unknown peptide which was present in the pars intermedia tissue sample at mass 1392.7 u was determined. The MALDI-PSD mass spectrum of precursor ion 1392.7 u contained sufficient structural information to uniquely identify the sequence by searching protein sequence databases. The determined amino acid sequence corresponds to the vasotocin peptide with a C-terminal extension of Gly-Lys-Arg ("vasotocinyl-GKR"), indicating incomplete processing of the vasotocin precursor protein in the pituitary pars intermediate of X. laevis. Both vasotocin and vasotocinyl-GKR are nonlinear peptides containing a disulfide (S-S) bridge between two cysteine residues. Interpretation of the spectra of these two peptides reveals three different forms of characteristic fragment ions of the cysteine side chain: peptide-CH2-SH (regular mass of Cys-containing fragment ions), peptide-CH2-S-SH (regular mass + 32 u) and peptide = CH2 (regular mass -34 u) due to cleavage on either side of the sulfur atoms.     

Molecular cloning and gene expression of chum salmon cystatin

A salmon cystatin cDNA clone was isolated from a chum salmon cDNA library. The clone encoded a full-length extracellular-type cystatin and its signal peptide and included 5'- and 3'-untranslated regions. The deduced amino acid sequence showed a high degree of sequence similarity to mammalian cystatin C, chicken egg cystatin, and chum salmon pituitary cystatin. By Northern blot analysis, the salmon cystatin was found to show apparently non-tissue specific expression. Because platyfish EHS cells transfected with a cystatin expression vector produced a 13 kDa mature cystatin in the culture medium, the salmon cystatin was considered to act as an extracellular type of cystatin in the fish cells. These findings indicate that the salmon cystatin is a homolog of mammalian cystatin C.     

Isolation and structural characterization of pituitary adenylate cyclase activating polypeptide (PACAP)-like peptide from the brain of a teleost, stargazer, Uranoscopus japonicus

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a novel neuropeptide consisting of 38-residue (PACAP 1-38) and a truncated form with 27 residues (PACAP 1-27) that plays several roles in tetrapods. We isolated a highly purified PACAP-like peptide from the brain of a teleost, the stargazer, by extracting of acetone-dried powder with acetic acid followed by high-performance liquid chromatography (HPLC) on gel-filtration, cation-exchange, and reverse-phase columns. Purification was monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting analysis using an anti-PACAP 1-27 antiserum. The PACAP-like peptide thus obtained had a molecular mass of 4,623, determined by mass spectrometry, and its amino acid sequence showed 89 and 87% identity with those of ovine and frog PACAPs, respectively. These results indicate that a PACAP-like peptide, which is a highly homologous with tetrapod PACAP, is present in the teleost brain.     

Human 15-lipoxygenase gene promoter: analysis and identification of DNA binding sites for IL-13-induced regulatory factors in monocytes

Cystatins are protein inhibitors of papain and related cysteine proteinases. A series of continuous synthetic peptides corresponding to the entire sequence of rat salivary cystatin was used to localize the binding domains of the cystatin to papain. Several synthetic peptides, one from the aminoterminal sequence (peptide 1-24) and others from the carboxylterminal (peptides 66-79, 66-90, 79-90, 79-114), showed binding to papain, but none of the peptides showed inhibition of papain activity. Three recombinant rat salivary cystatin variants (N-terminal truncated protein lacking amino acid residues 1-9; variant 49-53, in which amino acid residues QVVAG of rat salivary cystatin had been replaced with amino acid residues LVL in mutant protein; and variant 65-78, in which amino acid residues 65-78 had been replaced with amino acids PG in mutant protein) were produced using the Escherichia coli expression system pGex-4T. To generate N-terminal truncated protein the desired coding region of the cystatin gene was amplified by polymerase chain reaction (PCR). To produce the variants 49-53 and 65-78, a PCR-based approach of gene splicing by overlap extension was used. Recombinant cystatin proteins were produced as insoluble inclusion bodies as fusion proteins with a glutathione S-transferase (GST) carrier. After solubilization with urea the GST carrier was cleaved from the fusion protein with thrombin and cystatin variants purified by fast liquid chromatography on a MonoQ column. The purified proteins reacted with antibodies to rat salivary cystatin. The N-terminal truncated and variant 49-53 exhibited very little inhibitory activity towards papain, whereas variant 65-78 exhibited papain-inhibitory activity similar to the full-length recombinant cystatin.     

Automated interpretation of high-energy collision-induced dissociation spectra of singly protonated peptides by 'SeqMS', a software aid for de novo sequencing by tandem mass spectrometry

SeqMS, a software program designed for the automated interpretation of high-energy collision-induced dissociation (CID) mass spectra of singly protonated peptides ionized by fast atom bombardment, has been developed. The software is capable of probing the sequence of an unknown peptide, and even of certain modified peptides. The program, compiled for WINDOWS95 or NT, also permits the retrieval of raw data and the reconstruction of the spectra on a user-friendly graphical interface with the aid of several tools for processing the spectra, which include setting multiple threshold levels and automatic peak detection. SeqMS is capable of generating candidate sequences, based on the detected peaks, and of displaying the resulting assignments for each candidate in a spectrum or in tabular form. The software has the following capabilities: 1) the ions derived from backbone and side-chain fragmentations, internal and immonium ions, and side-chain loss ions can be used for calculation; 2) 18O-labeling of a peptide at the C terminus, a methodology which was developed to differentiate N-terminal from C-terminal ions, is applicable as an optional setting; 3) modified amino acids and N- or C-terminal blocking groups are taken into account for calculation according to the user's setting in a library; 4) amino acid composition and partial or complete amino acid sequence of a peptide can be used as input for calculation; 5) the assignments of signal output in a spectrum can be graphically edited, and then re-calculated based on the edited peaks. The efficacy of the program is demonstrated by testing 74 high-energy CID spectra, obtained using a four-sector instrument, of synthetic, proteolytic, and biologically active peptides, some of which contain modified groups.     

Characterization of monoclonal and polyclonal antibodies to human choline acetyltransferase and epitope analysis

Choline acetyltransferase (ChAT) was partially purified from human placenta and brain. In order to raise monoclonal antibodies, Balb/c mice were immunized with a preparation from placenta or with a mixture of eight synthetic peptides that were deduced from the primary structures of porcine and human ChAT. Polyclonal antibodies were raised in rabbits against five synthetic peptides deduced from the amino acid sequence of human ChAT. The monoclonal and polyclonal antibodies were characterized by their ability to recognize ChAT in various immunoassays: immunoblot, enzyme-linked immunosorbent assay (ELISA), two-side ELISA and immunohistochemistry. With one exception all monoclonal antibodies recognized ChAT on immunoblots, some were particularly sensitive; one bound active ChAT in ELISA when used as capture reagent; most antibodies recognized immobilized ChAT in ELISA. Two monoclonal antibodies out of nine gave particularly excellent results in staining cholinergic neurons and fibers on sections from rat and primate brain. With the help of nine synthetic peptides it was possible to evaluate two major binding sites for the monoclonal antibodies on the ChAT molecule, comprising amino acids 167-189 and 57-76, respectively.     

Proadrenomedullin-derived peptides

Posttranslational processing of the adrenomedullin gene product results in the formation of at least two biologically active peptides, adrenomedullin (AM) and proadrenomedullin N-20 terminal peptide (PAMP). Produced predominantly in the vasculature, both peptides are potent hypotensive agents, albeit via unique mechanisms of action. The gene is transcribed in a variety of other tissues including brain, pituitary, and kidney. Numerous actions have been reported most related to the physiologic control of fluid and electrolyte homeostasis. In the kidney, AM is diuretic and natriuretic, and both AM and PAMP inhibit aldosterone secretion by direct adrenal actions. In pituitary gland, both peptides at physiologically relevant doses inhibit basal ACTH secretion, again by apparently differing mechanisms. Additionally, AM antagonizes CRH-stimulated ACTH release. The peptides are produced in numerous brain sites, including hypothalamus and brainstem. Inhibition of AVP release has been reported and the physiologic significance of AM's ability to inhibit water drinking and salt appetite has been established. Thus the peptides appear to act in brain and pituitary gland to facilitate the loss of plasma volume, actions which complement their hypotensive effects in the blood vessel. Interestingly, direct cardiac effects (positive inotropism and chronotropism) and CNS actions (sympathostimulation) have been reported, leading to the hypothesis that these peptides also can exert important cardioprotective effects, helping to prevent vascular collapse during states of high AM secretion such as sepsis.     

Purification Technology and Antimicrobial Activity Analysis of Antimicrobiai Peptide from Ovotransferrin

Antibacterial peptides mixture purified from Ovotransferrin by pepsin digest was used as the raw material. Peptide sections with good antibacterial activity were determined after bacteriostasis experiments, its molecular weight and amino acid composition were analyzed. The results of experiments indicate that with Sephadex G-50 and distilled water as mobile phase, detection wavelength 220 nm, flow rate 1.5 mL/min, sample density 0.2 g/mL, and volume 0.2 mL are the optimal conditions. Bacteriostasis experiments of the fraction of purified peaks were carried out and the result was: peak 1>peak 3>peak 2; the molecular weight of peak 1 was about 3015 by high performance liquid chromatography; active peptide possessed positive charges by amino acid analysis, its cationic characteristics are in accordance with the nature of antimicrobial peptides.