Isolation and Characterization of a Novel Nucleoside from the Urines of Chronic Myelogenous Leukemia Patients

Abstract

From 24 hour collections of urines of chronic myelogenous leukemia (CML) patients, a novel nucleoside was isolated. It was assigned the structure, 5′-deoxyinosine (I) on the basis of UV, NMR and mass spectrometry and by comparison of the spectral data and HPLC and TLC mobilities with those of the authentic sample. Another nucleoside, 5′-deoxy-5′-methylthioadenosine sulfoxide previously isolated from the urines of immunodeficient children was also found in the urine of a CML patient. Possible origin and significance of both of these nucleosides are discussed.     

Immunodetection of pneumolysin in human urine by ELISA

An ELISA test has been employed for the detection of pneumolysin (PLY) in urine from 14 pneumococcal pneumonia patients and from 11 healthy adult volunteers. The urines of all the 11 healthy adult volunteers developed signals around the mean of the blanks, whereas all the pneumococcal pneumonia patient urines rendered signals at least five times this mean. Chemiluminescent Western blot analyses of these urines, carried out with the PLY-specific rabbit polyclonal IgG preparation used in ELISA, were negative. The 30-kDa filtrates of three high-signal urines were ELISA negative, suggesting that this ELISA test mainly detected high molecular weight forms in urine rather than free PLY-derived antigenic fragments. The urine sample, which rendered the highest ELISA signal, was then concentrated by filtration through a 10-kDa filter. When this concentrate was subjected to Western blot with the ELISA-capture monoclonal antibody, a major band was developed. Its relative molecular mass was similar to that of recombinant PLY and its peptide mass fingerprinting showed peptides corresponding to amino acid stretches from the four domains of the PLY molecule. When the pool of PLY-negative urines was sham-contaminated with purified recombinant pneumolysin, a conspicuous matrix effect was observed; nevertheless, this ELISA test was still reproducible and highly sensitive, detecting pneumolysin in the order of picograms per milliliter. A comparison was also made between this PLY-ELISA and the Binax NOW Streptococcus pneumoniae Urinary Antigen Test in analysing bacterial isolates. On the basis of the minimum number of pneumococci examined, both tests were shown to have similar potency, but strain-dependent discrepancies were observed. This ELISA could provide an alternative to the Binax NOW Streptococcus pneumoniae Urinary Antigen Test in the diagnosis of pneumococcal pneumonia.     

Pathophysiological role of nitric oxide and adrenomedullin in autism

Several studies indicate that nitric oxide (NO) is involved in the aetiopathogenesis of many neuropsychiatric disorders such as schizophrenia, bipolar disorder, depression, Alzheimer's disease, Hungtington disease and stroke. Although it has not been investigated yet, several recent studies proposed that NO may have a pathophysiological role in autism. Adrenomedullin (AM), a recently discovered 52-amino acid peptide hormone, induces vasorelaxation by activating adenylate cyclase and also by stimulating NO release. AM immune reactivity is present in the brain consistent with a role as a neurotransmitter. It has been stated that NO and AM do function in the regulation of many neurodevelopmental processes. We hypothesized that NO and AM activities have been affected in autistic patients and aimed to examine these molecules. Twenty-six autistic patients and 22 healthy control subjects were included in this study. AM and total nitrite (a metabolite of NO) levels have been measured in plasma. The mean values of plasma total nitrite and AM levels in the autistic group were significantly higher than control values, respectively (p < 0.001, p = 0.028). There is no correlation between total nitrite and AM levels (r = 0.11, p = 0.31). Certainly, this subject needs much further research investigating autistic patients in earlier periods of life and with subtypes of the disorder.     

Isolation and identification of alpha-methyloctanylcarnitines from human urine

Medium-chain acylcarnitines were isolated from human urine using a combination of chloroform-methanol extraction, silicic acid column and molecular sieving chromatography and preparative HPLC. Three purified acylcarnitines were analyzed by fast atom bombardment mass spectrometry and were also saponified and the free fatty acids analyzed by gas chromatography and mass spectrometry. Combined electron impact mass spectrometry and fast atom bombardment mass spectrometry and periodate oxidation for location of double bonds, demonstrated the occurrence of delta 6-octenylcarnitine, 2-methyloctanylcarnitine and 2-methyl-delta 6-octenylcarnitine. These acylcarnitines were present in the thirteen urines obtained from normal humans, but were not detected in urines from three individuals who had been on total parenteral nutrition for more than a year. The occurrence of alpha-methyl medium-chain acylcarnitines in human urine indicates a role for carnitine in excretion (detoxification) of these acyl derivatives.     

Rapid and sensitive determination of enzymatic degradation products of isomeric chondroitin sulfates by high-performance liquid chromatography

The separation and quantitative analysis of enzymatic degradation products of isomeric chondroitin sulfates by high-performance liquid chromatography (HPLC) are described. The substituted unsaturated disaccharides which result from digestion of chondroitin sulfates with chondroitinase are quickly separated on polar adsorbents such as silica gel. The UV absorption properties of these unsaturated disaccharides permit UV measurement with detection limits of approximately 100 ng. Their separation by HPLC facilitates the use of enzymatic methods for the determination of chondroitin sulfates A, B and C.

The potential of this method in clinical application is demonstrated by quantitative assays of glycosaminoglycans from a normal urine and urine from a patient with Hunter syndrome. The results are consistent with amount of isomeric chondroitin sulfates found in comparable urines by others.     

Isolation and Identification of α-Methyloctanylcarnitines from Human Urine

Medium-chain acylcarnitines were isolated from human urine using a combination of chloroform-methanol extraction, silicic acid column and molecular sieving chromatography and preparative HPLC. Three purified acylcarnitines were analyzed by fast atom bombardment mass spectrometry and were also saponified and the free fatty acids analyzed by gas chromatography and mass spectrometry. Combined electron inpact mass spectrometry and fast atom bombardment mass spectrometry and periodate oxidation for location of double bonds, demonstrated the occurrence of δ⁶-octenylcarnitine, 2-methyloct-anylcarnitine and 2-methyl-δ⁶-octenylcarnitine. These acylcarnitines were present in the thirteen urines obtained from normal humans, but were not detected in urines from three individuals who had been on total parenteral nutrition for more than a year. The occurrence of α-methyl medium-chain acylcarnitines in human urine indicates a role for carnitine in excretion (detoxification) of these acyl derivatives.     

A vasotocin-like peptide in Aplysia kurodai ganglia: HPLC and RIA evidence for its identity with Lys-conopressin G.

The presence of a vasopressin (VP)- or vasotocin (VT)-like peptide in the central nervous system of the gastropod mollusc Aplysia has been indicated previously. In the case of Aplysia californica, HPLC and RIA evidence suggested the peptide was VT-like but not identical with the nonmammalian vertebrate peptide [Arg8]VT (AVT). In the present study, anterior ganglia extracts from the related species Aplysia kurodai were analyzed by HPLC followed by RIA. Further analysis of the major AVT-IR peak showed it to be indistinguishable, in three distinct solvent systems, from the sea snail venom peptide Lys-conopressin G, but to be different from the vertebrate peptides [Arg8]VP (AVP), [Lys8]VP (LVP), AVT, oxytocin (OT), mesotocin, isotocin, aspargtocin, glumitocin, and valitocin, from the sea snail venom peptide Arg-conopressin S, and from the peptides [Lys8]VT and [Gln8]OT. In addition, the carboxymethylated (CM) A. kurodai peptide had the same HPLC retention time as CM-Lys-conopressin G. The HPLC/RIA results suggest that (i) based on the properties of the solvent systems used, the A. kurodai peptide has two basic amino acids (like the conopressins but unlike the vertebrate peptides), and (ii) there is a high probability that the A. kurodai peptide is identical with Lys-conopressin G.     

Ion-exchange high-performance liquid-chromatography steps in peptide purifications

Ion-exchange high-performance liquid chromatography (HPLC; on Ultropac TSK DEAE and CM) is compared with conventional soft-gel ion-exchange chromatography in identical peptide purifications. The results show that separating properties are similar, but as expected~ ion-exchange HPLC has a much higher resolving capacity and a higher sensitivity, and allows a con-siderably shorter total separation time. The same buffer systems as for conventional ion-exchange chromatography can be used, including urea to solubilize large peptides, if care is taken not to exceed the pH limits set by the column matrix.A complete purification scheme by HPLC in the nanomolar range, utilizing exclusion, ion-exchange, and reverse-phase chromatographies, is given with a complex peptide mixture from a digest of a large protein. Similar steps as in conventional soft-gel schemes can be utilized. It is concluded that ion-exchange HPLC is a suitable complement to commonly used reverse-phase HPLC steps and t h a t it permits high speed and sensitivity over wide ranges of peptide sizes and amounts.     

Exploring the hidden human urinary proteome via ligand library beads

The human urinary proteome has been reassessed and re-evaluated via a novel concentration/equalization technique, exploiting beads coated with hexameric peptide ligand libraries. These beads act by capturing the whole protein spectra contained in the sample, by drastically reducing the level of the most abundant species, while strongly concentrating the more dilute and rare ones. In a control urine sample, 134 unique proteins could be identified. The first bead eluate (in thiourea, urea, and CHAPS) permitted the identification of 317 gene products, whereas the second eluate (in 9 M urea, pH 3.8) allowed the identification of another 95 unique proteins. By eliminating redundancies, a total of 383 unique gene products could be identified in human urines. This represents a major increment as compared to data reported in recent literature. By comparing our data with those reported to the present, an additional 251 proteins could be added to the list, thus bringing the total unique gene products so far identified in human urines to ca. 800 species.     

Urinary p-cresol is elevated in young French children with autism spectrum disorder: a replication study

Abstract

The aromatic compound p-cresol (4-methylphenol) has been found elevated in the urines of Italian autistic children up to 8 years of age. The present study aims at replicating these initial findings in an ethnically distinct sample and at extending them by measuring also the three components of urinary p-cresol, namely p-cresylsulfate, p-cresylglucuronate and free p-cresol. Total urinary p-cresol, p-cresylsulfate and p-cresylglucuronate were significantly elevated in 33 French autism spectrum disorder (ASD) cases compared with 33 sex- and age-matched controls (p?<?0.05). This increase was limited to ASD children aged ≤8 years (p?<?0.01), and not older (p?=?0.17). Urinary levels of p-cresol and p-cresylsulfate were associated with stereotypic, compulsive/repetitive behaviors (p?<?0.05), although not with overall autism severity. These results confirm the elevation of urinary p-cresol in a sizable set of small autistic children and spur interest into biomarker roles for p-cresol and p-cresylsulfate in autism.     

Use of proline-specific endopeptidase in the isolation of all four "native" disulfides of hen egg white lysozyme

The disulfide peptides from the tryptic digestion of cyanogen bromide-treated hen egg white lysozyme (HEWL) were isolated by reverse phase high performance liquid chromatography (HPLC) and identified by amino acid analysis. Three peptides containing the I-VIII, II-VII, and III-V + IV-VI disulfide bonds were obtained. The two-disulfide peptide was further digested with proline-specific endopeptidase (PCE) (EC 3.4.21.26). Amino acid analysis of digest peptides separated by HPLC showed four peptides with the IV-VI disulfide bond as well as a peptide with the III-V disulfide bond. The IV-VI peptides were produced by hydrolysis of several alanine-X bonds as well as the prolyl-cystine bond. Our studies show that alanyl peptide bonds to lysyl, seryl, and leucyl residues are susceptible to hydrolysis by PCE preparations, thus substantially extending its known specificity range. The two-disulfide peptide was also digested sequentially with thermolysin and PCE; the resulting IV-VI and III-V peptides were identified by HPLC and amino acid analysis. PCE showed substantial activity at pH 5.3 as well as at pH 8.3. The lower pH is useful in studies of proteins or peptides where base-catalyzed reactions must be limited.     

A novel HPLC procedure for detection and quantification of aminoacetone, a precursor of methylglyoxal, in biological samples

Increase in methylglyoxal is thought to be involved in different pathological conditions. Deamination of aminoacetone by semicarbazide-sensitive amine oxidase (SSAO) leads to production of methylglyoxal. We have synthesized aminoacetone and developed a novel HPLC procedure for its quantitative determination. The urinary excretion of aminoacetone is approximately 20-30 microg/mouse/day, and the concentration is about 0.5 microg/g in mouse liver and small intestine. SSAO inhibitor increases aminoacetone levels in both tissues and urines. Results confirm that aminoacetone is an endogenous substrate for SSAO. However, data also indicate that deamination is not the only catabolic pathway for aminoacetone.     

Synthesis of porcine C5a anaphylatoxin by the solution procedure and confirmation of the reported structure

Porcine C5a anaphylatoxin, the primary structure of which was first determined by Gerard and Hugli in 1980 as a 74-amino acid peptide having three intramolecular disulfide bonds, was synthesized by the solution procedure applying our maximum protection strategy. The fully deprotected peptide was subjected to air oxidation in an acetate buffer at pH 7.5, and the product was isolated as a single entity by HPLC. Amino acid analysis and biological activities of the synthetic peptide agreed well with the reported values. However, the retention time of the synthetic C5a was different from that of the natural product, supplied by Dr. Hugli, on both reversed phase (RP) and ion-exchange (IEX) HPLC systems. The tryptic peptide mapping on HPLC revealed that Gln which was incorporated into the peptide at position 65 was replaced by Glu in the natural product. The elution pattern of tryptic peptides containing three disulfide bonds was identical with natural and synthetic C5a. It was also identical with that of a peptide which was synthesized following the estimated secondary structure proposed by Zimmermann and Vogt in 1984.     

High performance liquid chromatography of pharmacologically active amines and peptides in biological materials

Precise and quantitative reversed-phase high performance liquid chromatographic (HPLC) procedures are described which can be used in biogenic amine and neuropeptide research. The amine procedure was applied to various pharmacological matrices including plasma, heart tissue and brain. The use of peptide HPLC as an analytical tool for various neuropeptides is illustrated by studies on des-tyrosine-gamma-endorphin (DT gamma E) metabolism in the brain and the stability of an ACTH (ORG-2766) analogue during a chronic infusion in rats. The power of HPLC as a research tool in peptide pharmacology is described, discussed and demonstrated as an aid in the understanding of the pharmacological effects of exogenous peptides and the function of the brain.     

Quantification and partial characterisation of regulatory peptides in the liver fluke, Fasciola hepatica, from different mammalian hosts.

1. Extracts of the liver fluke, Fasciola hepatica from three different hosts (cow, sheep, rat) have been subjected to radioimmunoassay using antisera to 6 mammalian regulatory peptides. 2. Immunoreactivity was measured to pancreatic polypeptide, substance P, peptide histidine isoleucine and gastrin-releasing peptide. Levels of each peptide varied considerably in flukes from different hosts. 3. Reverse-phase HPLC of rat and sheep fluke extracts revealed three molecular forms of tachykinin immunoreactivity and single peaks of pancreatic polypeptide and peptide histidine isoleucine immunoreactivity. No GRP-immunoreactivity was detected by RIA of HPLC fractions.     

Total synthesis and further characterization of the gamma-carboxyglutamate-containing "sleeper" peptide from Conus geographus venom

The total synthesis of the Gla-containing "sleeper" peptide (Gly-Glu-Gla-Gla-Leu-Gln-Gla-Asn-Gln-Gla-Leu-Ile-Arg-Gla-Lys-Ser-Asn-NH2 ) from Conus geographus is described. A new strategy for the synthesis of acid-sensitive peptide amides was developed, which allowed complete deprotection and cleavage of the L-gamma-carboxyglutamate-containing peptide from the 2,4-dimethoxybenzhydrylamine resin. Synthetic sleeper peptide, after preparative high-performance liquid chromatography (HPLC) purification, was shown to be identical with the native peptide by all criteria (coelution experiments of HPLC, sequence analysis, and biological activity). In addition, a developmental switch in the behavioral symptoms induced by the peptide after intracerebral administration in mice was documented. At low doses of the peptide (4-30 pmol/g), a sleeplike state was induced in mice under 2 weeks old; in contrast, older mice became markedly hyperactive. It is proposed that, in the presence of Ca2+, the sleeper peptide assumes an alpha-helical configuration in which all the gamma-carboxyglutamate residues are located on the same side of the alpha-helix.     

Formation of isoaspartate 99 in bovine and porcine somatotropins

Asparagine 99 in bovine (BST) and porcine somatotropins (PST) was converted to an isoaspartate residue during incubation at neutral or alkalinepH. Isoaspartate 99 BST or isoaspartate 99 PST was resolved from the normal somatotropin by reversed-phase high-performance liquid chromatography (HPLC). The altered peptide of residues 96–108 which contains isoaspartate 99 was detected by tryptic peptide mapping of the modified BST or PST. Amino acid sequencing, amino acid analysis, mass spectrometry, and co-elution with a chemically synthesized peptide containing isoaspartate 99 were used to demonstrate the existence of isoaspartate in the modified peptides. Peptide bond cleavage between Asn 99 and Ser 100 also occurred during incubation of BST and PST at neutral or alkalinepH. This chemically cleaved product was resolved on reversed-phase HPLC from both the isoaspartate 99 and normal somatotropin molecules.     

Double dimer peptide constructs are immunogenic and protective against Plasmodium falciparum in the experimental Aotus monkey model.

Multiple antigen peptide constructs (MAPs) have been used to obtain defined multimeric peptide molecules useful in the development of possible synthetic malaria vaccines. In this context, a method was developed, named double dimer constructs (DDCs), involving the direct synthesis of a dimeric peptide with a C-terminal cysteine. A tetrameric molecule was then obtained by oxidation of sulfhydryl groups. Dimer synthesis was optimized using a Fmoc/tBu strategy, dimers were purified by HPLC, oxidized with DMSO and characterized by HPLC and MALDI-TOF-MS. The tetramers or DDCs obtained by this method were used as immunogens in the search for a possible malaria vaccine. It was found that they were immunogenic in the experimental Aotus monkey model, and were able to induce protective immunity when challenged experimentally with a highly infective Plasmodium falciparum malaria strain.     

Expression of antihypertensive peptide, His-His-Leu, as tandem repeats in Escherichia coli

His-His-Leu (HHL), a tripeptide derived from a Korean soybean paste, is an angiotensin-I-converting enzyme (ACE) inhibitor. We report here a method of producing this tripeptide efficiently by expressing tandem multimers of the codons encoding the peptide in E. coli and purifying the HHL after hydrolysis of the peptide multiners. The HHL gene, tandemly multimerized to a 40-mer, was ligated with ubiquitin as a fusion gene (UH40). UH40 was inserted into vector pET29b; the UH40 fusion protein was then produced in E. coli BL21. The recombinant UH40 protein was purified by cation-exchange chromatography with a yield of 17.3 mg/l and analyzed by matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) mass spectrometry and protein N-terminal sequencing. Leucine aminopeptidase was used to cleave a 405-Da HHL monomer from the UH40 fusion protein and the peptide was purified using reverse-phase high-performance liquid chromatography (HPLC) on a C18 HPLC column, with a final yield of 6.2 mg/l. The resulting peptide was confirmed to be HHL with the aid of MALDI-TOF mass spectrometry, glutamine-TOF mass spectrometry, N-terminal sequencing, and measurement of ACE inhibiting activity. These results suggest that our production method is useful for obtaining a large quantity of recombinant HHL for functional antihypertensive peptide studies.