KSAYMRFamide (PF3/AF8) is present in the free-living nematode, Caenorhabditis elegans

To date, seven FMRFamide-related peptides (FaRPs) have been structurally characterized from C. elegans, of which one is structurally identical to the parasitic nematode peptide AF2 (KHEYLRFamide). The other six FaRPs have so far been identified in free-living forms only. in the present study an additional FaRP was isolated and structurally characterized from an ethanolic extract of C. elegans. The extract was screened using a C-terminally directed FaRP antiserum, and the FMRFamide-immunoreactive peptide purified to homogeneity using HPLC. Approximately 80 pmol of the peptide was subjected to Edman degradation and the unequivocal primary structure of the K-7-amide, KSAYMRFamide (PF3/AF8) was determined following a single gas-phase sequencing run. The molecular mass of the peptide was determined using a MALDI-TOF mass spectrometer and was found to be 919 (MH+), which is in agreement with the theoretical mass of C-terminally amidated PF3. A new flp-gene, designated flp-6, has recently been identified which encodes six copies of KSAYMRFamide (PF3/AF8). (C) 1998 Academic Press.

THE FMRFAMIDE-LIKE NEUROPEPTIDE AF2 (ASCARIS-SUUM) IS PRESENT IN THE FREE-LIVING NEMATODE, PANAGRELLUS-REDIVIVUS (NEMATODA, RHABDITIDA)

Available primary structural information suggests that the FMRFamide-related peptides (FaRPs) from parasitic and free-living nematodes are different, and that free-living forms may not represent appropriate models for the study of the neurochemistry of parasitic forms in the laboratory. However, here we report the isolation and unequivocal identification of AF2 (originally isolated from the parasite, Ascaris suum) from acidified alcoholic extracts of the free-living species, Panagrellus redivivus. While reverse-phase HPLC analysis of extracts revealed FMRFamide-immunoreactivity to be highly heterogeneous, AF2 was the predominant FMRFamide-immunoreactive peptide present (at least 26 pmol/g wet weight of worms). This peptide was also the major immunoreactant identified by an antiserum raised to the conserved C-terminal hexapeptide amide of mammalian pancreatic polypeptide (PP), which has been used previously to isolate neuropeptide F (NPF). These observations were confirmed by radioimmunoassay and chromatographic fractionation of an acidified alcoholic extract of A. suum heads. The FMRFamide-related peptides present in a nematode extract may be highly dependent on the extraction medium employed, and these data would suggest that this complement of neuropeptides may not be as different between parasitic and free-living nematodes as initial studies have suggested. Finally, all of the evidence suggests that NPF is not present in nematodes and that the PP-immunoreactant previously demonstrated immunochemically is probably AF2.

NEUROPEPTIDE-Y AND NEUROPEPTIDE-Y 3-36 - ISOLATION FROM HUMAN PANCREATIC ENDOCRINE TUMORS

Using an antiserum raised to the C-terminal region of neuropeptide Y (NPY) which does not cross-react with pancreatic polypeptide (PP), immunoreactivity has been detected in two different endocrine tumours of the human pancreas in concentrations permitting isolation and structural analysis. In a clinically-typical gastrinoma, resected from the head of pancreas, the concentration of NPY immunoreactivity was 3.4 nmol/g. Reverse phase HPLC analysis of extracts of this tumour resolved a single immunoreactive peptide coeluting with synthetic human NPY. The molecular mass of the isolated peptide, determined by mass spectroscopy, was 4270 Da, which was in close agreement with that derived from the deduced primary structure of human tumour NPY (4271.7 Da), obtained by gas-phase sequencing. A somatostatinoma, resected from the region of the ampulla of Vater, contained 3.8 nmol/g of NPY immunoreactivity and isolation of this immunoreactive peptide followed by structural analyses, indicated a molecular structure consistent with NPY 3-36. These data suggest that NPY immunoreactivity detected in human pancreatic endocrine tumours is molecularly heterogenous, a finding which may be of relevance in the symptomatology of such tumours as attenuation of the N-terminus of this peptide generates receptor selectivity.

THE PRIMARY STRUCTURE OF PANCREATIC-POLYPEPTIDE FROM A PRIMITIVE INSECTIVOROUS MAMMAL, THE EUROPEAN HEDGEHOG (ERINACEOUS-EUROPAEUS)

Pancreatic polypeptide (PP) has been isolated from extracts of the pancreas of the European hedgehog (Erinaceous europaeus) which is a representative of the order Insectivora, deemed to be the most primitive group of placental mammals. Pancreatic tissues were extracted in acidified ethanol and the peptide was purified chromatographically using a PP C-terminal hexapeptide amide specific radioimmunoassay to monitor purification. Two major PP-immunoreactive peptides were baseline-resolved following the final analytical reverse phase HPLC fractionation. Each was separately subjected to plasma desorption mass spectroscopy (PDMS) and gas-phase sequencing. The molecular masses of each peptide were similar: (I) 4237.6 +/- 4 Da and (II) 4238.2 +/- 4 Da. The full primary structures of each peptide were deduced and these were identical: VPLEPVYPGDNATPEQMAHYAAELRRYINMLTRPRY. The peptides were deemed to be amidated due to their full molar cross-reactivity with the amide-requiring PP antiserum employed in radioimmunoassay. The molecular mass (4233.8 Da) calculated from the sequence was in close agreemeent with PDMS estimates and the reason for the different retention times of each peptide is unknown at present. Hedgehog PP exhibits only 2 unique amino acid substitutions, at positions 1 (Val) and 19 (His), when compared with other mammalian analogues.

A novel Kazal-type trypsin inhibitor from the skin secretion of the Central American red-eyed leaf frog, Agalychnis callidryas.

The chemical complexity of the defensive skin secretion of the red-eyed leaf frog, (Agalychnis callidryas), has not been elucidated in detail. During a systematic study of the skin secretion peptidomes of phyllomedusine frogs, we discovered a novel Kazal-type protein with potent trypsin inhibitory activity (Ki = 1.9 nM) that displays the highest degree of structural similarity with Kazal proteins from bony fishes. The protein was located in reverse-phase HPLC fractions following a screen of such for trypsin inhibition and subsequent partial Edman degradation of the peak active fraction derived the sequence: ATKPR-QYIVL-PRILRPV-GT. The molecular mass of the major component in this fraction was established by MALDI-TOF MS as 5893.09 Da. This partial sequence (assuming blank cycles to be Cys residues) was used to design a degenerate primer pool that was employed successfully in RACE-PCR to clone homologous precursor-encoding cDNA that encoded a mature Kazal protein of 52 amino acid residues with a computed molecular mass of 5892.82 Da. The protein was named A. callidryas Kazal trypsin inhibitor (ACKTI). BLAST analysis revealed that ACKTI contained a canonical Kazal motif (C-x(7)-C-x(6)-Y-x(3)-C-x(2,3)-C). This novel amphibian skin Kazal trypsin inhibitor adds to the spectrum of trypsin inhibitors of Kunitz- and Bowman Birk-type reported from this amphibian source.

Parallel Peptidome and Transcriptome Analyses of Amphibian Skin Secretions Using Archived Frozen Acid-Solvated Samples

Amphibian skin secretions are unique sources of bioactive peptides and their donor species are currently rapidly disappearing from the biosphere. Here, we report that both peptides and polyadenylated mRNAs from skin granular glands remain amenable to study in samples of stimulated skin secretions following their storage in 0.1 % aqueous trifluoroacetic acid at -20 °C for many years. Frozen acidified solutions of toad (Bombina variegata) skin secretions, stored for 12 years, were thawed and samples removed for direct reverse phase HPLC fractionation. Additional samples were removed, snap frozen and lyophilised for construction of cDNA libraries following polyadenylated mRNA capture using magnetic oligo-dT beads and reverse transcription. Using the bombesin and bradykinin peptides found in bombinid toad skin as models, individual variant peptides of each type were located in reverse phase HPLC fractions and their corresponding biosynthetic precursor-encoding mRNA transcripts were cloned from the cDNA library using a RACE PCR strategy. This study illustrates unequivocally that both amphibian skin secretion peptides and their biosynthetic precursor-encoding polyadenylated mRNAs are stable in frozen acid-solvated skin secretion samples for considerable periods of time-a finding that may have fundamental implications in the study of archived materials but also in the wider field of molecular biology.

The wbbD gene of E. coli strain VW187 (O7:K1) encodes a UDP-Gal:GlcNAc{alpha}-pyrophosphate-R {beta}1,3-galactosyltransferase involved in the biosynthesis of O7-specific lipopolysaccharide

In this work, we demonstrate that the wbbD gene of the O7 lipopolysaccharide (LPS) biosynthesis cluster in Escherichia coli strain VW187 (O7:K1) encodes a galactosyltransferase involved in the synthesis of the O7-polysaccharide repeating unit. The galactosyltransferase catalyzed the transfer of Gal from UDP-Gal to the GlcNAc residue of a GlcNAc-pyrophosphate-lipid acceptor. A mutant strain with a defective wbbD gene was unable to form O7 LPS and lacked this specific galactosyltransferase activity. The normal phenotype was restored by complementing the mutant with the cloned wbbD gene. To characterize the WbbD galactosyltransferase, we used a novel acceptor substrate containing GlcNAcalpha-pyrophosphate covalently bound to a hydrophobic phenoxyundecyl moiety (GlcNAc alpha-O-PO(3)-PO(3)-(CH(2))(11)-O-phenyl). The WbbD galactosyltransferase had optimal activity at pH 7 in the presence of 2.5 mM MnCl(2). Detergents in the assay did not increase glycosyl transfer. Digestion of enzyme product by highly purified bovine testicular beta-galactosidase demonstrated a beta-linkage. Cleavage of product by pyrophosphatase and phosphatase, followed by HPLC and NMR analyses, revealed a disaccharide with the structure Gal beta1-3GlcNAc. Our results conclusively demonstrate that WbbD is a UDP-Gal: GlcNAcalpha-pyrophosphate-R beta1,3-galactosyltransferase and suggest that the novel synthetic glycolipid acceptor may be generally applicable to characterize other bacterial glycosyltransferases.

A PEPTIDE FROM THE CAUDAL NEUROSECRETORY-SYSTEM OF THE DOGFISH SCYLIORHINUS-CANICULA THAT IS STRUCTURALLY RELATED TO UROTENSIN-I

Using reversed-phase HPLC in combination with a radioimmunoassay for ovine corticotropin-releasing hormone (CRH), a peptide with CRH-like immunoreactivity was isolated in pure form from an extract of the caudal spinal cord region of the spotted dogfish, Scyliorhinus canicula. The primary structure of the peptide was established as Pro-Ala-Glu-Thr-Pro-Asn-Ser-Leu-Asp-Leu(10)-Thr-Phe-His-Leu-Leu-Arg-Glu-Met-Ile-Glu(20)-Ile-Ala-Lys-His-Glu-Asn-Gln-Gln-Met-Gln(30)-Ala-Asp-Ser-Asn-Arg-Arg-Ile-Met-Asp-Thr(40)-Ile . NH2. This amino acid sequence shows moderate structural similarity to Catostomus urotensin I (51%) and to human CRH (56%). The data provide, therefore, chemical evidence to support the conclusions of earlier immunohistochemical studies that the diffuse caudal neurosecretory system of elasmobranchs produces a peptide that is immunochemically related to teleost urotensin I peptides. However, the primary structure of urotensin I has been poorly conserved during evolution. (C) 1995 Academic Press, Inc.

Size-fitting of Intravaginal Rings for Macaques and in vitro Release Kinetics of Zinc Finger Inhibitors

Background: Small molecule inhibitors of the zinc finger domain (ZFI) in the nucleocapsid protein (NCp7) of HIV-1 are potent inhibitors of HIV and SIV
replication and may have utility as topical products to prevent infection. Furthermore, intravaginal rings (IVRs) were developed as coitally-independent,
sustained release devices which could be used for administration of HIV microbicides. The aims of these studies were to demonstrate that IVRs sized for
macaques are practical and compatible with the current generation of thioester-based NCp7 inhibitors.

Methods: Non-medicated silicone elastomer vaginal rings of various sizes thought to be applicable for macaques were prepared and tested for vaginal fit in Pigtailed and Chinese Rhesus macaques. Macaques were monitored for 8 weeks for mucosal disruption by colposcopy and proinflammatory cytokine markers in cervical vaginal lavages (CVL) using Luminex bead-based technology. Three different ZFIs (compounds 52, 89 and 122, each derived from an N-substituted S-acyl-2-mercaptobenzamide thioester scaffold) were loaded at 50 mg into an optimal matrix-type ring design. In vitro continuous release studies were then conducted over 28 days and analyzed by HPLC. Rate of release was determined by linear regression analysis.

Results: Qualitative evaluation at the time of ring insertion suggested that the 25 mm ring provided optimal fit in both macaque species. All rings remained in
place during the study period (2 to 4 weeks), and the animals did not attempt to remove the rings. No tissue irritation was observed, and no signs of physical
discomfort were noted. Also, no significant induction of cervicovaginal proinflammatory markers was observed during the 8-week period during and following ring insertion. One Pigtailed macaque showed elevated IL-8 levels in the CVL during the period when the ring was in place; however, these levels were comparable to those observed in two control macaques. In vitro release of the ZFIs peaked at day 1 and then continually declined to near steady-state rates between 20-30 mcg/day. The percent release after 14 days was 2.9, 2.0 and 0.9 for ZFI 89, 52 and 122, respectively.

Conclusions: IVRs of 25mm diameter, determined to be the optimal size for macaques, were well tolerated and did not induce inflammation. Release of all ZFI compounds followed t 0.5 kinetics. These findings suggest that efficacy testing in primate models is warranted to fully evaluate the potential to prevent
transmission.

The structures of four bombesins and their cloned precursor-encoding cDNAs from acid-solvated skin secretion of the European yellow-bellied toad, Bombina variegata

Four different bombesins (bombesin, His(6)-bombesin, Phe(13)-bombesin and Asp(2)-, Phe(4)-SAP-bombesin) have been identified by a systematic sequencing study of peptides in reverse phase HPLC fractions of the skin secretion of the European yellow-bellied toad, Bombina variegata, that had been solvated in 0.1% (v/v) aqueous trifluoroacetic acid (TFA) and stored frozen at -20°C for 12 years. By using a 3'- and 5'-RACE PCR strategy, the corresponding biosynthetic precursor-encoding cDNAs of all four peptides were cloned from a cDNA library made from the same long-term frozen, acid-solvated skin secretion sample following thawing and lyophilization. Canonical bombesin and His(6)-bombesin are classical bombesin sub-family members, whereas Phe(13)-bombesin and Asp(2)-, Phe(4)-SAP-bombesin, belong to the litorin/ranatensin sub-family of bombesin-like peptides (BLPs). Assignment of these peptides to respective sub-families, was based upon both their primary structural similarities and their comparative pharmacological activities. An interesting observation in this study, was that the nucleotide sequences of the open-reading frames of cloned cDNAs encoding bombesin and its His(6)-substituted analog, were identical except for a single base that was responsible for the change observed at the position 6 residue in the mature peptide from Asn to His. In contrast, the precursor cDNA nucleotide sequences encoding the Phe(13)-bombesins, exhibited 53 base differences. The pharmacological activities of synthetic replicates of each bombesin were compared using two different mammalian smooth muscle preparations and all four peptides were found to be active. However, there were significant differences in their relative potencies.

Development of a solid-phase extraction coupling chemiluminescent enzyme immunoassay for determination of organophosphorus pesticides in environmental water samples

Solid-phase extraction (SPE) and direct competitive chemiluminescence enzyme immunoassay (dcCL-EIA) were combined for the detection of organophosphorus pesticides (OPs) in environmental water samples. dcCL-EIA based on horseradish peroxidase labeled with a broad-specificity monoclonal antibody against OPs was developed, and the effects of several physicochemical parameters on dcCL-EIA performance were studied. SPE was used for the pretreatment of water samples to remove interfering substances and to concentrate the OP analytes. The coupling of SPE and dcCL-EIA can detect seven OPs (parathion, coumaphos, phoxim, quinalphos, triazophos, dichlofenthion, and azinphos-ethyl) with the limit of quantitation below 0.1 ng/mL. The recoveries of OPs from spiked water samples ranged from 62.5% to 131.7% by SPE-dcCL-EIA and 69.5% to 112.3% by SPE-HPLC-MS/MS. The screening of OP residues in real-world environmental water samples by the developed SPE-dcCL-EIA and their confirmatory analysis using SPE-HPLC-MS/MS demonstrated that the assay is ideally suited as a monitoring method for OP residues prior to chromatographic analysis.

Development of a Broad-Specificity Monoclonal Antibody-Based Immunoaffinity Chromatography Cleanup for Organophosphorus Pesticide Determination in Environmental Samples

An immunoaffinity chromatographic (IAC) method for the selective extraction and concentration of 13 organophosphorus pesticides (OPs, including coumaphos, parathion, phoxim, quinalphos, dichlofenthion, triazophos, azinphos-ethyl, phosalone, isochlorthion, parathion-methyl, cyanophos, disulfoton, and phorate) prior to analysis by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was developed. The IAC column was prepared by covalently immobilizing a monoclonal antibody with broad specificity for OPs on CNBr-activated Sephrose 4B. The column capacity ranged from 884 to 2641 ng/mL of gel. The optimum elution solvent was 0.01 M phosphate-buffered saline containing 80% methanol. The breakthrough volume of the IAC column was found to be 400 mL. Recoveries of OPs from spiked environmental samples by IAC cleanup and HPLC-MS/MS analysis ranged from 60.2 to 107.1%, with a relative standard deviation below 11.1%. The limit of quantitation for 13 OPs ranged from 0.01 to 0.13 ng/mL (ng/g). The application of IAC cleanup coupled to HPLC-MS/MS in real environmental samples demonstrated the potential of this method for the determination of OP residues in environmental samples at trace levels.

Identification of an arsenic tolerant double mutant with a thiol-mediated component and increased arsenic tolerance in phyA mutants

A genetic screen was performed to isolate mutants showing increased arsenic tolerance using an Arabidopsis thaliana population of activation tagged lines. The most arsenic-resistant mutant shows increased arsenate and arsenite tolerance. Genetic analyses of the mutant indicate that the mutant contains two loci that contribute to arsenic tolerance, designated ars4 and ars5. The ars4ars5 double mutant contains a single T-DNA insertion, ars4, which co-segregates with arsenic tolerance and is inserted in the Phytochrome A (PHYA) gene, strongly reducing the expression of PHYA. When grown under far-red light conditions ars4ars5 shows the same elongated hypocotyl phenotype as the previously described strong phyA-211 allele. Three independent phyA alleles, ars4, phyA-211 and a new T-DNA insertion allele (phyA-t) show increased tolerance to arsenate, although to a lesser degree than the ars4ars5 double mutant. Analyses of the ars5 single mutant show that ars5 exhibits stronger arsenic tolerance than ars4, and that ars5 is not linked to ars4. Arsenic tolerance assays with phyB-9 and phot1/phot2 mutants show that these photoreceptor mutants do not exhibit phyA-like arsenic tolerance. Fluorescence HPLC analyses show that elevated levels of phytochelatins were not detected in ars4, ars5 or ars4ars5, however increases in the thiols cysteine, gamma-glutamylcysteine and glutathione were observed. Compared with wild type, the total thiol levels in ars4, ars5 and ars4ars5 mutants were increased up to 80% with combined buthionine sulfoximine and arsenic treatments, suggesting the enhancement of mechanisms that mediate thiol synthesis in the mutants. The presented findings show that PHYA negatively regulates a pathway conferring arsenic tolerance, and that an enhanced thiol synthesis mechanism contributes to the arsenic tolerance of ars4ars5.

Inorganic arsenic contents in rice-based infant foods from Spain, UK, China and USA

Spanish gluten-free rice, cereals with gluten, and pureed baby foods were analysed for total (t-As) and inorganic As (i-As) using ICP-MS and HPLC-ICP-MS, respectively. Besides, pure infant rice from China, USA, UK and Spain were also analysed. The i-As contents were significantly higher in gluten-free rice than in cereals mixtures with gluten, placing infants with celiac disease at high risk. All rice-based products displayed a high i-As content, with values being above 60% of the t-As content and the remainder being dimethylarsinic acid (DMA). Approximately 77% of the pure infant rice samples showed contents below 150 µg kg(-1) (Chinese limit). When daily intake of i-As by infants (4-12 months) was estimated and expressed on a bodyweight basis (µg d(-1) kg(-1)), it was higher in all infants aged 8-12 months than drinking water maximum exposures predicted for adults (assuming 1 L consumption per day for a 10 µg L(-1) standard).

Arsenic accumulation and phosphorus status in two rice (Oryza sativa L.) cultivars surveyed from fields in South China

The consumption of paddy rice (Oryza sativa L.) is a major inorganic arsenic exposure pathway in S.E. Asia. A multi-location survey was undertaken in Guangdong Province, South China to assess arsenic accumulation and speciation in 2 rice cultivars, one an Indica and the other a hybrid Indica. The results showed that arsenic concentrations in rice tissue increased in the order grain <husk <straw <root. Rice grain arsenic content of 2 rice cultivars was significant different and correlated with phosphorus concentration and molar ratio of P/As in shoot, being higher for the Indica cultivar than for the hybrid Indica, which suggests altering shoot phosphorus status as a promising route for breeding rice cultivars with reduced grain arsenic. Speciation of grain arsenic, performed using HPLC-ICP-MS, identified inorganic arsenic as the dominant arsenic species present in the rice grain.