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Biomedical subjects

R J Nachman

Publications and source records attributed to R J Nachman.

At least 37 records · Page 2Linked to original sources
PubMed1999

Comparison of rates of penetration through insect cuticle of amphiphylic analogs of insect pyrokinin neuropeptides.

Rates of penetration through the cuticle of amphiphylic analogs, synthesized by addition of 6-phenylhexanoic acid or 9-fluoreneacetic acid or 1-pyrenebutyric acid to the amino terminus of the pentapeptide Phe-Thr-Pro-Arg-Leu-amide, were assessed by quantitative analysis using reversed phase liquid chromatography. The analogs effectively penetrated the cuticle of both the adult American cockroach and tobacco budworm moth. However, the amounts of analogs that penetrated the cuticle of the cockroach were significantly lower and the rates of penetration were slower than for moth cuticle. Penetration of the analogs through the cuticle was dependent upon the size of the lipidic attachment to the pentapeptide. The 6-phenylhexanoic acid analog penetrated most rapidly followed by the 9-fluoreneacetic acid analog and the 1-pyrenebutyric acid analog penetrated slowest. All of the analogs exhibited an initial rapid period of penetration lasting 2-3 h followed by the establishment of a steady slow release state which lasted between 9-24 h and was dependent upon both the size and surface area of the aromatic lipidic portion of the analog and species of insect to which the analog was applied. The results confirmed the hypothesis that the insect cuticle could be employed as a slow release device for delivery of analogs of insect neuropeptides.

Animals↗
PubMed1999

Effects of an allatostatin and a myosuppressin on midgut carbohydrate enzyme activity in the cockroach Diploptera punctata.

Neuropeptides of the cockroach allatostatin (AST) family are known for their ability to inhibit the production of juvenile hormone by the corpora allata of cockroaches. Since their discovery, they have also been shown to modulate myotropic activity in a range of insect species as well as to act as neurotransmitters in Crustaceans and possibly in insects. The midgut of cockroaches contains numerous endocrine cells, some of which produce AST whereas others produce the FMRFamide-related peptide, leucomyosuppressin (LMS). We have determined if ASTs and LMS are also able to influence carbohydrate-metabolizing enzyme activity in the midgut of the cockroach, Diploptera punctata. Dippu-AST 7 stimulates activity of both invertase and alpha-amylase in a dose-dependent fashion in the lumen contents of ligatured midguts in vitro, but not in midgut tissue, whereas the AST analog AST(b)phi2, a cyclopropyl-ala, hydrocinnamic acid analog of Dippu-AST 6, has no effect. Leucomyosuppressin also stimulates enzyme activity in lumen contents only, although the EC50 is considerably greater than for Dippu-AST. Dippu-AST is also able to inhibit proctolin-induced contractions of midgut muscle, and this action had already been described for LMS [18]. Thus, in this organ, AST and LMS have at least two distinct physiological effects.

Animals↗
PubMed1999

Differential distribution of pyrokinin-isoforms in cerebral and abdominal neurohemal organs of the American cockroach.

Different pyrokinin isoforms were identified from major neurohemal organs of the American cockroach. During their isolation they were recognized by bioassay using a hyperneural muscle preparation that is sensitive to pyrokinins. All structures were elucidated by sequence analysis and mass spectrometry. The primary structures of the novel peptides isolated from the retrocerebral complex are LVPFRPRL-NH2 (designated Pea-PK-3) and DHLPHDVYSPRL-NH2 (designated Pea-PK-4). A pyrokinin, labeled Pea-PK-5, was isolated from abdominal perisympathetic organs. Structural analysis of this peptide yielded the sequence GGGGSGETSGMWFGPRL-NH2. The threshold concentrations of the identified pyrokinins for an eliciting effect on contractions of the hyperneural muscle preparations differed dramatically. This indicates that the different distribution of pyrokinin-isoform observed in neurohemal organs may be associated with different functions. This is the first report of a differential distribution of peptide-isoforms in the neurohemal organs of insects.

Animals↗
PubMed1999

Detection of nascent polyproline II helices in solution by NMR in synthetic insect kinin neuropeptide mimics containing the X-Pro-Pro-X motif.

The conformations of three synthetic peptide analogs containing the dPro-dPro-dXaa motif (dXaa = dThr, dGlu, dAsn) in aqueous solution were studied by a combination of NMR and molecular modeling simulations. The three compounds were identified from a random D-amino acid tripeptide library on the basis of their ability to either mimic or block the diuretic activity of neuropeptides of the insect kinin family. TOCSY and ROESY correlations, as well as abnormal secondary chemical shifts for protons on the D-proline residues were employed to obtain conformational ensembles consistent with the experimental NMR data for the three analogs using an in vacuo simulated annealing protocol. Similar secondary structures were found for the three molecules after refinement, in agreement with the similarities observed between their NMR spectra. Unrestrained molecular dynamics simulations with explicit water representation indicate that the structural motifs found in vacuo are stable in aqueous solution. The three analogs can be considered initiators of right-handed poly D-proline II helices, mirror images of the poly L-proline II left-handed helical motifs normally found in proline-rich proteins. The role of these secondary folds on binding of the analogs to the kinin receptors is discussed.

Insect Proteins↗
PubMed1999

Post-translational modifications of the insect sulfakinins: sulfation, pyroglutamate-formation and O-methylation of glutamic acid.

We identified and chemically characterized the two major forms of sulfakinins from an extract of 800 corpora cardiaca/corpora allata complexes of the American cockroach, Periplaneta americana. Bioactivity during the purification was monitored by measuring heart beat frequency in a preparation in situ. By Edman degradation analysis and MS, these main forms were identified as having the primary structures Pea-SK [EQFDDY(SO(3)H)GHMRFamide] and Lem-SK-2 [pQSDDY(SO(3)H)GHMRFamide]. The sulfation was confirmed by UV, MS and peptide synthesis. In addition, post-translationally modified sulfakinins of both major forms were isolated and identified. Firstly, nonsulfated forms of these peptides are present in considerable amounts in the corpora cardiaca/allata. Secondly, the N-terminally blocked Pea-SK and the nonblocked Lem-SK-2 occur naturally in neurohaemal release sites. Thirdly, modified Pea-SK with O-methylated glutamic acid occurs which is not an artefact of peptide purification. The major forms of the sulfakinins were shown to be highly active on both the heart and hindgut with threshold concentrations of approximately 5 x 10(-10) M (heart) and 2 x 10(-9) M (hindgut).

Amino Acid Sequence↗
PubMed1999

Stimulation of alpha-amylase release in the scallop Pecten maximus by the myosuppressins. Structure-activity relationships.

The insect myosuppressin LMS (pGlu-Asp-Val-Asp-His-Val-Phe-Leu-Arg-Phe-NH2) elicits potent stimulation of the release of the digestive enzyme alpha-amylase from cell suspensions of the stomach-digestive gland complex of the scallop Pecten maximus. The myosuppressins are members of the FMRFamide-like peptide superfamily, which immunocytochemical data confirm is present in the scallop. Structure-activity studies indicated that the two most critical residues for bioactivity are Arg and Phe. Bioactivity of the peptide can be maintained if the basic, aromatic residue His is replaced by another basic residue (Lys) and another aromatic residue (Trp), but not the aromatic Tyr, indicating a sensitivity to the introduction of a phenolic OH group. A restricted-conformation analogue containing a cyclopropyl-Ala residue in position 8 (Cpa-MS) demonstrates an ability to antagonize the amylase secretion activity of LMS at microM concentrations. This result provides evidence that the myosuppressins adopt a tight turn in the C-terminal tetrapeptide active core region while binding to the scallop digestive gland receptor. Cpa-MS may provide a useful tool to neuroendocrinologists studying in vitro and in vivo digestive processes in mollusks and other invertebrates.

Animals↗
PubMed1999

Development of amphiphylic mimics of insect neuropeptides for pest control.

Insect neuropeptides regulate virtually all aspects of insect life and are excellent candidates for development of new methods for pest control. However, they do not penetrate insect cuticle and are degraded by enzymes both in the digestive system and hemolymph. We have designed mimics of model neuropeptides by attachment of various lipidic moieties to the amino terminus of the bioactive core of the neuropeptides. These mimics have amphiphylic characteristics that allowed them to penetrate the hydrophobic insect cuticle. The mimics also induced prolonged physiological responses (up to 20 hours) and were resistant to peptidase attack. This knowledge has been used to develop a novel, species-specific approach to insect control.

Animals↗
PubMed1999

Comparison of active conformations of the insectatachykinin/tachykinin and insect kinin/Tyr-W-MIF-1 neuropeptide family pairs.

A comparison of solution conformations of active, restricted-conformation analogues of two sequence-similar insect/vertebrate neuropeptide family pairs shed light on the potential existence of molecular evolutionary relationships. Analogues of the locustatachykinins and the mammalian tachykinin substance P, containing a sterically hindered Aib-NMePhe/Tyr residue block, share similar low-energy turn conformations incorporating a cis peptide bond. Conversely, restricted conformation analogues of the insect kinins and the mammalian opiate peptide Tyr-W-MIF-1, with near identical C-terminal tetrapeptide sequences, adopt different conformations. The insect kinins adopt a cisPro 1-4 beta-turn, in which the Phe1 is critical for bioactivity. Tyr-W-MIF-1 prefers a transPro 2-5 turn, and an additional N-terminal Phe severely inhibits mu-opiate receptor binding. Comparisons of the chemical/conformational requirements for receptor interaction are consistent with a distant evolutionary relationship between the insectatachykinins and tachykinins, but not between the insect kinins and Tyr-W-MIF-1. Therefore, analogues of the insect kinins with pest control potential can be readily designed to avoid mammalian interactions.

Amino Acid Sequence↗
PubMed1998

An aminoisobutyric acid-containing analogue of the cockroach tachykinin-related peptide, LemTRP-1, with potent bioactivity and resistance to an insect angiotensin-converting enzyme.

Nine tachykinin-related peptides (TRPs), designated LemTRP-1-9, were recently isolated from the cockroach, Leucopheae maderae. To obtain a LemTRP resistant to endo- and exoprotease-mediated hydrolysis, we synthesized a peptide with one of the carboxy terminus residues substituted for a sterically hindered aminoisobutyric acid (Aib) and with the amino terminus blocked with a pyroglutamate. The Aib-containing analogue of the nonapeptide LemTRP-1 (Aib-LemTRP-1) thus has the sequence pGlu-Ala-Pro-Ser-Gly-Phe-Leu-Aib-Val-Arg-NH2. This analogue was shown to be resistant to hydrolysis by recombinant angiotensin-converting enzyme (ACE), from Drosophila melanogaster. Endogenous LemTRP-1 on the other hand was rapidly hydrolysed by ACE at the Gly7-Val8 bond, resulting in a single heptapeptide. The Aib-LemTRP-1 has about the same potency as LemTRP-I in inducing contractions of the L. maderae hindgut muscle. It was also tested in intracellular recordings for ability to induce firing of action potentials in dorsal unpaired median (DUM) neurons in the metathoracic ganglion of the locust Locusta migratoria. The Aib-containing analogue was nearly as active as LemTRP-1 and the natural ligand locustatachykinin I. LemTRP-1 and Aib-LemTRP-1 had the same transient time course of action on the cockroach hindgut. This suggests that peptide degradation is not likely to be the cause of the transient action of TRPs.

Amino Acid Sequence↗
PubMed1998

Immunocytochemical localisation and biological activity of diuretic peptides in the housefly, Musca domestica.

The distribution of a CRF-related diuretic peptide (Musca-DP) and the diuretic/myotropic insect myokinins in the central nervous system of larval and adult houseflies was investigated using antisera raised against Locusta-DH and leucokinin-I, respectively. Two separate, small populations of immunoreactive neurons are present in the brain and fused thoracic-abdominal ganglion mass. There is no evidence for these immunoreactivities being colocalised either within single neurons or at neurohaemal release sites. Crude extracts of tissues containing immunoreactive material increase fluid secretion by isolated Malpighian tubules from adult flies. Diuretic activity is highest in tissues containing myokinin-immunoreactive material. Consistent with this observation, myokinin analogues produce a four- to five-fold increase in fluid secretion, which is more than twice the response to Musca-DP. These effects are mimicked by treatments that increase intracellular calcium and cyclic AMP, respectively. When tested at threshold concentrations, the two classes of diuretic peptide act synergistically to accelerate tubule secretion, and their separate localisation may be important for the precise control of diuresis.

Animals↗
PubMed1998

Isolation of periviscerokinin-2 from the abdominal perisympathetic organs of the American cockroach, Periplaneta americana.

Using the isolated hyperneural muscle as bioassay, a novel myotropin was isolated from the abdominal perisympathetic organs of Periplaneta americana. This is the second neuropeptide identified from insect perisympathetic organs. Peptide sequence analysis and mass spectrometry yielded the following structure: Gly-Ser-Ser-Ser-Gly-Leu-Ile-Ser-Met-Pro-Arg-Val-NH2. This peptide, named periviscerokinin-2, was confirmed to be amidated by chemical synthesis, bioassay, and comparison of retention times between native and synthetic peptides. A highly specific antiserum was used to determine sites of synthesis in the abdominal ganglia. Besides periviscerokinin-1, periviscerokinin-2 is the only putative myotropic neurohormone from the abdominal perisympathetic organs that is effective in the nanomolar range. This confirms the hypothesis that the neurohormonal system of the ventral nerve cord is remarkably different from that of the brain.

Abdomen↗
PubMed1998

Synthesis, biological activity, and conformational studies of insect allatostatin neuropeptide analogues incorporating turn-promoting moieties.

Allatostatins are 6-18 amino acid peptides synthezed by insects to control production of juvenile hormones, which in turn regulate functions including metamorphosis and egg production. Four insect allatostatin neuropeptide analogues incorporating turn-promoting pseudopeptide moieties in the region responsible for biological activity were prepared by solid phase peptide synthetic methods. Bioassay indicated that activities approached those of the natural neuropeptides, and molecular models based on NMR data showed similar conformations and the presence of a beta-turn in the active core region for the four analogues. Differences in activity are believed to be due to differences in bulk and relative position of atoms in the unnatural portion of the analogues, and their differing degrees of conformational freedom. The studies support the feasibility of development of neuropeptide-based insect control agents resistant to peptidase deactivation.

Animals↗
PubMed1998

Isolation and immunocytochemical characterization of three tachykinin-related peptides from the mosquito, Culex salinarius.

Three myotropic peptides belonging to the Arg-amide insect tachykinin family were isolated from whole-body extracts of the mosquito, Culex salinarius. The peptides, APSGFMGMR-NH2, APYGFTGMR-NH2 and APSGFFGMR-NH2 (designated culetachykinin I, II, and III) were isolated and purified on the basis of their ability to stimulate muscle contractions of isolated Leucophaea maderae hindgut. Biologically inactive methionine sulfoxides of two of the three peptides were isolated using an ELISA system based upon antiserum raised against APYGFTGMR-NH2 and identified with mass spectrometry. Immunocytochemistry localized these peptides in cells in the brain, antennae, subesophageal, thoracic and abdominal ganglion, proventriculus and midgut. Nerve tracts containing these peptides were found in the median nerve of the brain, central body, nervi corpus cardiaci, cervical nerve, antennal lobe and on the surface of the midgut.

Amino Acid Sequence↗
PubMed1997

Prolonged pheromonotropic activity of pseudopeptide mimics of insect pyrokinin neuropeptides after topical application or injection into a moth.

Amphiphilic pseudopeptide analogs of Phe-Thr-Pro-Arg-Leu-NH2, representing the active C-terminal core pentapeptide of the pyrokinin class of insect neuropeptides, were synthesized by replacement of phenylalanine with hydrocinnamic acid (Hca-Thr-Pro-Arg-Leu-NH2), or addition of 1-pyrenebutyric acid (Pba-Phe-Thr-Pro-Arg-Leu-NH2) or 9-fluoreneacetic acid (Fla-Phe-Thr-Pro-Arg-Leu-NH2). The pseudopeptides were found to stimulate sex pheromone biosynthesis when injected into females of the moth Heliothis virescens. Optimal pheromonotropic responses were obtained by injection of 0.25 pmol of Hca-Thr-Pro-Arg-Leu-NH2, 2.5 pmol of Pba-Thr-Pro-Arg-Leu-NH2 and 0.5 pmol of Fla-Thr-Pro-Arg-Leu-NH2. Topical application of each of the pseudopeptides in water to the cuticle of moths stimulated significant production of pheromone at a dose of 50 pmol with optimal stimulation occurring when 500 pmol were applied. The parent peptide, Phe-Thr-Pro-Arg-Leu-NH2, failed to stimulate significant production of pheromone when applied topically at a dose as high as 2000 pmol. Temporal studies indicated that Hca-Thr-Pro-Arg-Leu-NH2 stimulated significant production of pheromone for only 4 h after application where as continuous pheromone production for 18 h was observed when either Pba-Phe-Thr-Pro-Arg-Leu-NH2 or Fla-Phe-Thr-Pro-Arg-Leu-NH2 were applied to the abdomen. The results show that modification of the C-terminal active core of the insect pyrokinins, by addition of hydrophobic moieties, can result in production of pseudopeptides which effectively penetrate the insect cuticle and have prolonged physiological effects making them favorable candidates for use in development of alternative strategies for pest insect control.

Acetates↗
PubMed1997

Hydrolysis of insect neuropeptides by an angiotensin-converting enzyme from the housefly, Musca domestica.

The presence in insect tissues of peptides with structural similarities to angiotensin I and to bradykinin, the two best known substrates of mammalian angiotensin-converting enzyme, has not been reported. As part of our study to identify potential substrates for insect angiotensin-converting enzyme, we have investigated the susceptibility of a number of known insect peptide hormones and neurotransmitters to hydrolysis by Musca domestica angiotensin-converting enzyme. Insect peptides belonging to the red pigment-concentrating hormone, leucokinin, locust tachykinin, and depolarizing peptide families were hydrolyzed by housefly angiotensin-converting enzyme, whereas proctolin and crustacean cardioactive peptide were not substrates. Cus-DP II, LK I, LK II, and Lom-TK I were all cleaved at the penultimate C-terminal peptide bond to release a dipeptide amide as a major fragment with Km values of 94 +/- 11, 634 +/- 8, and 296 +/- 35 microM for Cus-DP II, LK I, and Lom-TK I, respectively. The ability of insect angiotensin-converting enzyme to hydrolyze C-terminally amidated peptides in vitro might be of functional significance because the enzyme has been localized to neuropile regions of the insect brain and is present in the hemolymph of houseflies.

Animals↗