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Biomedical subjects

M S Wright

Publications and source records attributed to M S Wright.

At least 37 records · Page 2Linked to original sources
PubMed1994

Culekinin depolarizing peptide: a mosquito leucokinin-like peptide that influences insect Malpighian tubule ion transport.

A peptide termed culekinin depolarizing peptide (CDP) was isolated from approximately 1.2 million mosquitos (94% Culex salinarius). The peptide was isolated on the basis of a rapid myotropic assay that utilized a hindgut preparation from Leucophaea maderae and a transepithelial voltage assay that used mosquito Malpighian tubules from Aedes aegypti. A 15% trifluoroacetic acid extraction from the mosquitos, two solid phase extraction steps, and six HPLC steps resulted in the isolation of 9.7 nmol of CDP. This value corresponds to approximately 8 fmol/mosquito. Edman degradation indicated the following sequence for CDP: Asn-Pro-Phe-His-Ser-Trp-Gly-NH2. The sequence was confirmed as the suspected C-terminal amide form of the peptide, since native and synthetic CDP had identical chemical and biological properties. CDP is a member of the leucokinin family of neuropeptides. The leucokinins have been found in three other insect species (Leucophaea maderae, Acheta domesticus and Locusta migratoria) where these peptides were isolated by their myotropic properties alone. CDP shares a C-terminal sequence homology (i.e., Phe-X-Ser-Trp-Gly-NH2) with the rest of the leucokinins. CDP corresponds to the strongest tubule depolarizing activity in the C. salinarius extract. These findings agree with previous structure-activity studies that suggest that mosquitos would contain a leucokinin-like factor that had Phe-His-Ser-Trp-Gly-NH2 as the C-terminal pentapeptide. This is the first leucokinin isolated from blood feeding or holometabolous insects.

Aedes↗
PubMed1994

Isolation and characterization of a diuretic peptide common to the house fly and stable fly.

An identical CRF-related diuretic peptide (Musca-DP) was isolated and characterized from whole-body extracts of the house fly, Musca domestica, and stable fly, Stomoxys calcitrans. The peptide stimulates cyclic AMP production in Manduca sexta Malpighian tubules and increases the rate of fluid secretion by isolated Musca domestica tubules. The 44-residue peptide, with a mol.wt. of 5180, is amidated, and has the primary structure: NKPSLSIVNPLDVLRQRLLLEIARRQMKENTRQVELNRAILKNV-NH2. Musca-DP has a high percentage of sequence identity with other characterized CRF-related insect diuretic peptides.

Amino Acid Sequence↗
PubMed1993

Characterization of an antiserum against an achetakinin I-analog and its use for the localization of Culekinin Depolarizing Peptide II in the mosquito, Culex salinarius.

ELISA experiments revealed that an antiserum raised against an achetakinin-analog could specifically detect the recently isolated Culekinin Depolarizing Peptide (CDP)-II from the mosquito, Culex salinarius. The characterization indicated that two different epitopes in the C-terminal region of achetakinin I and CDP-II are recognized. One epitope is the -F-Y-region, the other is the -P-W-region. Among the peptides isolated from C. salinarius, the antiserum reacts only with CDP-II. Pre-absorption tests of the antiserum with CDP-II in immunohistological stainings abolished the reaction, while tests with pre-immune sera did not cause any immunopositive reactions. In the mosquito head ganglia, immunoreactive neurons were detected in the pars lateralis, the optic lobe and the suboesophageal ganglion. Although some immunopositive axons extended into the nervi corporis cardiacii II, no immunoreactivity was observed in the retrocerebral complex. In the thoracic ganglia, immunoreactive neurons were found in the pro-, meso- and metathoracic neuromeres. No immunoreactivity was found elsewhere. With this study we demonstrate that CDP-II, isolated from a whole body extract, is truly a neuropeptide, and the data suggest that its function is neuromodulating or neurotransmitting rather than neurohormonal.

Amino Acid Sequence↗
PubMed1993

Molecular cloning of a functional human thyrotropin-releasing hormone receptor.

A cDNA encoding the human thyrotropin-releasing hormone receptor (hTRH-R) was isolated from a human brain cDNA library. Screening of 1.2 million clones resulted in 2 candidates. The largest clone contained TRH-R homologous sequences starting in the third transmembrane domain and included a long 3' untranslated sequence. The smaller clone contained a potential start of the open reading frame, but was interrupted by an intron in the sixth transmembrane domain. The two clones had 497 bp of overlapping identical sequences and it was possible to assemble a complete cDNA thus restoring the assumed coding sequence. Electrophysiological studies of frog oocytes injected with in vitro transcribed mRNA showed TRH-specific inward currents, demonstrating that the reconstituted cDNA encoded a functional receptor. The predicted amino acid sequence of the hTRH-R protein showed high homology with the rat and mouse TRH-Rs with the exception of their C-terminal region. The human TRH-R gene seems to contain two introns.

Amino Acid Sequence↗
PubMed1993

Use of Tn4560 to generate nikkomycin non-producing mutants of Streptomyces tendae.

Transposon Tn4560 was used to generate three nikkomycin non-producing mutants in Streptomyces tendae ATCC 31160. Southern hybridization confirmed that Tn4560 was present in 10-12-kb BamHI fragments of the chromosomes of the mutants. Biologically active nikkomycins were not detected in culture broths of the mutants as determined by bioassays and HPLC. Differences in the HPLC profiles of culture broths suggest that Tn4560 inserted into different genes in the mutants.

Aminoglycosides↗
PubMed1993

Detection and partial characterization of an anti-steroidogenic peptide from the humoral immune system of the chicken.

Phenomenological association of alterations of immune system function at the time of puberty (e.g. involution of the chicken bursa of Fabricius) has led to postulation that the humoral immune system may negatively affect the hypothalamo-adenohypophyseal-gonadal axis of the neonate. Presently, we examined the effect of an acidic aqueous bursa of Fabricius extract, derived from prepubescent chickens, on in vitro basal and LH-stimulated progesterone biosynthesis by isolated ovarian granulosa cells of the largest preovulatory chicken follicles (F1 and F2). Crude extracts of < 5kDa and > 3kDa inhibited LH-stimulated progesterone secretion (P < 0.05). The bioactive component was observed to be heat labile and is sensitive to the endopeptidases chymotrypsin, trypsin and papain. The peptide is not sensitive to the exopeptidase, aminopeptidase M. Partial purification by reversed phase HPLC resulted in a fraction capable of inhibiting in vitro steroidogenesis. This fraction suppressed LH-stimulated progesterone biosynthesis to approximately basal levels (79% suppression). Following removal of the peptide, granulosa cells were capable of LH-stimulated progesterone biosynthesis similar to control cells. Bursal extract significantly inhibited cAMP analog-stimulated progesterone biosynthesis. These data indicate that the anti-steroidogenic peptide derived from the chicken bursa of Fabricius is a single heat labile, amino terminally blocked peptide with bioactivity independent of the gonadotropin receptor of the granulosa cell.

Analysis of Variance↗
PubMed1993

Cloning of the long intracellular loop of the AMPA-selective glutamate receptor for phosphorylation studies.

It is now widely accepted that many receptors are regulated by phosphorylation. The glutamate receptor which belongs to a family of AMPA receptors has recently been cloned and contains in the long intracellular loop several consensus phosphorylation sites for Ca(2+)-calmodulin-dependent protein kinase type II and protein kinase C. In order to study the possible function of these phosphorylation sites antibodies are specifically raised against these sites. For this purpose the DNA encoding the long intracellular loop was synthesized by PCR amplification from rat cerebral cortex and hippocampus RNA. Primer sequences were taken from the published receptor cDNA and the resulting inserts showed after sequence determination the presence of different receptor isoforms. The proteins have been expressed by the T7 RNA polymerase system. These can then be used after purification to generate antibodies against the entire protein and partial peptides both in a phosphorylated and unphosphorylated form.

Amino Acid Sequence↗
PubMed1992

Cloning strategies for peptide hormone receptors.

With the advancement in molecular biology in the last decade many receptors have been cloned and the understanding of their mechanism of function has improved proportionally. In addition the concurrent discovery of receptor gene families has been used to design elegant cloning strategies which in turn have facilitated the characterization of new receptors and receptor subtypes. This review covers the most currently used cloning strategies and gives some examples.

Animals↗
PubMed1992

Validation of a self-reported shelf inventory to measure food purchase behavior.

A mailed, self-reported shelf inventory was validated for use as a tool in assessing the impact of a community nutrition intervention that included a point-of-purchase component. The self-reported inventory was evaluated for overall accuracy as well as for the effects of gender, age, and shopping responsibility on accuracy. In addition, the food-shelf inventory was compared with a specific food frequency questionnaire. Specificity and sensitivity were calculated for self-reported inventories using an interviewer-completed, same-day inventory as the gold standard. Overall sensitivity was 86% and 87%, and overall specificity was 92% and 90% in two validation studies. Results show that the mailed, self-reported shelf inventory is a valid measure of the presence or absence of particular foods in households. As such, it may be a useful tool for assessing the impact of point-of-purchase nutrition interventions.

Adult↗
PubMed1991

Use of a lacZ fusion to study transcriptional regulation of the Rhodobacter capsulatus hemA gene.

An EcoRI fragment containing the Rhodobacter capsulatus hemA promoter has been cloned into a lacZ translational fusion vector. The resulting plasmid produced a hemA-lacZ fusion protein with a molecular mass of 147,000. Expression of the hemA-lacZ fusion, as measured by production of beta-galactosidase, was regulated 2- to 3-fold by oxygen tension. The unexpectedly small change in beta-galactosidase levels suggests that transcriptional regulation of the hemA gene is not the major factor in oxygen-mediated control of porphyrin synthesis.

5-Aminolevulinate Synthetase↗
PubMed1991

Immunocytochemical localization of leucomyosuppressin-like peptides in the CNS of the cockroach, Leucophaea maderae.

Immunocytochemistry was used to determine sites of synthesis and pathways for the transport of the neuropeptide, Leucomyosuppressin (pQDVDHVFLRFamide) in the cockroach, Leucophaea maderae. This study led to identification of neurons in the brain and thoracic ganglia reactive to polyclonal antibodies raised against this peptide. No immunoreactive cells were found in the subesophageal or abdominal ganglia. Although the corpus cardiacum contained no intrinsic cells immunoreactive to LMS antibodies, the periphery of this organ and that of the nervi corporis allati contain an abundance of LMS-reactive terminals.

Amino Acid Sequence↗
PubMed1991

Localization of leucomyosuppressin-like peptides in the central nervous system of the stable fly with immunocytochemistry.

An antiserum raised against leucomyosuppressin (LMS), the first insect neuropeptide shown to inhibit contraction of both visceral and skeletal muscles of insects, revealed the presence of LMS-like material in neurons of the adult stable fly, Stomoxys calcitrans (L.). Cells and their processes immunoreactive to the LMS antiserum were found in the brain, subesophageal, and thoracico-abdominal ganglia, with the majority of these neurons in the thoracico-abdominal ganglion. Positive immunostaining after preabsorption of the antibodies with the molluscan tetrapeptide FMRFamide, which absorbs the antibodies directed against the C-terminal FLRFamide of LMS, indicates that structural similarities between LMS and the stable fly neuropeptide(s) extend beyond a similar C-terminal tetrapeptide sequence.

Animals↗
PubMed1990

Different-sized mRNAs from GH4C1 cells induce a TRH-dependent electrical response in Xenopus laevis oocytes.

Poly(A)+ RNA from the GH4C1 rat pituitary cell line elicited a thyrotropin releasing hormone response in Xenopus laevis oocytes which could be measured as a change in membrane current by the voltage-clamp method. Oocytes injected with Poly(A)+ RNA from GH12C1 cells which do not bind thyrotropin releasing hormone or with buffer solution alone did not show this response. Size fractionation of total poly(A)+ RNA by sucrose density-gradient centrifugation shows two response maximal representing various mRNA fractions larger than 18S. These results indicate the presence of thyrotropin releasing hormone receptor mRNA heterogeneity where the smallest mRNA is at least 2 kb.

Animals↗