Identification of His5,Trp7,Tyr8-GnRH (chicken GnRH II) in amphibian brain

GnRH immunoreactive and bioactive peptides in Xenopus laevis brain extract were investigated by high performance liquid chromatography (HPLC), radioimmunoassay with region-specific antisera raised against GnRH (mammalian), His5,Trp7,Tyr8-GnRH (chicken II) and Tyr3,Leu5,Glu6,Trp7,Lys8-GnRH (lamprey), and by assessment of biological activity. Two immunoreactive peptides eluted in the same positions as GnRH and His5,Trp7,Tyr8-GnRH respectively in HPLC systems which were specifically designed to separate four known natural vertebrate GnRHs (mammalian, chicken I and II, salmon). The immunological properties of these two immunoreactive peaks, determined by relative interaction with three region-specific antisera raised against mammalian GnRH and two specific His5,Trp7,Tyr8-GnRH antisera, were identical to those of GnRH and His5,Trp7,Tyr8-GnRH. The immunoreactive peak co-eluting with His5,Trp7,Tyr8-GnRH represented approximately one-third of the total brain GnRH. Both immunoreactive peaks stimulated luteinizing hormone (LH) release in a chicken dispersed pituitary cell bioassay, and the amounts of LH release stimulated by the two peaks were appropriate for these peaks being GnRH and His5,Trp7,Tyr8-GnRH. A small hydrophobic peak with GnRH immunoreactivity eluted in the same position as Trp7,Leu8-GnRH (salmon), while Gln8-GnRH (chicken I) and lamprey GnRH were not detected. Two additional rather hydrophilic peptides cross-reacted with a COOH-terminus-directed antiserum and had LH-releasing activity. LH-releasing activity was also detected in hydrophobic HPLC fractions. In summary, these data provide evidence for the presence of both GnRH and a second peptide with properties identical to His5,Trp7,Tyr8-GnRH in X. laevis brain.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural modifications of non-mammalian gonadotropin-releasing hormone (GnRH) isoforms: design of novel GnRH analogues

Three natural forms of vertebrate gonadotropin-releasing hormone (GnRH) provided the structural basis upon which to design new GnRH agonists: [His⁵,Trp⁷,Leu⁸]-GnRH, dogfish (df) GnRH; [His⁵,Asn⁸]-GnRH, catfish (cf) GnRH; and [His⁵,Trp⁷,Tyr⁸]-GnRH, chicken (c) GnRH-II. The synthetic peptides incorporated the position 6 dextro ( )-isomers -arginine ( -Arg) or -naphthylalanine ( -Nal) in combination with an ethylamide substitution of position 10. The in vitro potencies for LH and FSH release of these analogues were assessed using static cultures of rat anterior pituitary cells. Efficacious peptides were examined for their gonadotropin-II and growth hormone releasing abilities from perifused goldfish pituitary fragments. Rat LH and FSH release was measured using homologous radioimmunoassays, whereas goldfish growth hormone and gonadotropin-II release were determined using heterologous carp hormone radioimmunoassays. The receptor binding of the most potent analogues was determined in bovine pituitary membrane preparations. Substitution of -Nal⁶ into [His⁵,Asn⁸]-GnRH increased the potency over 2200-fold compared with the native ligand (cfGnRH) in cultured rat pituitary cells. This was equivalent to a 55-fold greater potency than that of the native mammal (m) GnRH peptide. Substitution of -Nal⁶ or -Arg⁶ into dfGnRH or cGnRH-II resulted in potencies that were related to the overall hydrophobicity of the analogues. The [ -Nal⁶,Pro⁹NEt]-cfGnRH bound to the bovine membrane preparation with an affinity statistically similar to that of [ -Nal⁶,Pro⁹NEt]-mGnRH (k_d = 0.40 ± 0.04 and 0.55 ± 0.10 nM, respectively) in cultured rat pituitary cells. All analogues tested released the same ratio of FSH to LH. In goldfish, the analogues did not possess superagonistic activity but instead desensitized the pituitary fragments at lower analogue doses than that of the sGnRH standard suggesting differences in receptor affinity or signal transduction.     

Multiple molecular forms of gonadotropin-releasing hormone in teleost fish brain

Gonadotropin-releasing hormone (GnRH) immunoreactive peptides in extracts of hake (Merluccius capensis) and tilapia (Tilapia sparrmanii) brain were investigated by high performance liquid chromatography (HPLC) and radioimmunoassay with region-specific antisera. In hake brain, content and concentration of GnRH was higher in the pituitary gland than in the hypothalamic lobes or extrahypothalamic brain. Hake pituitary gland GnRH was purified by six consecutive HPLC systems. The major GnRH molecular form co-eluted with salmon brain GnRH (Trp7, Leu8-GnRH) in four different HPLC systems which were specifically designed to separate the four natural vertebrate GnRHs (mammalian, salmon, chicken I and II). The immunoreactive peak in the final purification step had a retention time identical to that of Trp7, Leu8-GnRH and an UV absorbance (280 nm) peak appropriate for two tryptophan residues in the peptide, as in Trp7, Leu8-GnRH. Six additional less hydrophobic forms of GnRH were detected. Tilapia brain extract contained two major GnRH molecular forms which had identical retention times to chicken GnRH I (Gln8-GnRH) and Trp7, Leu8-GnRH in an HPLC system which separates the natural vertebrate GnRHs. The immunological properties of these two immunoreactive peaks, determined by relative interaction with four region-specific GnRH antisera raised against vertebrate GnRHs, were identical to those of Gln8-GnRH and Trp7, Leu8-GnRH. Additional GnRH molecular forms were also detected. In summary, these findings indicate that a major GnRH molecule in hake pituitary gland is Trp7, Leu8-GnRH, while tilapia brain contains both Trp7, Leu8-GnRH and Gln8-GnRH. Additional GnRH molecular forms were detected in both species.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of gonadotropin-releasing hormone and associated binding substances in the blood serum of a holocephalan (Hydrolagus colliei)

The identity of the gonadotropin-releasing hormone (GnRH) form and the presence of GnRH-binding substances in the blood serum of the holocephalan, spotted ratfish (Hydrolagus colliei), were investigated. The GnRH-like peptides in the serum were identified on the basis of relative hydrophobicity using reverse-phase HPLC. [His⁵,Trp⁷,Tyr⁸]GnRH (chicken GnRH-II) was the only GnRH form detected in the serum. It has been previously shown to be the only GnRH form in the brain of this species. The presence of GnRH-binding substances was inferred by anomalous HPLC elution of GnRH, ultrafiltration behavior, and by the direct binding of iodinated GnRH analogues by blood serum components. The mean GnRH concentration in the extracted blood serum was 125 ± 11 pg ml⁻¹ (n = 5) in males and 64 ± 48 pg ml⁻¹ (n = 4) and 155 ± 26 (n = 4) in two separate groups of females. Measurement of GnRH in the blood serum is complicated by the presence of GnRH-binding substances, which may cause the coprecipitation of GnRH during extraction with organic solvents. The high concentration of GnRH and the presence of GnRH-binding substances suggest that systemic blood is the route by which GnRH reaches the gonadotropes and/or that GnRH may have a hormonal role in H. colliei.     

Identification of N-acetylglucosamine binding residues in Griffonia simplicifolia lectin II

Primary structure and crystallographic data of several legume lectins were used to predict the involvement in carbohydrate binding of six amino acid residues (Asp⁸⁸, Glu¹⁰⁸, Tyr¹³⁴, Asn¹³⁶, Leu²²⁶ and Gln²²⁷) in Griffonia simplicifolia lectin II (GS-II). The functional involvement of these residues was evaluated by assessing GlcNAc binding of modified forms of GS-II in which these residues were eliminated in truncated peptides or systematically substituted with other amino acids by site-specific mutations. Mutations at (Asp⁸⁸, Tyr¹³⁴ or Asn¹³⁶ eliminated GlcNAc binding activity by GS-II, while those at Glut¹⁰⁸, Leu²²⁶ or Gln²²⁷ did not alter the activity. The former three amino acids were functionally essential for carbohydrate binding by GS-II presumably through hydrogen bonding to and hydrophobic interactions with GlcNAc. Although an Asp or Gly substitution for Tyr¹³⁴ eliminated GlcNAc affinity, substitution with Phe did not appreciably affect binding. Despite the fact that mutations to Leu²²⁶ and Gln²²⁷ did not alter carbohydrate binding, a truncated form of GS-II lacking these residues no longer exhibited carbohydrate binding affinity.     

Trp7,Leu8]LH-RH in reptilian brain

Luteinizing hormone-releasing hormone (LH-RH) immunoreactive peptides in acetic acid extracts of lizard (Cordylis nigra) brain were studied by high performance liquid chromatography (HPLC) and radioimmunoassay with region-specific antisera. Four different LH-RH immunoreactive peptides were detected. The major form co-eluted with salmon brain LH-RH, [Trp7,Leu8]LH-RH, in a cation exchange and three reverse phase HPLC systems which were specifically designed to separate a range of LH-RH analogues. The interaction of this major LH-RH immunoreactive peptide with a number of antisera directed against different regions of mammalian, chicken and salmon LH-RH was similar to the relative interaction of [Trp7,Leu8]LH-RH with these antisera. These data strongly indicate that the major form of lizard brain LH-RH is identical to salmon brain LH-RH [( Trp7,Leu8]LH-RH). The three additional molecular forms of immunoreactive LH-RH in lizard brain appear to differ from mammalian LH-RH in the middle to C-terminal region of the molecule.     

Gonadotropin-releasing hormone in elasmobranch (electric ray, Torpedo marmorata) brain and plasma: chromatographic and immunological evidence for chicken GnRH II and novel molecular forms.

Gonadotropin-releasing hormone (GnRH) peptides in the brain, testis and plasma of an electric ray (Torpedo marmorata) were investigated by gel filtration chromatography, reverse phase high performance liquid chromatography and radioimmunoassay with region-specific antisera. In the brain, two major forms of GnRH were demonstrated. One form had identical chromatographic and immunological properties to chicken GnRH II, and the second, novel, molecular form had structural features in common with mammalian, chicken II and salmon GnRHs. A minor, early-eluting immunoreactive peak, possibly also a novel GnRH, was also evident. Immunoreactive GnRH was not detected in the testis. In the plasma, a single major early-eluting immunoreactive peak was demonstrated. This peak, identical to the minor peak observed in the brain, is likely to represent a novel form of GnRH which has immunological properties in common with mammalian, chicken II and salmon GnRHs. Immunoreactive GnRH was not detected in the plasma of species from other vertebrate classes, including rabbit, chicken, monitor lizard, clawed toad, frog, cichlid fish and lamprey. The finding of chicken GnRH II in a species of Chondrichthyes adds further support to our hypothesis that this widespread structural variant may represent an early-evolved and conserved form of GnRH. The presence of a GnRH molecular form in the plasma of the electric ray suggests that GnRH may reach target organs (pituitary and gonads) via the general circulation in some species of Chondrichthyes.     

A radioimmunoassay specific for [Gln]LH-RH: Application in the confirmation of the structure of chicken hypothalamic luteinizing hormone-releasing hormone

In the first report on the chemical structure of a nonmammalian LH-RH, chicken hypothalamic LH-RH was demonstrated to be [Gln⁸]LH-RH [2–4]. However, these studies and subsequent reports [7,8] did not totally exclude the possibility of a reverse sequence of the two amino acids Leu-Gln. In view of the recently described structure of salmon brain LH-RH as [Trp⁷,Leu⁸]LH-RH [9], we undertook to confirm our earlier conclusion that chicken LH-RH is [Gln⁸]LH-RH and not [Gln⁷,Leu⁸]LH-RH. The immunologic, chromatographic and biological properties of natural chicken hypothalamic LH-RH were compared with those of the two synthetic peptides, [Gln⁸]LH-RH and [Gln⁷,Leu⁸]LH-RH. A radioimmunoassay highly specific for [Gln⁸]LH-RH was developed. Natural chicken LH-RH cross-reacted fully with the antiserum which requires the COOH-terminal Gln⁸ to Gly¹⁰-NH₂ for binding, while [Gln⁷,Leu⁸]LH-RH showed less than 0.1% cross-reaction. On a high resolution reverse phase high performance liquid chromatography system, natural chicken LH-RH co-eluted with [Gln⁸]LH-RH and was well separated from [Gln⁷,Leu⁸]LH-RH. In a chicken anterior pituitary cell bioassay, natural chicken LH-RH and [Gln⁸]LH-RH were equipotent in stimulating luteinizing hormone release, while the relative potency of [Gln⁷,Leu⁸]LH-RH was 4.4%. These data, in particular the use of a specific [Gln⁸]LH-RH antiserum, provide conclusive evidence that chicken LH-RH is [Gln⁸]LH-RH.     

Tachykinins (Substance P, Neurokinin A and Neuropeptide γ) and Neurotensin from the Intestine of the Burmese Python, Python molurus

Conlon, J. M., T. E. Adrian and S. M. Secor. Tachykinins (substance P, neurokinin A and neuropeptide γ,) and neurotensin from the intestine of the burmese python, Python molurus. Peptides 18(10) 1505–1510, 1997.—Peptides with substance P-like immunoreactivity, neurokinin A-like immunoreactivity and neurotensin-like immunoreactivity were isolated in pure form from an extract of the intestine of the Burmese python (Python molurus). The primary structure of python substance P (Arg-Pro-Arg-Pro-Gln-Gln-Phe-Tyr-Gly-Leu-Met-NH₂) shows one amino acid substitution (Phe⁸ → Tyr) compared with chicken/alligator substance P and an additional substitution (Lys³ → Arg) as compared with mammalian substance P. The neurokinin A-like immunoreactivity was separated into two components. Python neuropeptide γ (Asp-Ala-Gly-Tyr-Ser-Pro-Leu-Ser-His-Lys-Arg-His-Lys-Thr-Asp-Ser-Phe-Val-Gly-Leu-Met-NH₂ shows three substitutions (Gly⁵ → Ser, Gln⁶ → Pro and Ile⁷ → Leu) compared with alligator neuropeptide γ and an additional substitution (His⁴ → Tyr) compared with mammalian neuropeptide γ. Python neurokinin A (His-Lys-Thr-Asp-Ser-Phe-Val-Gly-Leu-Met.NH₂) is identical to human/chicken/alligator neurokinin A. Python neurotensin (pGlu-Leu-Val-His-Asn-Lys-Ala-Arg-Pro-Tyr-Ile-Leu) is identical to chicken/alligator neurotensin. The data are indicative of differential evolutionary pressure to conserve the amino acid sequences of reptilian gastrointestinal peptides.     

Identification of Gln8-GnRH and His5,Trp7,Tyr8-GnRH in the hypothalamus and extrahypothalamic brain of the ostrich (Struthio camelus)

Gonadotropin-releasing hormone (GnRH) molecular forms were studied in extracts of ostrich hypothalamus and extrahypothalamic brain using high performance liquid chromatography, radioimmunoassay with region-specific antisera and assessment of luteinizing hormone (LH)-releasing activity using chicken dispersed pituitary cells. Two molecular forms of GnRH with chromatographic, immunological and biological properties identical to those of Gln8-GnRH and His5,Trp7,Tyr8-GnRH were demonstrated in both the hypothalamic and extrahypothalamic brain extracts. A greater proportion of His5,Trp7,Tyr8-GnRH was present in the hypothalamus than in extrahypothalamic brain. It is likely that these two forms of GnRH are present in all bird species, since the chicken and the ostrich have evolved separately.     

Chromatographic and immunological evidence for mammalian GnRH and chicken GnRH II in eel (Anguilla anguilla) brain and pituitary

Gonadotropin-releasing hormone (GnRH) peptides in the brain and pituitary of the European eel (Anguilla anguilla) were investigated by reverse phase high performance liquid chromatography (HPLC) and radioimmunoassay with region-specific antisera. Two GnRH molecular forms were demonstrated in brain and pituitary extracts. One form eluted in the same position as synthetic mammalian GnRH on HPLC and was recognized by antibodies directed against the NH2 and COOH termini of mammalian GnRH as well as by antibodies to the middle region. The second form eluted in the same position as synthetic chicken GnRH II and was recognized by specific antibodies to this molecule. Salmon GnRH and chicken GnRH I were not detected. The occurrence of mammalian GnRH in teleost fish suggests that this molecular form is more ancient than was previously suspected and arose earlier than in primitive tetrapods, or that it has arisen in the eel through random mutation of salmon GnRH. The lack of salmon GnRH in the eel brain indicates that this molecular form is not common to all teleost species. The finding in eel brain of chicken GnRH II, which has previously been described in species of Mammalia, Aves, Reptilia, Amphibia, Osteichthyes, and Chondrichthyes, supports our hypothesis that this widespread structural variant may represent an early evolved and conserved form of GnRH.     

Differential regional distribution of gonadotropin-releasing hormones in amphibian (clawed toad, Xenopus laevis) brain

In most vertebrate species two forms of gonadotropin-releasing hormone (GnRH) are present in the brain, and their differential distribution suggests they have different functional roles. The regional distribution and relative concentrations of GnRH molecular forms in the brain of adult clawed toad (Xenopus laevis) were determined using high performance liquid chromatography and radioimmunoassay with a library of region-specific GnRH antisera. Four immunoreactive forms of GnRH were detected: mammalian, hydroxyproline mammalian, chicken II, and an unidetified form of GnRH. Mammalian GnRH was distributed throughout the brain, and hydroxyproline mammalian was present in the forebrain, midbrain (excluding hypothalamus), and hypothalamus. Chicken GnRH II also occurred throughout the brain, but was present in greater amounts in the hindbrain and midbrain (excluding hypothalamus). An unidentified form of GnRH with properties of salmon GnRH was detected in the forebrain. Considering the relative proportions of mammalian GnRH and chicken GnRH II in the major brain areas, the concentration of mammalian GnRH was high in the forebrain, midbrain (excluding hypothalamus), and in particular in the hypothalamus, and very little chicken GnRH II was present in these areas. In the hindbrain, chicken GnRH II predominated and the concentration of chicken GnRH II was highest in the medulla. These findings suggest: (1) mammalian GnRH is the prime regulator of gonadotropin release from the pituitary, and (2) chicken GnRH II has an extrapituitary role.     

Fluorescence studies of endothelin-1

Fluorescence measurements and singlet singlet energy transfer experiments on endothelin-1 provide information on the conformation of this peptide in dilute aqueous solution. The tyrosine fluorescence quantum yield in the absence of transfer (in [Phe²¹]endothelin-1) is relatively large (Φ_tyr = 0.39), indicating the side-chain is oriented away from fluorescence quenching groups such as the two disulfide bonds of the peptide. The fluorescence emission maximum (λ = 351 nm) and quantum yield (Φ_trp = 0.099) of tryptophan in endothelin-I suggests that this residue is fully accessible to the solvent and that the indole ring is not located near the fluorescence quenching histidinium moiety or the disulfide bonds.

Singlet-singlet fluorescence energy transfer measurements of the Tyr¹³/Trp²¹ intramolecular distance by both donor fluorescence quenching and relative enhancement of acceptor fluorescence yield a distance of about 12.8 ± 0.6 Å. Molecular modeling of a fully extended C-terminal hexapeptide indicates a Tyr¹³/Trp²¹ distance of about 25 Å. Thus, the C-terminal residues must bend back towards the bicyclic portion of the molecule.     

Primary structure of two forms of gonadotropin-releasing hormone from brains of the American alligator (Alligator mississippiensis)

Two forms of gonadotropin-releasing hormone (GnRH) have been purified from brains of the American alligator, Alligator mississippiensis, using reverse-phase high-pressure liquid chromatography (HPLC). The concentration of total GnRH was 8.8 ng/g of frozen brain tissue or 21.1 ng per brain. The amino acid sequence of each form of GnRH was determined using automated Edman degradation. The presence of the N-terminal pGlu residue was established by digestion studies with bovine pyroglutamyl aminopeptidase and coelution with synthetic forms of the native peptide. The primary structure of alligator GnRH I is pGlu-His-Trp-Ser-Tyr-Gly-Leu-Gln-Pro-Gly-NH2 and alligator GnRH II is pGlu-His-Trp-Ser-His-Gly-Trp-Tyr-Pro-Gly-NH2.     

Structure-activity relationship in vasoconstrictor effects of sarafotoxins andendothelin-1

Sarafotoxins (SRTa, SRTb and SRTc) as well as endothelin-1 (ET-1) produced vasoconstrictions in rat thoracic aorta, rat isolated perfused mesentery and pithed rat in various of extents. The potency was ET-1> SRTb> SRTa> SRTc at lower doses, but SRTb> ET-1> SRTa> SRTc at higher doses. [Nitrophenylsulfenylated Trp²¹]SRTb and SRTb(1-19) caused no vasoconstriction. Either the reduction and carboxymethylation of Cys residues, the destruction of the intramolecular loop or the production of the non-natural disulfide bond, eliminated the constrictor activity. These results indicate that Trp²¹ and the intramolecular loop structure are essential, and Lys⁹ and Tyr¹³ may play some important roles for the vasoconstrictor activity of these peptides.     

Presence of corazonin in three insect species, and isolation and identification of [His7]corazonin from Schistocerca americana.

An ELISA for corazonin, a cardioactive neuropeptide from the American cockroach, Periplaneta americana, was developed. It was used to isolate corazonin from the cockroach Nauphoeta cinerea, the locust Schistocerca americana, and the hawkmoth Manduca sexta. The peptides from Nauphoeta and Manduca had the same retention times as Periplaneta corazonin, and their amino acid compositions also suggested that these peptides are identical with corazonin. The corazonin-immunoreactive peptide from Schistocerca eluted slightly earlier on HPLC than corazonin, and its structure was determined to be [His⁷]corazonin, or pGlu-Thr-Phe-Gln-Tyr-Ser-His-Gly-Trp-Thr-Asn-amide. These results indicate that corazonin is generally present in insects and that its structure has been well conserved.     

Divalent cation coordination and mode of membrane interaction in cyclotides: NMR spatial structure of ternary complex Kalata B7/Mn2+/DPC micelle

The cyclotides are the family of hydrophobic bioactive plant peptides, characterized by a circular protein backbone and three knot forming disulfide bonds. It is believed that membrane activity of the cyclotides underlines their antimicrobial, cytotoxic and hemolytic properties, but the specific interactions with divalent cations can be also involved. To assess the mode of membrane interaction and divalent cation coordination in cyclotides, the spatial structure of the Möbius cyclotide Kalata B7 from the African perennial plant Oldenlandia affinis was determined in the presence of anisotropic membrane mimetic (dodecylphosphocholine micelles). The model of peptide/cation/micelle complex was built using 5-doxylstearate and Mn²⁺ relaxation probes. Results show that the peptide binds to the micelle surface with relatively high affinity by two hydrophobic loops (loop 2 – Thr⁶-Leu⁷ and loop 5 – Trp¹⁹-Ile²¹). The partially hydrated divalent cation is coordinated by charged side-chain of Glu³, aromatic side chain of Tyr¹¹ and free carbonyls of Thr⁴ and Thr⁹, and is located in direct contact with the polar head-groups of detergent. The comparison with data about other cyclotides indicates that divalent cation coordination is the invariant property of all cyclotides, but the mode of peptide/membrane interactions is varied. Probably, the specific cation/peptide interactions play a major, but yet not known, role in the biological activity of the cyclotides.     

Partial characterization of four forms of immunoreactive gonadotropin-releasing hormone in the brain and terminal nerve of the spiny dogfish (Elasmobranchii; Squalus acanthias).

Four forms of immunoreactive GnRH have been detected in tissue extracts of both whole brains and terminal nerves from the spiny dogfish (Squalus acanthias). The GnRH forms were characterized using reverse-phase high pressure liquid chromatography (HPLC) and immunological recognition with four different antisera. Three of these forms possess immunological and chromatographic properties consistent with known forms of GnRH: mammalian GnRH, chicken GnRH-II and salmon GnRH. An additional form, with an HPLC elution position intermediate between chicken GnRH-II and salmon GnRH appears to be a new structure of GnRH. The presence of all four GnRH forms in the terminal nerve suggests a lack of regional specificity of the expressed forms of GnRH in the brain.     

Molecular cloning of a phospholipid-hydroperoxide glutathione peroxidase gene from the tick, Boophilus microplus (Acari: Ixodidae)

Phospholipid-hydroperoxide glutathione peroxidase (PHGPx) enzymes are associated with cellular protection by the role they play in reducing hydroperoxides of phospholipids, thereby preventing membrane lipoperoxidation. As part of their toxic effect, some pesticides stimulate peroxidation of cellular membranes. We isolated and sequenced a PHGPx gene from the cattle tick Boophilus microplus that encodes a protein of 169 amino acids, including a TGA-encoded selenocysteine at residue 46 and active site residues Gln⁸² and Trp¹³⁵ that interact with the selenocysteine. The motif that directs the insertion of selenocysteine at the opal codon is found in the 3′-untranslated region. PHGPx sequences from pesticide-resistant and susceptible B. microplus ticks show nucleotide differences at eight positions among the strains, with five resulting in amino acid substitutions in the deduced protein sequence. Two distinct PHGPx alleles were identified in an organophosphate-resistant tick strain. Real-time PCR quantification of gene expression revealed increased PHGPx in two strains resistant to a single acaricide class. Strains resistant to two or more classes showed a reduction in PHGPx.     

Characterization and localization of gonadotropin-releasing hormone in the brain and pituitary of the three-spined stickleback,Gasterosteus aculeatus

Radioimmunoassay (RIA) studies on highperformance liquid chromatography (HPLC) fractions of brain extracts of the three-spined stickleback, Gasterosteus aculeatus, provided evidence for at least two forms of gonadotropin-releasing hormone (GnRH). One form showed chromatographic and immunological properties similar to that of synthetic salmon GnRH (sGnRH). A second, unidentified form of GnRH eluted in the same position as chicken GnRH I (cGnRH-I); however, it did not cross-react in a cGnRH-I RIA. Furthermore, it cannot be excluded that chicken GnRH II (cGnRH-II) and maybe one other unidentified form are present in the stickleback. The distribution of GnRH in the brain of breeding adult male sticklebacks was studied by use of immunohistochemistry. Two antisera against sGnRH and antisera against mGnRH and cGnRH-II were applied on cryosections and visualized using the peroxidase-antiperoxidase method. Staining patterns were similar after incubations with all four antisera. Immunoreactive fibers were found in most parts of the brain. Three distinct groups of GnRH-immunoreactive perikarya were found in the nucleus olfactoretinalis, in the nucleus anterior periventricularis, and in the nucleus lateralis tuberis. Moreover, weakly stained cells occurred in a periventricular position in the midbrain. The proximal pars distalis of the pituitary, housing the gonadotropic cells, was richly innervated by GnRH-positive fibers. In the pars intermedia and in the rostral pars distalis, immunoreactive fibers were absent.