Identification, by RT-PCR, of eight novel I₂-conotoxins from the worm-hunting cone snails Conus brunneus, Conus nux, and Conus princeps from the eastern Pacific (Mexico).

Marine snails of the genus Conus (∼500 species) are tropical predators that produce venoms for capturing prey, defense and competitive interactions. These venoms contain 50-200 different peptides ("conotoxins") that generally comprise 7-40 amino acid residues (including 0-5 disulfide bridges), and that frequently contain diverse posttranslational modifications, some of which have been demonstrated to be important for folding, stability, and biological activity. Most conotoxins affect voltage- and ligand-gated ion channels, G protein-coupled receptors, and neurotransmitter transporters, generally with high affinity and specificity.

Purification and structural characterization of lectins from the cnidarian Bunodeopsis antilliensis [corrected].

Purification and characterization of two different lectins from the Mexican anemone Bunodeopsis antilliensis are reported. These two lectins named Bunodeopsis antilliensis agglutinin-A (BAA-A) and -B (BAA-B) presented the following characteristics: BAA-A was resolved as a component, with haemagglutinating activity for human blood type A (N-acetylgalactosamine-galactose-fucose), with a molecular weight of 28,900 obtained by means of mass spectrometry, showed an isoelectric point of 5.04 with a higher carbohydrate specificity for N-acetylgalactosamine (GalNAc).

Combined solid-phase/solution synthesis of a 31-residue vasoactive intestinal peptide analog: general method for repetitive coupling of fragments without isolation and purification of intermediates.

A novel analog of vasoactive intestinal peptide (VIP) has been reported which exhibits high potency and enhanced duration of in vivo biological activity. This VIP analog, cyclo-(Lys21-Asp25)Ac[Glu8 Lys12 Nle17 Ala19, Asp25 Leu26,Lys27,28,Gly29,30,Thr31]-VIP, which also has a lactam bridge, has been reported to have relaxant effects that are significantly more potent than other beta-agonists such as salbutamol and salmeterol. Because it has potential use for the treatment of bronchial asthma in humans, various convergent syntheses were evaluated to enable the economic preparation of large quantities of this medium-sized hentriacontapeptide.

Pegylated peptides. V. Carboxy-terminal PEGylated analogs of growth hormone-releasing factor (GRF) display enhanced duration of biological activity in vivo.

In the present study, human growth hormone-releasing factor (hGRF) and analogs were successfully pegylated at the carboxy-terminus using a novel solid- and solution-phase strategy. Following synthesis, these pegylated hGRF analogs were evaluated for in vitro and in vivo biological activity. Specifically, hGRF (1-29)-NH2, [Ala15]-hGRF (1-29)-NH2, [desNH2Tyr1, D-Ala2, Ala15]-hGRF(1-29)-NH2 and [His1, Val2, Gln8, Ala15, Leu27]-hGRF(1-32)-OH were each C-terminally extended using a Gly-Gly-Cys-NH2 spacer (previously demonstrated not to alter intrinsic biological activity), and then monopegylated via coupling to an activated dithiopyridyl-PEG reagent.

Ras-dependent maturation of Xenopus oocytes is blocked by modified peptides of GTPase activating protein (GAP).

Guanosine triphosphatase activating protein (GAP) is an important modulator of p21ras (Ras)-dependent signal transduction in mammalian cells and in insulin-induced maturation of Xenopus oocytes. A synthetic octapeptide from the catalytic domain of GAP, residues 899-906 (F899VFLRLIC906), inhibited GAP-stimulated hydrolysis of GTP to GDP by Ras in an in vitro biochemical assay (IC50 = 12 microM). The peptide was assayed for its ability to block insulin- (Ras-dependent) and progesterone- (Ras-independent) induced maturation of stage VI Xenopus laevis oocytes, marked by germinal vesicle breakdown (GVBD).

Membrane topology of the rat kidney neutral and basic amino acid transporter.

A recently cloned rat kidney protein (NBAT) mediates the sodium-independent transport of neutral as well as basic amino acids and cystine when expressed in Xenopus laevis oocytes. The human equivalent of this transporter may be the one that is defective in cystinuria. Immunocytochemical studies have indicated that NBAT is primarily localized in the brush border membranes of rat kidney and intestinal epithelial cells, a localization consistent with its proposed role in amino acid transport.

Peptide synthesis catalyzed by the Glu/Asp-specific endopeptidase. Influence of the ester leaving group of the acyl donor on yield and catalytic efficiency.

We recently described a two-step enzymatic semisynthesis of the superpotent analog of human growth hormone releasing factor, [desNH2Tyr1,D-Ala2,Ala15]-GRF(1-29)-NH2 (4), from the precursor, [Ala15,29]-GRF(4-29)-OH (1). C-Terminal amidation of 1 to form [Ala15]-GRF(4-29)-NH2 (2) was achieved by carboxypeptidase-Y-catalyzed exchange of Ala29-OH for Arg-NH2. The target analog 4 was then obtained by acylation of segment 2 with desNH2Tyr-D-Ala-Asp(OH)-OR (3) (R = CH3CH2- or 4-NO2C6H4CH2-) catalyzed by the V8 protease.

Effect of daily replacement therapy with recombinant bovine somatotropin on somatotropin, insulin-like growth factor I, and onset of puberty in beef heifers immunized against growth hormone-releasing factor.

Two experiments examined whether replacement therapy with recombinantly derived bovine somatotropin (rbST) would induce puberty in heifers that had been actively immunized at 6 mo of age against growth hormone-releasing factor (GRF). Heifers received daily i.m.

Concentrations of hormones and metabolites, estimates of metabolism, performance, and reproductive performance of sows actively immunized against growth hormone-releasing factor.

Cyclic females actively immunized against growth hormone-releasing factor (GRF; n = 5) or human serum albumin (HSAi; n = 4) were used to determine the effects of reduced serum somatotropin (ST) and IGF-I on metabolism and production in gestating and lactating sows. Sows farrowed, pigs were weaned at 28 d of lactation, and sows were observed for estrus after weaning. Blood samples were collected at 15-min intervals for 5 to 6 h on d 110 of gestation and d 21 of lactation.

Radioimmunoassays for the C-terminus of prothymosin alpha and the N-terminus of parathymosin alpha for the measurement of the levels of alpha-thymosins in human cancer.

A radioimmunoassay specific for the C-terminus of human prothymosin alpha was developed using the synthetic peptide [Cys-Aca degrees]-human prothymosin alpha (90-109)-OH coupled to KLH as antigen and the analogue [Tyr-Aca degrees]-human prothymosin alpha (90-109)-OH labelled with 125I as tracer. The radioimmunoassay measured intact prothymosin alpha, in the range of 2-100 pmol and does not cross-react with the partly homologous polypeptide parathymosin alpha. A major epitope was located in the segment 95-107.

Recent applications of enzymatic peptide synthesis.

Recent applications of enzyme catalysis in peptide synthesis are reviewed. A brief history of the development of these techniques is presented, and existing strategies and tactics of regio- and stereospecific peptide bond syntheses catalyzed by proteolytic enzymes are summarized. The recent literature (ca.

Enhanced stability and potency of novel growth hormone-releasing factor (GRF) analogues derived from rodent and human GRF sequences.

Native human GRF(1-44)-NH2(hGRF44) is subject to biological inactivation by both enzymatic and chemical routes. In plasma, hGRF44 is rapidly degraded via dipeptidylpeptidase IV (DPP-IV) cleavage between residues Ala2 and Asp3. The hGRF44 is also subject to chemical rearrangement (Asn8-->Asp8, beta-Asp8 via aminosuccinimide formation) and oxidation [Met27-->Met(O)27] in aqueous environments, greatly reducing its bioactivity.

Changes in serum somatotropin, somatotropin mRNA, and serum and follicular insulin-like growth factor-I in response to feed restriction in cows actively immunized against growth hormone-releasing factor.

Cyclic cows immunized against growth hormone-releasing factor (GRFi, n = 19), human serum albumin (HSAi, n = 10), or not immunized (CON, n = 18) were used to investigate the effects of feed restriction on serum and pituitary somatotropin (ST), pituitary ST mRNA, and serum and follicular IGF-I. Cows were either fed 2.7 kg/d cottonseed hulls (R) or given ad libitum access to feed (AL) for 15 d.

Feedlot performance, carcass characteristics, hormones, and metabolites in steers actively immunized against growth hormone-releasing factor.

Large-framed Simmental and Charolais steers were actively immunized against growth hormone-releasing factor (GRF) to evaluate the effect on growth, carcass characteristics (especially intramuscular fat deposition), and concentrations of somatotropin (ST) and IGF-I. Primary immunizations of 1.5 mg of GRF-(1-29)-Gly-Gly-Cys-NH2 conjugated to 1.

Effect of feed restriction on serum somatotropin, insulin-like growth factor-I-(IGF-I) and IGF binding proteins in cyclic heifers actively immunized against growth hormone releasing factor.

Feed restriction often increases serum somatotropin (ST) and decreases insulin-like growth factor-I (IGF-I) in ruminants; however, the mechanisms responsible for this change in ST and IGF-I are not well defined. We investigated the effects of feed restriction on serum ST, IGF-I, IGF binding proteins (IGFBP), insulin and nonesterified fatty acids (NEFA) in cyclic Angus and Charolais heifers (n = 15) previously immunized against growth hormone releasing factor (GRFi) or human serum albumin (HSAi). Cows were fed a concentrate diet ad libitum (AL) or were restricted to 2 kg cotton seed hulls (R) for 4 d.

Characterization of the rat neutral and basic amino acid transporter utilizing anti-peptide antibodies.

High-titer, site-specific antibodies have been produced against the rat kidney broad-spectrum, sodium-independent neutral and basic amino acid transporter (NBAA-Tr) whose cDNA we cloned earlier. These antibodies have allowed us to characterize the transporter protein in normal rat tissues and in various cellular and in vitro expression systems. Western analysis detected 84- to 87-kDa glycosylated species enriched in rat renal and jejunal epithelial cell brush border membranes.

Endocrine events prior to puberty in heifers: role of somatotropin, insulin-like growth factor-I and insulin-like growth factor binding proteins.

We have utilized active immunization against growth hormone releasing factor (GRF) to investigate relationships among somatotropin (ST), insulin-like growth factor-I (IGF-I), IGF binding proteins (IGFBP) and ovarian function in heifers. Active immunization against GRF (GRFi) has been demonstrated to abolish episodic release of ST and decrease serum concentrations of IGF-I. In initial experiments investigating onset of puberty, breeds of heifers differing in growth rate and reproductive traits (Angus, Charolais and Simmental) were immunized against GRF or served as controls (immunized against carrier protein, human serum albumin, HSAi).

Enzymatic semisynthesis of a superpotent analog of human growth hormone-releasing factor.

A superpotent analog of human growth hormone-releasing factor, [desNH2Tyr1,D-Ala2,Ala15]-GRF(1-29)-NH2 (4), was prepared from the precursor, [Ala15,29]-GRF(4-29)-OH (1), by a two-step enzymatic semisynthesis. The amidated C-terminus, essential for high biological potency, was obtained via a carboxypeptidase Y-catalyzed exchange of Ala29-OH for Arg29-NH2 to produce [Ala15]-GRF(4-29)-NH2 (2). The N-terminal desNH2Tyr-D-Ala moiety, which greatly increases in vivo duration of action, was then incorporated by V8 protease-catalyzed condensation of segment 2 with desNH2Tyr-D-Ala-Asp(OH)-OR [R = CH3CH2- (3a) or 4-NO2C6H4CH2-(3b)].

Semisynthesis of human growth hormone-releasing factor by trypsin catalyzed coupling of leucine amide to a C-terminal acid precursor.

Human growth hormone-releasing factor, GRF(1-44)-NH2, was synthesized by trypsin catalyzed coupling of Leu-NH2 to Arg43 of the precursor, GRF(1-43)-OH, prepared by solid phase peptide synthesis. The semisynthetic GRF(1-44)-NH2 was fully characterized and showed full potency in the rat pituitary in vitro bioassay. Conversion to GRF(1-44)-NH2 was limited to 60-70% in both 75% v:v N,N'-dimethylacetamide and 95% v:v 1,4-butanediol due to competing transpeptidations at Arg41 and Arg38 generating [Leu42]-GRF(1-42)-NH2 and [Leu39]-GRF(1-39)-NH2 side-products, respectively.

Kinetics of dipeptidyl peptidase IV proteolysis of growth hormone-releasing factor and analogs.

The kinetics and selectivity of proteolysis of synthetic human growth hormone-releasing factor and analogs by purified human placental dipeptidyl peptidase IV (DPP IV) were studied by HPLC. The initial rates of Ala2-Asp3 cleavage (pH 7.8, 37 degrees C, So = 0.

Synthesis and biological activity of novel C-terminal-extended and biotinylated growth hormone-releasing factor (GRF) analogs.

A series of novel hGRF(1-29)-NH2 analogs were synthesized and biotinylated. The immunological and biological activities of these analogs were then characterized. To distance the biotin moiety from the putative bioactive core, a C-terminal spacer arm consisting of -Gly-Gly-Cys-NH2 (-GGC) was added to hGRF(1-29)-NH2 (hGRF29) and analogs, with subsequent biotinylation performed at the cysteine residue.

Semisynthesis of human growth hormone-releasing factors by alpha-amidating enzyme catalyzed oxidation of glycine-extended precursors.

Recombinant alpha-amidating enzyme was used in the semisynthesis (1-5 mg scale) of human growth hormone-releasing factor, GRF(1-44)-NH2, by in vitro enzymatic oxidation of the glycine-extended precursor, GRF(1-44)-Gly-OH, prepared by solid-phase synthesis. The equipotent analog, GRF(1-29)-NH2, and the superactive analog, [Ala15]-GRF(1-29)-NH2, were also prepared by this route and were fully characterized. Isolated yields of about 75% were obtained, and the products each possessed full potency in an in vitro rat pituitary bioassay and full receptor-binding affinity.

Solution structures of cyclic and dicyclic analogues of growth hormone releasing factor as determined by two-dimensional NMR and CD spectroscopies and constrained molecular dynamics.

Solution structures were determined for a linear analogue of growth hormone releasing factor (GRF), and cyclic and dicyclic analogues in which the side chains of aspartyl and lysyl residues spaced at positions i-(i + 4) were joined to form a lactam. The four analogues were [Ala15]-GRF-(1-29)-NH2 and its cyclo8-12, cyclo21-25, and dicyclo8-12;21-25 derivatives. The peptides were studied in two solvent systems: 75% methanol/25% water at pH 6.

A sensitive and specific competition microELISA for the immunoactive polypeptide parathymosin and detection of this peptide in porcine tissues.

A sensitive and specific microELISA assay is described for the immunoactive polypeptide parathymosin. Antibodies against a synthetic peptide corresponding to the rat parathymosin sequence 5-30 were raised in rabbits immunised with this peptide conjugated to keyhole limpet hemocyanin (KLH). The useful range of the assay was 0.

Effect of active immunization against growth hormone releasing factor on concentrations of somatotropin and insulin-like growth factor I in lactating beef cows.

Two experiments were conducted to determine the effects of immunoneutralization of growth hormone-releasing factor [GRF(1-29)-NH2] on concentrations of somatotropin (ST) and insulin-like growth factor I (IGF-I) in lactating beef cows. In Experiment 1, multiparous Hereford cows were immunized against 2 mg GRF(1-29)-(Gly)4-Cys-NH2 conjugated to human serum albumin (GRFi, n = 3) or 2 mg human serum albumin (HSAi, n = 3) at 52 +/- 1 d prior to parturition. Boosters (1 mg) were administered on days 12, 40 and 114 postpartum (pp).